Research ArticleSesquiterpene Lactones Inhibit Advanced OxidationProtein Product-Induced MCP-1 Expression in Podocytes viaan IKKNF-120581B-Dependent Mechanism

Yan Zhao12 Si-jia Chen1 Jian-cheng Wang1 Hong-xin Niu1

Qian-qian Jia3 Xiao-wen Chen1 Xiao-yan Du1 Lu Lu4 Bo Huang1 Quan Zhang5

Yue Chen5 and Hai-bo Long1

1Division of Nephrology Zhujiang Hospital Southern Medical University Guangzhou Guangdong 510280 China2Department of Nephrology Xiangyang Central Hospital Hubei University of Arts and Science Xiangyang Hubei 441021 China3Department of Nephrology Zhumadian Central Hospital Zhumadian Henan 463000 China4Department of Traditional Chinese Medicine Zhujiang Hospital Southern Medical University GuangzhouGuangdong 510280 China5College of Pharmacy The State Key Laboratory of Elemento-Organic Chemistryand Tianjin Key Laboratory of Molecular Drug Research Nankai University Tianjin 300071 China

Correspondence should be addressed to Hai-bo Long longhb1966163com

Received 7 September 2014 Accepted 10 December 2014

Academic Editor Vittorio Calabrese

Copyright copy 2015 Yan Zhao et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Inflammation is a relevant factor in the pathogenesis of diabetes nephropathy (DN) Sesquiterpene lactones (SLs) originally isolatedfrom Tanacetum parthenium have been reported to exhibit anti-inflammatory effects but few studies have examined their effectson DN To determine whether advanced oxidation protein products (AOPPs) can induce the expression of chemokine monocytechemoattractant protein- (MCP-) 1 in cultured mouse podocytes and to explore the mechanisms of the potential renoprotectionof SLs we treated podocytes with AOPPs and SLs (parthenolide and its derivatives micheliolide compound 1 and compound 2)MCP-1mRNAandprotein expressionwere tested using quantitative real-timePCR andELISA respectively and the protein levels ofIKK120573 phospho-IKK120573 I120581B120572 NF-120581B p65 phospho-NF-120581B p65 and tubulin were analyzed byWestern blotting AOPPs activated theexpression of MCP-1 mRNA and protein in a dose- and time-dependent manner activated IKK120573 and NF-120581B p65 and promotedI120581B120572 degradation The IKKNF-120581B inhibitor parthenolide decreased AOPP-induced MCP-1 expression Pretreatment with SLsinhibited MCP-1 mRNA and protein expression and suppressed IKK120573 and NF-120581B p65 phosphorylation and I120581B120572 degradationTaken together these findings provide a novel explanation for the anti-inflammatory effects of SLs that will ultimately benefit DNand potentially other inflammatory and immune renal diseases

1 Introduction

The latest data from the International Diabetes Federationreveal that there are currently 371 million people livingwith diabetes and another 280 million are at high risk ofdeveloping the disease Half of a billion people are expectedto be livingwith diabetes by 2030 DN is a commonmicrovas-cular complication of diabetes leading to premature deathand end-stage renal disease (ESRD) Remarkably the excessrisk of death from any cause with type 1 or 2 diabetes is

associated almost entirely with the presence of kidney disease[1ndash3] Recent studies have shown that kidney inflammationis crucial in promoting the development and progression ofDN Inflammation may be a key factor which is activatedby the metabolic biochemical and hemodynamic derange-ments known to exist in the diabetic kidney [4ndash6] MCP-1 also known as C-C motif chemokine (CCL) 2 increasesprogressively in diabetic kidneys in animal models [7] MCP-1 is involved in the direction of macrophage migration intothe diabetic kidney upregulates the expression of adhesion

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 934058 13 pageshttpdxdoiorg1011552015934058

2 Oxidative Medicine and Cellular Longevity

molecules and promotes the expression of other proinflam-matory cytokines in DN [8] MCP-1 can be synthesizedby mononuclear cells and renal resident cells includingpodocytes [5 9] The podocyte seems to be a suitable choicefor further investigation asMCP-1 gene expression appears tobe predominantly localized to podocytes in the glomeruli ofdiabetic mice [7] The latest data show the urinary sedimentpodocalyxin to creatinine ratio had a positive correlationwith the urinary albumin to creatinine ratio and the urinaryMCP-1 to creatinine ratio in patients with type 2 diabetes[10] Other research has shown that once MCP-1 is secretedit can diffuse back to the podocyte and act in a loop byactivating the CCR2 receptor which then initiates profoundbiological effects [11] suggesting podocytes as a potentialtarget of inflammation in DN Considering that podocytesare related to increased proteinuria and contribute to renalprogression in DN [12] therapies aimed at preventing orlimiting podocyte injury andor promoting podocyte repairor regeneration may have major clinical and economicbenefits [13]

Advanced oxidation protein products (AOPPs) were firstdiscovered and reported as uremic toxins by Witko-Sarsat etal in 1996 [14] and were found in association with diabetes inlater studies [15 16] AOPPs are the dityrosine-containing andcross-linking protein products formed during oxidative stressby the reaction of serum protein with chlorinated oxidantsand are often carried by albumin in vivo [14 17] Recentstudies have found that the chronic accumulation of AOPPspromotes inflammation in both the diabetic and nondiabetickidney by significantly increasing macrophage infiltrationand the overexpression of MCP-1 in the remnant kidney andduring experimental diabetic nephropathy [18 19] AOPPscan also decrease the expression of nephrin and podocin inpodocytes resulting in podocyte apoptosis and deletion [20ndash22] Although these studies suggest that the accumulationof AOPPs has an important role in the progression of DNthe mechanism underlying the pathogenic effect of AOPPsremains to be further investigated

Sesquiterpene lactones originally isolated from medic-inal herbs of the Asteraceae family have been reported toexhibit a variety of anti-inflammatory immunomodulatoryand anticancer effects mostly based on their alkylatingcapabilities [23 24] Parthenolide (PTL) and other SLs havebeen reported to exhibit anti-inflammatory effects by inhibit-ing IKK and I120581B120572 phosphorylation andor affecting DNAbinding ability [25 26] Other studies have demonstrated thatPTL blocks MCP-1 mRNA and protein expression by inhibit-ing NF-120581B activity in experimental glomerulonephritis andhuman renal mesangial cells [27 28] as well as in podocytes[9 29]

Recently we demonstrated that SLs including PTLmicheliolide (MCL) and several synthetic analogs of thesemolecules could effectively attenuate the high glucose-stimulated and AOPP-induced activation of NF-120581B thedegradation of I120581B120572 and the expression of MCP-1 in ratmesangial cells (MCs) [30 31] These findings showed thatSLs can exert a beneficial role in high glucose-stimulatedand AOPPs-induced inflammation and ECM deposition bypreventing NF-120581B activation in MCs However whether

SLs have anti-inflammatory effects in podocytes under theaccumulation of AOPPs has not yet been clarified and ourprevious studies were focused on the screening of anti-inflammatory effects of SLs but the underlying mechanismof action has not yet been elucidated Thus the presentstudy aimed to characterize the inflammatory effect ofAOPPsin cultured mouse podocytes and more importantly weselected MCL compound 1 and compound 2 according toour previous experiments to further explore the mechanismof the potential anti-inflammatory effects of SLs

2 Materials and Methods

21 AOPPs-MSA Preparation and Determination AOPPs-MSA was prepared in vitro according to a previouslydescribed method [21] Briefly MSA solution (200mgmL)was incubated with 200mM HClO (Fluke Buchs Switzer-land) at an optimal ratio (MSA to HClO = 1 140) for 30minat 37∘C and dialyzed overnight against PBS to remove freeHClO The preparation of AOPPs was passed through aDetoxi-Gel column (Pierce Rockford IL USA) to removeany contaminated endotoxin Endotoxin levels in the prepa-ration were measured with a Limulus Amoebocyte Lysatekit and were found to be below 0025 EUmL The AOPPcontent was determined by measuring absorbance at 340 nmin acidic conditions andwas calibratedwith chloramines-T inthe presence of potassium iodide The content of AOPPs was7240 plusmn 98 nmolmg protein in prepared AOPPs-MSA and02 plusmn 006 nmolmg protein in native MSA

22 Cell Culture of Podocytes and Drugs Conditionallyimmortalized mouse podocytes were generously providedby Shankland et al (Harvard Medical School CharlestownMA USA) and cultured as previously described [32]Briefly undifferentiated podocytes were grown in RPMI1640 containing 10 FBS (BRL Co Ltd USA) peni-cillin (100UmL) streptomycin (100mgmL) and 50 IUmLrecombinant murine IFN-120574 (Pepro Tech USA) at 33∘C (per-missive conditions) To acquire a differentiated phenotypepodocytes were cultured at 37∘C in the absence of IFN-120574 (nonpermissive conditions) for 10ndash14 days Differentiatedpodocytes were starved in RPMI 1640 for 24 h and thentreated with various reagents Podocytes between passages10 and 20 were used in all experiments PTL and MCLwere purchased from Accendatech Co Ltd (Tianjin China)compound 1 and compound 2 were synthesized followingthe procedure provided in [33 34] by Accendatech Co Ltd(Tianjin China) and the purity of those four compoundswas more than 98 (Figure 1) Depending on the experimentdemands podocytes were divided into different groups andthen treated with the drugs orand AOPPs

23 Quantitative Real-Time PCR (qPCR) Analysis TotalRNA from podocytes in all samples was extracted anddissolved in RNA-free water and quantified using UV-clear microplates Aliquots of each RNA extraction werereverse-transcribed simultaneously into cDNA using M-MLV reverse transcriptase according to the manufacturerrsquos

Oxidative Medicine and Cellular Longevity 3

PTL

O O

O

MCL

O

O

OHH

Compound 1

OO

H

HH

H

Compound 2

OO

O

Figure 1 Structures of natural SLs and synthetic derivatives

protocol (Invitrogen USA) The qPCR assay was performedusing SYBR Green assays (Applied Biosystems USA) Thethermal cycling conditions comprised a 30-second step at95∘C followed by 40 cycles with denaturation at 95∘C for5 s annealing at 60∘C for 30 s and extension at 72∘C for60 s The primers for qPCR which were designed withPrimer Express software were as follows MCP-1 (125 bp)forward 51015840-CCCAATGAGTAGGCTGGAGA-31015840 reverse51015840-TCTGGACCCATTCCTTCTTG-31015840 and GAPDH (102 bp)forward 51015840-ATTGTCAGCAATGCATCCTG-31015840 reverse 51015840-ATGGACTGTGGtcATGAGCC-31015840 GAPDH was used as anendogenous reference and each sample was normalized to itsGAPDHcontentThe results were analyzed using the 2-DDCtmethod and shown as fold change in expression with respectto the controls (unstimulated cells) for all samples

24 Measurements of MCP-1 by ELISA To quantify the levelof MCP-1 protein under various experimental conditionsthe level of supernatant MCP-1 was measured using a solid-phase quantitative sandwich enzyme-linked immunosorbentassay (ELISA) kit (eBioscience Inc USA) for MCP-1 thatwas specific for mouse MCP-1 and sensitive to 22 pgmLand the standard curve range was 156ndash1000 pgmL Theconcentration in the culture supernatant was normalized tothe total protein content

25 Western Blot Analysis All samples were washed threetimes with ice-cold PBS and dissolved in RIPA lysis buffercontaining 1mM PMSF for 30min on ice The lysates werecentrifuged (12000timesg) at 4∘C for 15minTheprotein concen-tration was then determined using a BCA protein assay kitProtein samples (40 120583g per well) were separated by 12 SDS-PAGE and transferred onto PVDF membranes (MilliporeBedford MA) After being blocked in 5 nonfat milk inTris-buffered saline (TBS) solution for 1 h the membraneswere incubated overnight at 4∘C with the following pri-mary antibodies (Cell Signaling Technologies DanversMA)IKK120573 rabbit mAb (Product 2370) phospho-IKK120572120573 rabbitmAb (Product 2697) NF-120581B p65 rabbit mAb (Product8242) phospho-NF-120581B p65 rabbit mAb (Product 3033)I120581B120572 mouse mAb (Product 4814) and tubulin mousemAb (Beyotime Institute of Biotechnology Shanghai ChinaProduct AT819) Next the corresponding HRP-conjugatedsecondary antibodies (Cell Signaling Technologies DanversMA product 7074 product 7076) were used for 1 h atroom temperature Electrochemiluminescence detection was

performed and the protein bands were captured and doc-umented using a CCD system (Image Station 2000MMKodak Rochester NY USA) The intensities of the pro-tein bands were quantified by Molecular Imaging SoftwareVersion 40 which was provided with the Kodak 2000MMSystem The optical density was normalized against tubulinprotein expression levels

26 Statistical Analysis Results are expressed as the meansplusmn SEM and represent assays from at least three independentexperiments Statistical significance was estimated using theone-way analysis of variance (ANOVA) Values with P lt 005were considered statistically significant Statistical analyseswere performed using SPSS 130 (SPSS Chicago IL USA)

3 Results

31 AOPPs Increased the Expression of MCP-1 mRNA andProtein in Cultured Podocytes To investigate whether AOPPsinduce the expression of MCP-1 in podocytes we tested theexpression levels of MCP-1 mRNA by qPCR and MCP-1protein by ELISAThe data showed that AOPPs can promotethe expression ofMCP-1 protein in a dose-dependentmannerand reach a maximum level at a concentration of 200120583gmLAt the same time AOPPs can enhance the expression ofMCP-1 protein in a time-dependentmanner and a significantincrease in MCP-1 protein was observed after a 24 h incu-bation qPCR revealed that AOPPs significantly increasedthe expression of MCP-1 and peaked at 200120583gmL at 24 hThe expression of MCP-1 was unchanged in cells incubatedwith native MSA or medium alone suggesting that theupregulation of MCP-1 was associated with an oxidativemodification of MSA (Figure 2)

32 The Impact of AOPPs on the Expression of IKK120573 I120581B120572andNF-120581BProtein in Podocytes To examine the involvementof the proinflammatory signaling pathways of IKKNF-120581Bin AOPP-induced MCP-1 expression we first examined thephosphorylation of IKK120573 degradation of I120581B120572 and activa-tion of NF-120581B by Western blotting As shown above thelevels of phospho-IKK120572120573 and phospho-NF-120581B p65 proteinwere upregulated by 200120583gmL AOPPs (Figures 3(a) and3(c)) whereas I120581B120572 protein expression declined (Figure 3(b))when compared to unstimulated cells or cells treated withnative MSA Total levels of IKK120573 and NF-120581B p65 did notsignificantly change among each group

4 Oxidative Medicine and Cellular Longevity

0

100

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400

lowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

0

100

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lowastlowastlowastlowastlowastlowast

lowastlowastlowast

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CON MSA 3 6 12 24 48AOPP (h)

MCP

-1se

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(pg

mLmiddot120583

gpr

otei

nminus1

(

(b)

0

1

2

3

4

lowast

lowastlowastlowastlowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

0

1

2

3

4

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(d)

Figure 2 AOPPs stimulation increased the expression of MCP-1 in cultured podocytes Podocytes were incubated with the indicatedconcentration of AOPPs for 24 h or 200120583gmL of AOPPs for the indicated time and subjected to MCP-1 mRNA and protein expressionanalysis AOPP treatment increased the expression of MCP-1 at both the mRNA (c d) and protein levels (a b) in a dose- and time-dependentmanner Data are expressed as the means plusmn SD of three independent experiments ANOVA lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versusCON CON untreated cells MSA mouse serum albumin

To further confirm the role of activated IKKNF-120581B inthe AOPP-induced upregulation of MCP-1 the expressionof MCP-1 was measured in podocytes treated with AOPPsin the presence of PTL an inhibitor of the IKKNF-120581Bsignaling pathway As shown in Figure 3(d) PTL significantlydecreased the AOPP-induced upregulation of MCP-1

These data suggest that the AOPP-induced upregulationof MCP-1 was related to the activation of IKK120573 and NF-120581Bp65 and the degradation of I120581B120572

33 The Effect of SLs (MCL Compound 1 and Compound2) on the AOPP-Induced Expression of MCP-1 mRNA andProtein Next we tested the hypothesis that SLs (MCL com-pound 1 and compound 2) as derivatives of PTL may havethe same anti-inflammatory response in podocytes Afterpreincubation with SLs cells were treated with 200120583gmLAOPPs for 24 h PTL (5120583M) was used as a positive controlThe expression of MCP-1 mRNA was determined by qPCRand MCP-1 protein expression was measured by ELISA Theresults presented in Figures 4(a) 4(b) and 4(c) and Figures

5(a) 5(b) and 5(c) demonstrated that MCL compound 1and compound 2 responded in a dose-dependent mannerto prevent the expression of MCP-1 mRNA and proteinAdditionally MCL compound 1 and compound 2 at a con-centration of 5120583M 10 120583M and 25 120583M respectively producedan almost equal inhibitory effect on MCP-1 as 5 120583M of PTL

To further examine the effect of treatment time whenusing SLs 200120583gmL of AOPPs was used to treat podocytesfor 24 h along with the addition of SLs for the indicated timeAs shown in Figures 4(d) 4(e) and 4(f) and Figures 5(d)5(e) and 5(f) MCL compound 1 and compound 2 treatmentresulted in a time-dependent prevention of MCP-1 mRNAand protein expression and a longer exposure time of SLsduring a 24 h period showed a greater inhibition of MCP-1expression

Our data reveal that these three compounds have anactivity profile similar to that of PTL MCL compound 1and compound 2 could inhibit the expression of MCP-1mRNA and protein induced by AOPPs in a dose- and time-dependent manner in podocytes

Oxidative Medicine and Cellular Longevity 5

00

05

10

15

CON MSA AOPP

p-IK

K120572120573

IKK120573

lowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

00

05

10

15

CON MSA AOPP

39kDa

50kDa

lowastlowast

I120581B120572

tubu

lin

I120581B120572

Tubulin

(b)

00

05

10

15

CON MSA AOPP

lowastlowast

p-N

F-120581

B p6

5N

F-120581

B p6

5

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

(c)

CON MSA AOPP PTL

lowastlowastlowast

0

100

200

300

400

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(d)

Figure 3 AOPP-increasedMCP-1 expression in podocytes was mainly mediated by the IKKNF-120581B pathwayThe overnight serum-deprivedpodocytes were treated with 200 120583gmL AOPPs for 30min The expression of p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was examined by Westernblotting (a b and c) To verify the role of the IKKNF-120581B pathway involved in the AOPP-induced MCP-1 expression protein levels of MCP-1were determined by ELISA using cell supernatants exposed to AOPPs for 24 h in the presence of the IKKNF-120581B inhibitor PTL (d) Data areexpressed as the means plusmn SEM of three independent experiments ANOVA (a b and c) lowastP lt 005 lowastlowastP lt 001 versus CON (d) P lt 0001versus CON lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

34 The Effects of SLs (MCL Compound 1 and Compound 2)on the Activation of IKK120573 and NF-120581B and the Degradationof I120581B120572 by AOPPs To further analyze whether factors of theNF-120581B activation cascade are influenced by SLs we pretreatedpodocytes with different concentrations of MCL compound1 and compound 2 for 1 h before incubation with 200120583gmLof AOPPs or native MSA for 30min or 200120583gmL AOPPsfor 30min and we also added SLs for the indicated times Asshown in Figures 6(a) 6(b) and 6(c) Figures 7(a) 7(b) and7(c) and Figures 8(a) 8(b) and 8(c) MCL compound 1 and

compound 2 prevented the protein expression of phospho-IKK120572120573 and phospho-NF-120581B p65 and increased the proteinexpression of I120581B120572 in a dose-dependent manner The effectof MCL compound 1 and compound 2 on inhibiting theactivation of IKK120573 degradation of I120581B120572 and activation ofNF-120581B peaked at 10 120583M 20120583M and 5 120583M respectively andalmost reached the effect of 10 120583M of PTL Figures 6(d)6(e) and 6(f) Figures 7(d) 7(e) and 7(f) and Figures8(d) 8(e) and 8(f) suggest that these three compoundsprevented the phosphorylation of IKK120573 and NF-120581B p65

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

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MCP

-1se

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ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

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lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

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100

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MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

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(

MCL ( h)

(d)

lowast

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CON MSA AOPP 3 6 12 240

100

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MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

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CON MSA AOPP 3 6 12 240

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MCP

-1se

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(pg

mLmiddot120583

gpr

otei

nminus1

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Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

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05

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1 25 5

MCP

-1m

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se)

MCL (120583M)

(a)

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-1m

RNA

leve

l(fo

ld in

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CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

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(c)

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(d)

lowastlowast

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MCP

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(e)

lowastlowast

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MCP

-1m

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CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

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p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

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20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

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(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

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p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Oxidative Medicine and Cellular Longevity

molecules and promotes the expression of other proinflam-matory cytokines in DN [8] MCP-1 can be synthesizedby mononuclear cells and renal resident cells includingpodocytes [5 9] The podocyte seems to be a suitable choicefor further investigation asMCP-1 gene expression appears tobe predominantly localized to podocytes in the glomeruli ofdiabetic mice [7] The latest data show the urinary sedimentpodocalyxin to creatinine ratio had a positive correlationwith the urinary albumin to creatinine ratio and the urinaryMCP-1 to creatinine ratio in patients with type 2 diabetes[10] Other research has shown that once MCP-1 is secretedit can diffuse back to the podocyte and act in a loop byactivating the CCR2 receptor which then initiates profoundbiological effects [11] suggesting podocytes as a potentialtarget of inflammation in DN Considering that podocytesare related to increased proteinuria and contribute to renalprogression in DN [12] therapies aimed at preventing orlimiting podocyte injury andor promoting podocyte repairor regeneration may have major clinical and economicbenefits [13]

Advanced oxidation protein products (AOPPs) were firstdiscovered and reported as uremic toxins by Witko-Sarsat etal in 1996 [14] and were found in association with diabetes inlater studies [15 16] AOPPs are the dityrosine-containing andcross-linking protein products formed during oxidative stressby the reaction of serum protein with chlorinated oxidantsand are often carried by albumin in vivo [14 17] Recentstudies have found that the chronic accumulation of AOPPspromotes inflammation in both the diabetic and nondiabetickidney by significantly increasing macrophage infiltrationand the overexpression of MCP-1 in the remnant kidney andduring experimental diabetic nephropathy [18 19] AOPPscan also decrease the expression of nephrin and podocin inpodocytes resulting in podocyte apoptosis and deletion [20ndash22] Although these studies suggest that the accumulationof AOPPs has an important role in the progression of DNthe mechanism underlying the pathogenic effect of AOPPsremains to be further investigated

Sesquiterpene lactones originally isolated from medic-inal herbs of the Asteraceae family have been reported toexhibit a variety of anti-inflammatory immunomodulatoryand anticancer effects mostly based on their alkylatingcapabilities [23 24] Parthenolide (PTL) and other SLs havebeen reported to exhibit anti-inflammatory effects by inhibit-ing IKK and I120581B120572 phosphorylation andor affecting DNAbinding ability [25 26] Other studies have demonstrated thatPTL blocks MCP-1 mRNA and protein expression by inhibit-ing NF-120581B activity in experimental glomerulonephritis andhuman renal mesangial cells [27 28] as well as in podocytes[9 29]

Recently we demonstrated that SLs including PTLmicheliolide (MCL) and several synthetic analogs of thesemolecules could effectively attenuate the high glucose-stimulated and AOPP-induced activation of NF-120581B thedegradation of I120581B120572 and the expression of MCP-1 in ratmesangial cells (MCs) [30 31] These findings showed thatSLs can exert a beneficial role in high glucose-stimulatedand AOPPs-induced inflammation and ECM deposition bypreventing NF-120581B activation in MCs However whether

SLs have anti-inflammatory effects in podocytes under theaccumulation of AOPPs has not yet been clarified and ourprevious studies were focused on the screening of anti-inflammatory effects of SLs but the underlying mechanismof action has not yet been elucidated Thus the presentstudy aimed to characterize the inflammatory effect ofAOPPsin cultured mouse podocytes and more importantly weselected MCL compound 1 and compound 2 according toour previous experiments to further explore the mechanismof the potential anti-inflammatory effects of SLs

2 Materials and Methods

21 AOPPs-MSA Preparation and Determination AOPPs-MSA was prepared in vitro according to a previouslydescribed method [21] Briefly MSA solution (200mgmL)was incubated with 200mM HClO (Fluke Buchs Switzer-land) at an optimal ratio (MSA to HClO = 1 140) for 30minat 37∘C and dialyzed overnight against PBS to remove freeHClO The preparation of AOPPs was passed through aDetoxi-Gel column (Pierce Rockford IL USA) to removeany contaminated endotoxin Endotoxin levels in the prepa-ration were measured with a Limulus Amoebocyte Lysatekit and were found to be below 0025 EUmL The AOPPcontent was determined by measuring absorbance at 340 nmin acidic conditions andwas calibratedwith chloramines-T inthe presence of potassium iodide The content of AOPPs was7240 plusmn 98 nmolmg protein in prepared AOPPs-MSA and02 plusmn 006 nmolmg protein in native MSA

22 Cell Culture of Podocytes and Drugs Conditionallyimmortalized mouse podocytes were generously providedby Shankland et al (Harvard Medical School CharlestownMA USA) and cultured as previously described [32]Briefly undifferentiated podocytes were grown in RPMI1640 containing 10 FBS (BRL Co Ltd USA) peni-cillin (100UmL) streptomycin (100mgmL) and 50 IUmLrecombinant murine IFN-120574 (Pepro Tech USA) at 33∘C (per-missive conditions) To acquire a differentiated phenotypepodocytes were cultured at 37∘C in the absence of IFN-120574 (nonpermissive conditions) for 10ndash14 days Differentiatedpodocytes were starved in RPMI 1640 for 24 h and thentreated with various reagents Podocytes between passages10 and 20 were used in all experiments PTL and MCLwere purchased from Accendatech Co Ltd (Tianjin China)compound 1 and compound 2 were synthesized followingthe procedure provided in [33 34] by Accendatech Co Ltd(Tianjin China) and the purity of those four compoundswas more than 98 (Figure 1) Depending on the experimentdemands podocytes were divided into different groups andthen treated with the drugs orand AOPPs

23 Quantitative Real-Time PCR (qPCR) Analysis TotalRNA from podocytes in all samples was extracted anddissolved in RNA-free water and quantified using UV-clear microplates Aliquots of each RNA extraction werereverse-transcribed simultaneously into cDNA using M-MLV reverse transcriptase according to the manufacturerrsquos

Oxidative Medicine and Cellular Longevity 3

PTL

O O

O

MCL

O

O

OHH

Compound 1

OO

H

HH

H

Compound 2

OO

O

Figure 1 Structures of natural SLs and synthetic derivatives

protocol (Invitrogen USA) The qPCR assay was performedusing SYBR Green assays (Applied Biosystems USA) Thethermal cycling conditions comprised a 30-second step at95∘C followed by 40 cycles with denaturation at 95∘C for5 s annealing at 60∘C for 30 s and extension at 72∘C for60 s The primers for qPCR which were designed withPrimer Express software were as follows MCP-1 (125 bp)forward 51015840-CCCAATGAGTAGGCTGGAGA-31015840 reverse51015840-TCTGGACCCATTCCTTCTTG-31015840 and GAPDH (102 bp)forward 51015840-ATTGTCAGCAATGCATCCTG-31015840 reverse 51015840-ATGGACTGTGGtcATGAGCC-31015840 GAPDH was used as anendogenous reference and each sample was normalized to itsGAPDHcontentThe results were analyzed using the 2-DDCtmethod and shown as fold change in expression with respectto the controls (unstimulated cells) for all samples

24 Measurements of MCP-1 by ELISA To quantify the levelof MCP-1 protein under various experimental conditionsthe level of supernatant MCP-1 was measured using a solid-phase quantitative sandwich enzyme-linked immunosorbentassay (ELISA) kit (eBioscience Inc USA) for MCP-1 thatwas specific for mouse MCP-1 and sensitive to 22 pgmLand the standard curve range was 156ndash1000 pgmL Theconcentration in the culture supernatant was normalized tothe total protein content

25 Western Blot Analysis All samples were washed threetimes with ice-cold PBS and dissolved in RIPA lysis buffercontaining 1mM PMSF for 30min on ice The lysates werecentrifuged (12000timesg) at 4∘C for 15minTheprotein concen-tration was then determined using a BCA protein assay kitProtein samples (40 120583g per well) were separated by 12 SDS-PAGE and transferred onto PVDF membranes (MilliporeBedford MA) After being blocked in 5 nonfat milk inTris-buffered saline (TBS) solution for 1 h the membraneswere incubated overnight at 4∘C with the following pri-mary antibodies (Cell Signaling Technologies DanversMA)IKK120573 rabbit mAb (Product 2370) phospho-IKK120572120573 rabbitmAb (Product 2697) NF-120581B p65 rabbit mAb (Product8242) phospho-NF-120581B p65 rabbit mAb (Product 3033)I120581B120572 mouse mAb (Product 4814) and tubulin mousemAb (Beyotime Institute of Biotechnology Shanghai ChinaProduct AT819) Next the corresponding HRP-conjugatedsecondary antibodies (Cell Signaling Technologies DanversMA product 7074 product 7076) were used for 1 h atroom temperature Electrochemiluminescence detection was

performed and the protein bands were captured and doc-umented using a CCD system (Image Station 2000MMKodak Rochester NY USA) The intensities of the pro-tein bands were quantified by Molecular Imaging SoftwareVersion 40 which was provided with the Kodak 2000MMSystem The optical density was normalized against tubulinprotein expression levels

26 Statistical Analysis Results are expressed as the meansplusmn SEM and represent assays from at least three independentexperiments Statistical significance was estimated using theone-way analysis of variance (ANOVA) Values with P lt 005were considered statistically significant Statistical analyseswere performed using SPSS 130 (SPSS Chicago IL USA)

3 Results

31 AOPPs Increased the Expression of MCP-1 mRNA andProtein in Cultured Podocytes To investigate whether AOPPsinduce the expression of MCP-1 in podocytes we tested theexpression levels of MCP-1 mRNA by qPCR and MCP-1protein by ELISAThe data showed that AOPPs can promotethe expression ofMCP-1 protein in a dose-dependentmannerand reach a maximum level at a concentration of 200120583gmLAt the same time AOPPs can enhance the expression ofMCP-1 protein in a time-dependentmanner and a significantincrease in MCP-1 protein was observed after a 24 h incu-bation qPCR revealed that AOPPs significantly increasedthe expression of MCP-1 and peaked at 200120583gmL at 24 hThe expression of MCP-1 was unchanged in cells incubatedwith native MSA or medium alone suggesting that theupregulation of MCP-1 was associated with an oxidativemodification of MSA (Figure 2)

32 The Impact of AOPPs on the Expression of IKK120573 I120581B120572andNF-120581BProtein in Podocytes To examine the involvementof the proinflammatory signaling pathways of IKKNF-120581Bin AOPP-induced MCP-1 expression we first examined thephosphorylation of IKK120573 degradation of I120581B120572 and activa-tion of NF-120581B by Western blotting As shown above thelevels of phospho-IKK120572120573 and phospho-NF-120581B p65 proteinwere upregulated by 200120583gmL AOPPs (Figures 3(a) and3(c)) whereas I120581B120572 protein expression declined (Figure 3(b))when compared to unstimulated cells or cells treated withnative MSA Total levels of IKK120573 and NF-120581B p65 did notsignificantly change among each group

4 Oxidative Medicine and Cellular Longevity

0

100

200

300

400

lowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

0

100

200

300

400

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(b)

0

1

2

3

4

lowast

lowastlowastlowastlowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

0

1

2

3

4

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(d)

Figure 2 AOPPs stimulation increased the expression of MCP-1 in cultured podocytes Podocytes were incubated with the indicatedconcentration of AOPPs for 24 h or 200120583gmL of AOPPs for the indicated time and subjected to MCP-1 mRNA and protein expressionanalysis AOPP treatment increased the expression of MCP-1 at both the mRNA (c d) and protein levels (a b) in a dose- and time-dependentmanner Data are expressed as the means plusmn SD of three independent experiments ANOVA lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versusCON CON untreated cells MSA mouse serum albumin

To further confirm the role of activated IKKNF-120581B inthe AOPP-induced upregulation of MCP-1 the expressionof MCP-1 was measured in podocytes treated with AOPPsin the presence of PTL an inhibitor of the IKKNF-120581Bsignaling pathway As shown in Figure 3(d) PTL significantlydecreased the AOPP-induced upregulation of MCP-1

These data suggest that the AOPP-induced upregulationof MCP-1 was related to the activation of IKK120573 and NF-120581Bp65 and the degradation of I120581B120572

33 The Effect of SLs (MCL Compound 1 and Compound2) on the AOPP-Induced Expression of MCP-1 mRNA andProtein Next we tested the hypothesis that SLs (MCL com-pound 1 and compound 2) as derivatives of PTL may havethe same anti-inflammatory response in podocytes Afterpreincubation with SLs cells were treated with 200120583gmLAOPPs for 24 h PTL (5120583M) was used as a positive controlThe expression of MCP-1 mRNA was determined by qPCRand MCP-1 protein expression was measured by ELISA Theresults presented in Figures 4(a) 4(b) and 4(c) and Figures

5(a) 5(b) and 5(c) demonstrated that MCL compound 1and compound 2 responded in a dose-dependent mannerto prevent the expression of MCP-1 mRNA and proteinAdditionally MCL compound 1 and compound 2 at a con-centration of 5120583M 10 120583M and 25 120583M respectively producedan almost equal inhibitory effect on MCP-1 as 5 120583M of PTL

To further examine the effect of treatment time whenusing SLs 200120583gmL of AOPPs was used to treat podocytesfor 24 h along with the addition of SLs for the indicated timeAs shown in Figures 4(d) 4(e) and 4(f) and Figures 5(d)5(e) and 5(f) MCL compound 1 and compound 2 treatmentresulted in a time-dependent prevention of MCP-1 mRNAand protein expression and a longer exposure time of SLsduring a 24 h period showed a greater inhibition of MCP-1expression

Our data reveal that these three compounds have anactivity profile similar to that of PTL MCL compound 1and compound 2 could inhibit the expression of MCP-1mRNA and protein induced by AOPPs in a dose- and time-dependent manner in podocytes

Oxidative Medicine and Cellular Longevity 5

00

05

10

15

CON MSA AOPP

p-IK

K120572120573

IKK120573

lowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

00

05

10

15

CON MSA AOPP

39kDa

50kDa

lowastlowast

I120581B120572

tubu

lin

I120581B120572

Tubulin

(b)

00

05

10

15

CON MSA AOPP

lowastlowast

p-N

F-120581

B p6

5N

F-120581

B p6

5

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

(c)

CON MSA AOPP PTL

lowastlowastlowast

0

100

200

300

400

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(d)

Figure 3 AOPP-increasedMCP-1 expression in podocytes was mainly mediated by the IKKNF-120581B pathwayThe overnight serum-deprivedpodocytes were treated with 200 120583gmL AOPPs for 30min The expression of p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was examined by Westernblotting (a b and c) To verify the role of the IKKNF-120581B pathway involved in the AOPP-induced MCP-1 expression protein levels of MCP-1were determined by ELISA using cell supernatants exposed to AOPPs for 24 h in the presence of the IKKNF-120581B inhibitor PTL (d) Data areexpressed as the means plusmn SEM of three independent experiments ANOVA (a b and c) lowastP lt 005 lowastlowastP lt 001 versus CON (d) P lt 0001versus CON lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

34 The Effects of SLs (MCL Compound 1 and Compound 2)on the Activation of IKK120573 and NF-120581B and the Degradationof I120581B120572 by AOPPs To further analyze whether factors of theNF-120581B activation cascade are influenced by SLs we pretreatedpodocytes with different concentrations of MCL compound1 and compound 2 for 1 h before incubation with 200120583gmLof AOPPs or native MSA for 30min or 200120583gmL AOPPsfor 30min and we also added SLs for the indicated times Asshown in Figures 6(a) 6(b) and 6(c) Figures 7(a) 7(b) and7(c) and Figures 8(a) 8(b) and 8(c) MCL compound 1 and

compound 2 prevented the protein expression of phospho-IKK120572120573 and phospho-NF-120581B p65 and increased the proteinexpression of I120581B120572 in a dose-dependent manner The effectof MCL compound 1 and compound 2 on inhibiting theactivation of IKK120573 degradation of I120581B120572 and activation ofNF-120581B peaked at 10 120583M 20120583M and 5 120583M respectively andalmost reached the effect of 10 120583M of PTL Figures 6(d)6(e) and 6(f) Figures 7(d) 7(e) and 7(f) and Figures8(d) 8(e) and 8(f) suggest that these three compoundsprevented the phosphorylation of IKK120573 and NF-120581B p65

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

MCL ( h)

(d)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

lowastlowastlowast

lowastlowast

CON MSA AOPP 3 6 12 240

50

100

150

200

250

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 3

PTL

O O

O

MCL

O

O

OHH

Compound 1

OO

H

HH

H

Compound 2

OO

O

Figure 1 Structures of natural SLs and synthetic derivatives

protocol (Invitrogen USA) The qPCR assay was performedusing SYBR Green assays (Applied Biosystems USA) Thethermal cycling conditions comprised a 30-second step at95∘C followed by 40 cycles with denaturation at 95∘C for5 s annealing at 60∘C for 30 s and extension at 72∘C for60 s The primers for qPCR which were designed withPrimer Express software were as follows MCP-1 (125 bp)forward 51015840-CCCAATGAGTAGGCTGGAGA-31015840 reverse51015840-TCTGGACCCATTCCTTCTTG-31015840 and GAPDH (102 bp)forward 51015840-ATTGTCAGCAATGCATCCTG-31015840 reverse 51015840-ATGGACTGTGGtcATGAGCC-31015840 GAPDH was used as anendogenous reference and each sample was normalized to itsGAPDHcontentThe results were analyzed using the 2-DDCtmethod and shown as fold change in expression with respectto the controls (unstimulated cells) for all samples

24 Measurements of MCP-1 by ELISA To quantify the levelof MCP-1 protein under various experimental conditionsthe level of supernatant MCP-1 was measured using a solid-phase quantitative sandwich enzyme-linked immunosorbentassay (ELISA) kit (eBioscience Inc USA) for MCP-1 thatwas specific for mouse MCP-1 and sensitive to 22 pgmLand the standard curve range was 156ndash1000 pgmL Theconcentration in the culture supernatant was normalized tothe total protein content

25 Western Blot Analysis All samples were washed threetimes with ice-cold PBS and dissolved in RIPA lysis buffercontaining 1mM PMSF for 30min on ice The lysates werecentrifuged (12000timesg) at 4∘C for 15minTheprotein concen-tration was then determined using a BCA protein assay kitProtein samples (40 120583g per well) were separated by 12 SDS-PAGE and transferred onto PVDF membranes (MilliporeBedford MA) After being blocked in 5 nonfat milk inTris-buffered saline (TBS) solution for 1 h the membraneswere incubated overnight at 4∘C with the following pri-mary antibodies (Cell Signaling Technologies DanversMA)IKK120573 rabbit mAb (Product 2370) phospho-IKK120572120573 rabbitmAb (Product 2697) NF-120581B p65 rabbit mAb (Product8242) phospho-NF-120581B p65 rabbit mAb (Product 3033)I120581B120572 mouse mAb (Product 4814) and tubulin mousemAb (Beyotime Institute of Biotechnology Shanghai ChinaProduct AT819) Next the corresponding HRP-conjugatedsecondary antibodies (Cell Signaling Technologies DanversMA product 7074 product 7076) were used for 1 h atroom temperature Electrochemiluminescence detection was

performed and the protein bands were captured and doc-umented using a CCD system (Image Station 2000MMKodak Rochester NY USA) The intensities of the pro-tein bands were quantified by Molecular Imaging SoftwareVersion 40 which was provided with the Kodak 2000MMSystem The optical density was normalized against tubulinprotein expression levels

26 Statistical Analysis Results are expressed as the meansplusmn SEM and represent assays from at least three independentexperiments Statistical significance was estimated using theone-way analysis of variance (ANOVA) Values with P lt 005were considered statistically significant Statistical analyseswere performed using SPSS 130 (SPSS Chicago IL USA)

3 Results

31 AOPPs Increased the Expression of MCP-1 mRNA andProtein in Cultured Podocytes To investigate whether AOPPsinduce the expression of MCP-1 in podocytes we tested theexpression levels of MCP-1 mRNA by qPCR and MCP-1protein by ELISAThe data showed that AOPPs can promotethe expression ofMCP-1 protein in a dose-dependentmannerand reach a maximum level at a concentration of 200120583gmLAt the same time AOPPs can enhance the expression ofMCP-1 protein in a time-dependentmanner and a significantincrease in MCP-1 protein was observed after a 24 h incu-bation qPCR revealed that AOPPs significantly increasedthe expression of MCP-1 and peaked at 200120583gmL at 24 hThe expression of MCP-1 was unchanged in cells incubatedwith native MSA or medium alone suggesting that theupregulation of MCP-1 was associated with an oxidativemodification of MSA (Figure 2)

32 The Impact of AOPPs on the Expression of IKK120573 I120581B120572andNF-120581BProtein in Podocytes To examine the involvementof the proinflammatory signaling pathways of IKKNF-120581Bin AOPP-induced MCP-1 expression we first examined thephosphorylation of IKK120573 degradation of I120581B120572 and activa-tion of NF-120581B by Western blotting As shown above thelevels of phospho-IKK120572120573 and phospho-NF-120581B p65 proteinwere upregulated by 200120583gmL AOPPs (Figures 3(a) and3(c)) whereas I120581B120572 protein expression declined (Figure 3(b))when compared to unstimulated cells or cells treated withnative MSA Total levels of IKK120573 and NF-120581B p65 did notsignificantly change among each group

4 Oxidative Medicine and Cellular Longevity

0

100

200

300

400

lowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

0

100

200

300

400

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(b)

0

1

2

3

4

lowast

lowastlowastlowastlowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

0

1

2

3

4

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(d)

Figure 2 AOPPs stimulation increased the expression of MCP-1 in cultured podocytes Podocytes were incubated with the indicatedconcentration of AOPPs for 24 h or 200120583gmL of AOPPs for the indicated time and subjected to MCP-1 mRNA and protein expressionanalysis AOPP treatment increased the expression of MCP-1 at both the mRNA (c d) and protein levels (a b) in a dose- and time-dependentmanner Data are expressed as the means plusmn SD of three independent experiments ANOVA lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versusCON CON untreated cells MSA mouse serum albumin

To further confirm the role of activated IKKNF-120581B inthe AOPP-induced upregulation of MCP-1 the expressionof MCP-1 was measured in podocytes treated with AOPPsin the presence of PTL an inhibitor of the IKKNF-120581Bsignaling pathway As shown in Figure 3(d) PTL significantlydecreased the AOPP-induced upregulation of MCP-1

These data suggest that the AOPP-induced upregulationof MCP-1 was related to the activation of IKK120573 and NF-120581Bp65 and the degradation of I120581B120572

33 The Effect of SLs (MCL Compound 1 and Compound2) on the AOPP-Induced Expression of MCP-1 mRNA andProtein Next we tested the hypothesis that SLs (MCL com-pound 1 and compound 2) as derivatives of PTL may havethe same anti-inflammatory response in podocytes Afterpreincubation with SLs cells were treated with 200120583gmLAOPPs for 24 h PTL (5120583M) was used as a positive controlThe expression of MCP-1 mRNA was determined by qPCRand MCP-1 protein expression was measured by ELISA Theresults presented in Figures 4(a) 4(b) and 4(c) and Figures

5(a) 5(b) and 5(c) demonstrated that MCL compound 1and compound 2 responded in a dose-dependent mannerto prevent the expression of MCP-1 mRNA and proteinAdditionally MCL compound 1 and compound 2 at a con-centration of 5120583M 10 120583M and 25 120583M respectively producedan almost equal inhibitory effect on MCP-1 as 5 120583M of PTL

To further examine the effect of treatment time whenusing SLs 200120583gmL of AOPPs was used to treat podocytesfor 24 h along with the addition of SLs for the indicated timeAs shown in Figures 4(d) 4(e) and 4(f) and Figures 5(d)5(e) and 5(f) MCL compound 1 and compound 2 treatmentresulted in a time-dependent prevention of MCP-1 mRNAand protein expression and a longer exposure time of SLsduring a 24 h period showed a greater inhibition of MCP-1expression

Our data reveal that these three compounds have anactivity profile similar to that of PTL MCL compound 1and compound 2 could inhibit the expression of MCP-1mRNA and protein induced by AOPPs in a dose- and time-dependent manner in podocytes

Oxidative Medicine and Cellular Longevity 5

00

05

10

15

CON MSA AOPP

p-IK

K120572120573

IKK120573

lowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

00

05

10

15

CON MSA AOPP

39kDa

50kDa

lowastlowast

I120581B120572

tubu

lin

I120581B120572

Tubulin

(b)

00

05

10

15

CON MSA AOPP

lowastlowast

p-N

F-120581

B p6

5N

F-120581

B p6

5

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

(c)

CON MSA AOPP PTL

lowastlowastlowast

0

100

200

300

400

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(d)

Figure 3 AOPP-increasedMCP-1 expression in podocytes was mainly mediated by the IKKNF-120581B pathwayThe overnight serum-deprivedpodocytes were treated with 200 120583gmL AOPPs for 30min The expression of p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was examined by Westernblotting (a b and c) To verify the role of the IKKNF-120581B pathway involved in the AOPP-induced MCP-1 expression protein levels of MCP-1were determined by ELISA using cell supernatants exposed to AOPPs for 24 h in the presence of the IKKNF-120581B inhibitor PTL (d) Data areexpressed as the means plusmn SEM of three independent experiments ANOVA (a b and c) lowastP lt 005 lowastlowastP lt 001 versus CON (d) P lt 0001versus CON lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

34 The Effects of SLs (MCL Compound 1 and Compound 2)on the Activation of IKK120573 and NF-120581B and the Degradationof I120581B120572 by AOPPs To further analyze whether factors of theNF-120581B activation cascade are influenced by SLs we pretreatedpodocytes with different concentrations of MCL compound1 and compound 2 for 1 h before incubation with 200120583gmLof AOPPs or native MSA for 30min or 200120583gmL AOPPsfor 30min and we also added SLs for the indicated times Asshown in Figures 6(a) 6(b) and 6(c) Figures 7(a) 7(b) and7(c) and Figures 8(a) 8(b) and 8(c) MCL compound 1 and

compound 2 prevented the protein expression of phospho-IKK120572120573 and phospho-NF-120581B p65 and increased the proteinexpression of I120581B120572 in a dose-dependent manner The effectof MCL compound 1 and compound 2 on inhibiting theactivation of IKK120573 degradation of I120581B120572 and activation ofNF-120581B peaked at 10 120583M 20120583M and 5 120583M respectively andalmost reached the effect of 10 120583M of PTL Figures 6(d)6(e) and 6(f) Figures 7(d) 7(e) and 7(f) and Figures8(d) 8(e) and 8(f) suggest that these three compoundsprevented the phosphorylation of IKK120573 and NF-120581B p65

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

MCL ( h)

(d)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

lowastlowastlowast

lowastlowast

CON MSA AOPP 3 6 12 240

50

100

150

200

250

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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4 Oxidative Medicine and Cellular Longevity

0

100

200

300

400

lowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

0

100

200

300

400

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(b)

0

1

2

3

4

lowast

lowastlowastlowastlowastlowastlowast

CON MSA 50 100 200 400AOPP (120583gmL)

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(c)

lowastlowastlowastlowastlowastlowast

lowastlowast

CON MSA 3 6 12 24 48AOPP (h)

0

1

2

3

4

(fold

incr

ease

)M

CP-1

mRN

A le

vel

(d)

Figure 2 AOPPs stimulation increased the expression of MCP-1 in cultured podocytes Podocytes were incubated with the indicatedconcentration of AOPPs for 24 h or 200120583gmL of AOPPs for the indicated time and subjected to MCP-1 mRNA and protein expressionanalysis AOPP treatment increased the expression of MCP-1 at both the mRNA (c d) and protein levels (a b) in a dose- and time-dependentmanner Data are expressed as the means plusmn SD of three independent experiments ANOVA lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versusCON CON untreated cells MSA mouse serum albumin

To further confirm the role of activated IKKNF-120581B inthe AOPP-induced upregulation of MCP-1 the expressionof MCP-1 was measured in podocytes treated with AOPPsin the presence of PTL an inhibitor of the IKKNF-120581Bsignaling pathway As shown in Figure 3(d) PTL significantlydecreased the AOPP-induced upregulation of MCP-1

These data suggest that the AOPP-induced upregulationof MCP-1 was related to the activation of IKK120573 and NF-120581Bp65 and the degradation of I120581B120572

33 The Effect of SLs (MCL Compound 1 and Compound2) on the AOPP-Induced Expression of MCP-1 mRNA andProtein Next we tested the hypothesis that SLs (MCL com-pound 1 and compound 2) as derivatives of PTL may havethe same anti-inflammatory response in podocytes Afterpreincubation with SLs cells were treated with 200120583gmLAOPPs for 24 h PTL (5120583M) was used as a positive controlThe expression of MCP-1 mRNA was determined by qPCRand MCP-1 protein expression was measured by ELISA Theresults presented in Figures 4(a) 4(b) and 4(c) and Figures

5(a) 5(b) and 5(c) demonstrated that MCL compound 1and compound 2 responded in a dose-dependent mannerto prevent the expression of MCP-1 mRNA and proteinAdditionally MCL compound 1 and compound 2 at a con-centration of 5120583M 10 120583M and 25 120583M respectively producedan almost equal inhibitory effect on MCP-1 as 5 120583M of PTL

To further examine the effect of treatment time whenusing SLs 200120583gmL of AOPPs was used to treat podocytesfor 24 h along with the addition of SLs for the indicated timeAs shown in Figures 4(d) 4(e) and 4(f) and Figures 5(d)5(e) and 5(f) MCL compound 1 and compound 2 treatmentresulted in a time-dependent prevention of MCP-1 mRNAand protein expression and a longer exposure time of SLsduring a 24 h period showed a greater inhibition of MCP-1expression

Our data reveal that these three compounds have anactivity profile similar to that of PTL MCL compound 1and compound 2 could inhibit the expression of MCP-1mRNA and protein induced by AOPPs in a dose- and time-dependent manner in podocytes

Oxidative Medicine and Cellular Longevity 5

00

05

10

15

CON MSA AOPP

p-IK

K120572120573

IKK120573

lowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

00

05

10

15

CON MSA AOPP

39kDa

50kDa

lowastlowast

I120581B120572

tubu

lin

I120581B120572

Tubulin

(b)

00

05

10

15

CON MSA AOPP

lowastlowast

p-N

F-120581

B p6

5N

F-120581

B p6

5

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

(c)

CON MSA AOPP PTL

lowastlowastlowast

0

100

200

300

400

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(d)

Figure 3 AOPP-increasedMCP-1 expression in podocytes was mainly mediated by the IKKNF-120581B pathwayThe overnight serum-deprivedpodocytes were treated with 200 120583gmL AOPPs for 30min The expression of p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was examined by Westernblotting (a b and c) To verify the role of the IKKNF-120581B pathway involved in the AOPP-induced MCP-1 expression protein levels of MCP-1were determined by ELISA using cell supernatants exposed to AOPPs for 24 h in the presence of the IKKNF-120581B inhibitor PTL (d) Data areexpressed as the means plusmn SEM of three independent experiments ANOVA (a b and c) lowastP lt 005 lowastlowastP lt 001 versus CON (d) P lt 0001versus CON lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

34 The Effects of SLs (MCL Compound 1 and Compound 2)on the Activation of IKK120573 and NF-120581B and the Degradationof I120581B120572 by AOPPs To further analyze whether factors of theNF-120581B activation cascade are influenced by SLs we pretreatedpodocytes with different concentrations of MCL compound1 and compound 2 for 1 h before incubation with 200120583gmLof AOPPs or native MSA for 30min or 200120583gmL AOPPsfor 30min and we also added SLs for the indicated times Asshown in Figures 6(a) 6(b) and 6(c) Figures 7(a) 7(b) and7(c) and Figures 8(a) 8(b) and 8(c) MCL compound 1 and

compound 2 prevented the protein expression of phospho-IKK120572120573 and phospho-NF-120581B p65 and increased the proteinexpression of I120581B120572 in a dose-dependent manner The effectof MCL compound 1 and compound 2 on inhibiting theactivation of IKK120573 degradation of I120581B120572 and activation ofNF-120581B peaked at 10 120583M 20120583M and 5 120583M respectively andalmost reached the effect of 10 120583M of PTL Figures 6(d)6(e) and 6(f) Figures 7(d) 7(e) and 7(f) and Figures8(d) 8(e) and 8(f) suggest that these three compoundsprevented the phosphorylation of IKK120573 and NF-120581B p65

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

MCL ( h)

(d)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

lowastlowastlowast

lowastlowast

CON MSA AOPP 3 6 12 240

50

100

150

200

250

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 5

00

05

10

15

CON MSA AOPP

p-IK

K120572120573

IKK120573

lowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

00

05

10

15

CON MSA AOPP

39kDa

50kDa

lowastlowast

I120581B120572

tubu

lin

I120581B120572

Tubulin

(b)

00

05

10

15

CON MSA AOPP

lowastlowast

p-N

F-120581

B p6

5N

F-120581

B p6

5

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

(c)

CON MSA AOPP PTL

lowastlowastlowast

0

100

200

300

400

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(d)

Figure 3 AOPP-increasedMCP-1 expression in podocytes was mainly mediated by the IKKNF-120581B pathwayThe overnight serum-deprivedpodocytes were treated with 200 120583gmL AOPPs for 30min The expression of p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was examined by Westernblotting (a b and c) To verify the role of the IKKNF-120581B pathway involved in the AOPP-induced MCP-1 expression protein levels of MCP-1were determined by ELISA using cell supernatants exposed to AOPPs for 24 h in the presence of the IKKNF-120581B inhibitor PTL (d) Data areexpressed as the means plusmn SEM of three independent experiments ANOVA (a b and c) lowastP lt 005 lowastlowastP lt 001 versus CON (d) P lt 0001versus CON lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

34 The Effects of SLs (MCL Compound 1 and Compound 2)on the Activation of IKK120573 and NF-120581B and the Degradationof I120581B120572 by AOPPs To further analyze whether factors of theNF-120581B activation cascade are influenced by SLs we pretreatedpodocytes with different concentrations of MCL compound1 and compound 2 for 1 h before incubation with 200120583gmLof AOPPs or native MSA for 30min or 200120583gmL AOPPsfor 30min and we also added SLs for the indicated times Asshown in Figures 6(a) 6(b) and 6(c) Figures 7(a) 7(b) and7(c) and Figures 8(a) 8(b) and 8(c) MCL compound 1 and

compound 2 prevented the protein expression of phospho-IKK120572120573 and phospho-NF-120581B p65 and increased the proteinexpression of I120581B120572 in a dose-dependent manner The effectof MCL compound 1 and compound 2 on inhibiting theactivation of IKK120573 degradation of I120581B120572 and activation ofNF-120581B peaked at 10 120583M 20120583M and 5 120583M respectively andalmost reached the effect of 10 120583M of PTL Figures 6(d)6(e) and 6(f) Figures 7(d) 7(e) and 7(f) and Figures8(d) 8(e) and 8(f) suggest that these three compoundsprevented the phosphorylation of IKK120573 and NF-120581B p65

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

MCL ( h)

(d)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

lowastlowastlowast

lowastlowast

CON MSA AOPP 3 6 12 240

50

100

150

200

250

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 05 1 25 5MCL (120583M)

0

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP PTL 05 1 25 5 100

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (120583M)

(b)

lowastlowastlowast lowastlowastlowastlowastlowast

lowast

CON MSA AOPP PTL 025001 05 1 250

50

100

150

200

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (120583M)

(c)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

MCL ( h)

(d)

lowast

lowastlowastlowast

lowastlowastlowast

CON MSA AOPP 3 6 12 240

100

200

300

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 1 (h)

(e)

lowastlowastlowast

lowastlowast

CON MSA AOPP 3 6 12 240

50

100

150

200

250

MCP

-1se

cret

ion

(pg

mLmiddot120583

gpr

otei

nminus1

(

Compound 2 (h)

(f)

Figure 4 SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent manner Podocytes werepreincubated with the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPsfor 24 h PTL (5120583M)was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and thenMCL (5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1protein were determined by ELISA SLs decreased the AOPP-induced expression of MCP-1 protein in a dose- and time-dependent mannerThe data are expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 andlowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serum albumin

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 7

lowastlowastlowast

lowastlowast

CON MSA AOPP PTL 025 0500

05

10

15

20

25

1 25 5

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

MCL (120583M)

(a)

lowastlowastlowastlowastlowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 05 1 25 5 10Compound 1 (120583M)

(b)

lowastlowastlowastlowastlowastlowastlowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP PTL 025001 05 1 25Compound 2 (120583M)

(c)

lowastlowastlowast

lowastlowast

0

1

2

3

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24MCL (h)

(d)

lowastlowast

lowastlowast

lowast

00

05

10

15

20

25

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 1 (h)

(e)

lowastlowast

lowast

0

1

2

3

4

MCP

-1m

RNA

leve

l(fo

ld in

crea

se)

CON MSA AOPP 3 6 12 24Compound 2 (h)

(f)

Figure 5 SLs decreased theAOPP-induced expression ofMCP-1mRNA in a dose- and time-dependentmanner Podocyteswere preincubatedwith the indicated concentration of MCL compound 1 or compound 2 for 1 h before treatment with 200120583gmL of AOPPs for 24 h PTL(5120583M) was used as a positive control (a b and c) Aliquots of 200120583gmL AOPPs were used to treat podocytes for 24 h and then MCL(5120583M) compound 1 (10 120583M) or compound 1 (25 120583M) was added for the indicated times (d e and f) The expression levels of MCP-1 mRNAwere determined by qPCR SLs decreased the AOPP-induced expression of MCP-1 mRNA in a dose- and time-dependent manner The dataare expressed as the means plusmn SEM of three independent experiments ANOVA P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt0001 versus AOPP CON untreated cells MSA mouse serum albumin

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Oxidative Medicine and Cellular Longevity

lowastlowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(a)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15Tubulin

I120581B120572

I120581B120572

tubu

lin

39kDa

50kDa

(b)

lowastlowastlowastlowastlowast

CON MSA AOPP PTL 525 10120583M)MCL (

00

05

10

15

20

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(c)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

20p-

IKK120572

120573IK

K120573

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

00

05

10

15

(e)

lowastlowastlowast

CON MSA AOPP 3015 60MCL (min)

00

05

10

15

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

(f)

Figure 6 MCL prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependent mannerin podocytes After 24 h of serum starvation podocytes were pretreated with MCL (25120583M 5 120583M and 10 120583M) for 1 h and then exposed to200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocytes were treatedwith 200 120583gmL AOPPs for 30min and then MCL (10120583M) was added for a preset time (d e and f) The protein expression of p-IKK120572120573p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independent experimentsANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 and lowastlowastlowastP lt 0001 versus AOPP CON untreated cells MSA mouse serumalbumin

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 9

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowast lowastlowastlowast

p-IK

K120572120573

IKK120573

00

05

10

15

20

25

CON MSA AOPP 105 20PTLCompound 1 (120583M)

(a)

lowastlowastlowastlowastlowast

00

02

04

06

08

CON MSA AOPP 105 20PTL

I120581B120572

tubu

lin

Compound 1 (120583M)

39kDa

50kDaTubulin

I120581B120572

(b)

lowastlowastlowastlowast

lowast

00

05

10

15

CON MSA AOPP 105 20PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 1 (120583M)

65kDa

65kDa

(c)

00

05

10

15

20p-

IKK120572

120573IK

K120573

CON MSA AOPP 3015 60Compound 1 (min)

lowastlowast

p-IKK120572120573

IKK120573

87kDa

87kDa

(d)

00

05

10

15

CON MSA AOPP 3015 60

lowastlowastlowast

I120581B120572

tubu

lin

Tubulin

I120581B120572 39kDa

50kDa

Compound 1 (min)

(e)

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60

lowast

Compound 1 (min)

(f)

Figure 7 Compound 1 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 1 (5120583M 10120583M and 20 120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmL AOPPs for 30min and then compound 1 (20 120583M) was added for a preset time (d e f) The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 001 P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSA mouse serumalbumin

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Oxidative Medicine and Cellular Longevity

p-IKK120572120573

IKK120573

87kDa

87kDa

lowastlowastlowastlowast

lowastlowast

p-IK

K120572120573

IKK120573

CON MSA AOPP 2500

05

10

15

20

1 5PTLCompound 2 (120583M)

(a)

lowast lowast

CON MSA AOPP 251 5PTL00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

Compound 2 (120583M)

(b)

lowastlowast lowastlowast lowastlowast

0

1

2

3

CON MSA AOPP 251 5PTL

p-NF-120581B p65

NF-120581B p65

p-N

F-120581

B p6

5N

F-120581

B p6

5

Compound 2 (120583M)

65kDa

65kDa

(c)

lowast lowast

00

05

10

15

CON MSA AOPP 3015 60

p-IK

K120572120573

IKK120573

p-IKK120572120573

IKK120573

87kDa

87kDa

Compound 2 (min)

(d)

lowastlowastlowast

00

05

10

15Tubulin

I120581B120572 39kDa

50kDa

I120581B120572

tubu

lin

CON MSA AOPP 3015 60Compound 2 (min)

(e)

lowastlowastlowast

00

05

10

15

20

25

p-NF-120581B p65

NF-120581B p65

65kDa

65kDa

p-N

F-120581

B p6

5N

F-120581

B p6

5

CON MSA AOPP 3015 60Compound 2 (min)

(f)

Figure 8 Compound 2 prevented AOPP-induced IKK120573 and NF-120581B p65 activation and I120581B120572 degradation in a dose- and time-dependentmanner in podocytes After 24 h of serum starvation podocytes were pretreated with compound 2 (1120583M 25 120583M and 5120583M) for 1 h and thenexposed to 200 120583gmL AOPPs or native MSA for 30min PTL (10 120583M) was used as a positive control (a b and c) Alternatively podocyteswere treated with 200 120583gmLAOPPs for 30min and then compound 2 (5 120583M)was added for a preset time (d e and f)The protein expressionof p-IKK120572120573 p-NF-120581B p65 and I120581B120572 was analyzed by Western blotting The data are expressed as the means plusmn SEM of three independentexperiments ANOVA P lt 005 P lt 001 and P lt 0001 versus CON lowastP lt 005 lowastlowastP lt 001 versus AOPP CON untreated cells MSAmouse serum albumin

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 11

and the degradation of I120581B120572 protein in a time-dependentmanner

4 Discussion

Our data clearly showed that AOPPs upregulated the mRNAand protein expression of MCP-1 and activated proteinsclosely related to the NF-120581B signaling pathway PTL as apotent inhibitor of NF-120581B prevented this effect of AOPPsThe application of SLs (MCL compound 1 and compound 2)in podocytes downregulated AOPP-induced MCP-1 expres-sion and inhibited the proteins closely associated with theNF-120581B signaling pathway In summary the findings indicatethat AOPPs stimulate the expression of the chemokineMCP-1 through an IKKNF-120581B-dependent signaling pathway incultured differentiated mouse podocytes More importantlywe have demonstrated that SLs and their derivatives were ableto significantly decrease AOPP-inducedMCP-1 expression inpodocytes by inhibiting the IKKNF-120581B pathway suggestingthat SLsmayprotect againstDNaswell as other inflammatoryand immune renal diseases mainly throughNF-120581B inhibitionand anti-inflammatory effects

Recent studies on AOPPs as a class of potential renalpathogenic mediators have highlighted the importance ofdetermining the mechanisms by which AOPPs might induceor promote the progression of glomerulopathy Accordingto previous studies there are several major pathogenicmechanisms for AOPPs (1) Central link the PKC-NADPHoxidase-dependent activation of ROS [21 22 35 36] (2)an AOPPs-RAGE interaction [22 37] and (3) a CD36-dependent pathway involving the activation of NF-120581BAP-1 [36 38] However the role of IKKNF-120581B has not yetbeen clearly illuminated Increasing evidence supports thenotion that the IKKNF-120581B pathway plays a role in theinduction and maintenance of the state of inflammationthat underlies metabolic diseases such as obesity and type2 diabetes [39 40] NF-120581B normally resides in the cytosolin an inactive complex with an I120581B family member such asI120581B120572 and this interaction prevents NF-120581B from entering thenucleus and activating DNA transcription However oncethe IKK complex is activated the IKK phosphorylation ofI120581B molecules promotes their degradation and the release ofNF-120581B which then translocates to the nucleus to promotethe transcription of target genes IKK120573 as a subunit of theIKK complex has been shown to be an essential mediator ofthe inflammatory process [39ndash41] Researchers have reportedthat AOPPs could activate NF-120581B which plays a critical rolein the regulation of inflammatory cytokines [36 42] Wefound that the accumulation of AOPPs increased NF-120581B p65phosphorylation Notably we also demonstrated that AOPPsactivated IKK120573 and I120581B120572 in cultured podocytes Consistentwith this observation our previous study demonstrated thatthe administration of AOPPs induced a similar reaction incultured rat MCs [31]

Enough evidence has accumulated to prove a closerelationship between inflammation andDN [4 43 44]MCP-1 expression is significantly increased inDN andmacrophageinfiltration into the glomeruli is associated with glomerularinjury MCP-1-null mice are protected against DN [33] In the

present study we found that AOPPs significantly increasedMCP-1 expression in podocytes In addition our resultsdemonstrate that AOPP-inducedMCP-1 expression is mainlymediated by the IKKNF-120581B-dependent signaling pathwaybecause PTL a potent inhibitor of NF-120581B decreased thisAOPP-induced MCP-1 expression

PTL have been proved to gradually show its anti-inflammatory effect [25 26] Studies have shown that PTLblocks the expression of MCP-1 mRNA and protein throughthe inhibition of IKK activity thereby preventing I120581B degra-dation and inhibiting NF-120581B translocation [27 28] In ourprevious studies we found that PTL could inhibit highglucose-induced and AOPP-induced NF-120581B activation andMCP-1 expression in MCs [30 31] Here we found thatPTL could suppress the MCP-1 expression upregulated byAOPPs via the IKKNF-120581B pathway Notably other stud-ies have demonstrated that PTL derivatives have similaranticancerous effects as PTL moreover the water-solubleform of MCL DMAMCL demonstrated superior efficacywhen compared to DMAPT in the treatment of an acuteleukemia mouse model [34 45] Similarly our previousstudies indicated that certain analogs of PTL have distinctlyanti-inflammatory effects [30 31] The data from the presentstudy show that MCL compound 1 and compound 2 as PTLanalogs exhibit varying degrees of anti-inflammatory effectsthrough the inhibition of the IKKNF-120581B pathway Interest-ingly in addition to the similar bioactivities between PTLand the synthetic compounds the synthetic compounds weresubstantially more stable and presented fewer side effectscompared to PTL Therefore the synthetic SL derivativesmay have a broad scope in future clinical applications withregard to inflammatory and immune renal diseases includingDN

In summary the accumulation of AOPPs promotes aninflammatory response in podocytes and this response ismainly mediated by IKKNF-120581B activation Remarkably wefound that SLs are able to inhibit AOPP-induced MCP-1 expression in podocytes and the salutary effects of SLsare likely mediated by their anti-inflammatory propertiesthrough the inhibition of the IKKNF-120581Bpathway As amajorcomplication of diabetes DN often leads to ESRD and highmortality and finding an effective treatment and preventionfor DN has been a major challenge facing modern medicineThe observations presented in this study demonstrate thepotential renoprotective utility of SLs and suggest a thera-peutic agent for the treatment of inflammation in DN andpotentially other inflammatory and immune renal diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (NSFC) (no 81072848 to Hai-bo Longnos 21072106 and 21372129 to Yue Chen nos 81001377 and81370086 to Quan Zhang and no 81170682 to Hong-xin

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Oxidative Medicine and Cellular Longevity

Niu) and by the Fok Ying Tong Education Foundation (no122037)

References

[1] Z-H Liu ldquoNephrology in Chinardquo Nature Reviews Nephrologyvol 9 no 9 pp 523ndash528 2013

[2] P-H GroopM CThomas J LMoran et al ldquoThe presence andseverity of chronic kidney disease predicts all-causemortality intype 1 diabetesrdquo Diabetes vol 58 no 7 pp 1651ndash1658 2009

[3] M Afkarian M C Sachs B Kestenbaum et al ldquoKidney diseaseand increased mortality risk in type 2 diabetesrdquo Journal of theAmerican Society of Nephrology vol 24 no 2 pp 302ndash308 2013

[4] J Wada and HMakino ldquoInflammation and the pathogenesis ofdiabetic nephropathyrdquo Clinical Science vol 124 no 3 pp 139ndash152 2013

[5] J F Navarro-Gonzalez C Mora-Fernandez M M de Fuentesand J Garcıa-Perez ldquoInflammatory molecules and pathwaysin the pathogenesis of diabetic nephropathyrdquo Nature ReviewsNephrology vol 7 no 6 pp 327ndash340 2011

[6] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts oxidative stress and diabetic nephropathyrdquo OxidativeMedicine and Cellular Longevity vol 3 no 2 pp 101ndash108 2010

[7] F Chow E Ozols D J Nikolic-Paterson R C Atkins and GH Tesch ldquoMacrophages in mouse type 2 diabetic nephropathycorrelation with diabetic state and progressive renal injuryrdquoKidney International vol 65 no 1 pp 116ndash128 2004

[8] C Viedt and S R Orth ldquoMonocyte chemoattractant protein-1 (MCP-1) in the kidney does it more than simply attractmonocytesrdquoNephrology Dialysis Transplantation vol 17 no 12pp 2043ndash2047 2002

[9] L Gu S Hagiwara Q Fan et al ldquoRole of receptor for advancedglycation end-products and signalling events in advanced glyca-tion end-product-induced monocyte chemoattractant protein-1 expression in differentiated mouse podocytesrdquo NephrologyDialysis Transplantation vol 21 no 2 pp 299ndash313 2006

[10] Y Xing S Ye YHu andY Chen ldquoPodocyte as a potential targetof inflammation role of pioglitazone hydrochloride in patientswith type 2 diabetesrdquo Endocrine Practice vol 18 no 4 pp 493ndash498 2012

[11] E Y Lee C H Chung C C Khoury et al ldquoThe monocytechemoattractant protein-1CCR2 loop inducible by TGF-120573increases podocyte motility and albumin permeabilityrdquo Amer-ican Journal of PhysiologmdashRenal Physiology vol 297 no 1 ppF85ndashF94 2009

[12] G R Reddy K Kotlyarevska R F Ransom and R K MenonldquoThe podocyte and diabetes mellitus is the podocyte the keyto the origins of diabetic nephropathyrdquo Current Opinion inNephrology and Hypertension vol 17 no 1 pp 32ndash36 2008

[13] P W Mathieson ldquoThe podocyte as a target for therapies-newand oldrdquo Nature Reviews Nephrology vol 8 no 1 pp 52ndash562012

[14] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[15] A Piwowar M Knapik-Kordecka andMWarwas ldquoMarkers ofoxidative protein damage in plasma and urine of type 2 diabeticpatientsrdquo British Journal of Biomedical Science vol 66 no 4 pp194ndash199 2009

[16] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[17] V Witko-Sarsat M Friedlander T N Khoa et al ldquoAdvancedoxidation protein products as novel mediators of inflammationand monocyte activation in chronic renal failurerdquo The Journalof Immunology vol 161 no 5 pp 2524ndash2532 1998

[18] H Y Li F F Hou X Zhang et al ldquoAdvanced oxidation proteinproducts accelerate renal fibrosis in a remnant kidney modelrdquoJournal of the American Society of Nephrology vol 18 no 2 pp528ndash538 2007

[19] Y S Xiao F H Fan X N Hong et al ldquoAdvanced oxidationprotein products promote inflammation in diabetic kidneythrough activation of renal nicotinamide adenine dinucleotidephosphate oxidaserdquoEndocrinology vol 149 no 4 pp 1829ndash18392008

[20] L Yang M Liang Q Zhou et al ldquoAdvanced oxidation pro-tein products decrease expression of nephrin and podocinin podocytes via ROS-dependent activation of p38 MAPKrdquoScience China Life Sciences vol 53 no 1 pp 68ndash77 2010

[21] L Li Zhou F F Hou G B Wang et al ldquoAccumulation ofadvanced oxidation protein products induces podocyte apop-tosis and deletion through NADPH-dependent mechanismsrdquoKidney International vol 76 no 11 pp 1148ndash1160 2009

[22] L L Zhou W Cao C Xie et al ldquoThe receptor of advancedglycation end products plays a central role in advanced oxi-dation protein products-induced podocyte apoptosisrdquo KidneyInternational vol 82 no 7 pp 759ndash770 2012

[23] V B Mathema Y S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[24] M Chadwick H Trewin F Gawthrop and C Wagstaffldquo Sesquiterpenoids lactones benefits to plants and peoplerdquoInternational Journal of Molecular Sciences vol 14 no 6 pp12780ndash12805 2013

[25] A Saadane S Masters J Di Donato J Li and M BergerldquoParthenolide inhibits I120581B kinase NF-120581B activation andinflammatory response in cystic fibrosis cells and micerdquo Amer-ican Journal of Respiratory Cell and Molecular Biology vol 36no 6 pp 728ndash736 2007

[26] V B Mathema Y-S Koh B C Thakuri and M SillanpaaldquoParthenolide a sesquiterpene lactone expresses multiple anti-cancer and anti-inflammatory activitiesrdquo Inflammation vol 35no 2 pp 560ndash565 2012

[27] O Lopez-Franco Y Suzuki G Sanjuan et al ldquoNuclear factor-kappa B inhibitors as potential novel anti-inflammatory agentsfor the treatment of immune glomerulonephritisrdquo The Ameri-can Journal of Pathology vol 161 no 4 pp 1497ndash1505 2002

[28] W-J Wang F Wu Y Qian et al ldquoInhibition of inflammatoryfactors by parthenolide in human renal mesangial cells underhyperglycemic conditionrdquoAfrican Journal of Biotechnology vol9 no 23 pp 3458ndash3463 2010

[29] M D Sanchez-Nino J Poveda A B Sanz et al ldquoFn14in podocytes and proteinuric kidney diseaserdquo Biochimica etBiophysica ActamdashMolecular Basis of Disease vol 1832 no 12pp 2232ndash2243 2013

[30] Q-Q Jia J-C Wang J Long et al ldquoSesquiterpene lactones andtheir derivatives inhibit high glucose-inducedNF-120581B activation

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 13

and MCP-1 and TGF-1205731 expression in rat mesangial cellsrdquoMolecules vol 18 no 10 pp 13061ndash13077 2013

[31] J C Wang Y Zhao S J Chen et al ldquoAOPPs induce MCP-1expression by increasing ROS-mediated activation of the NF-kappaB pathway in rat mesangial cells inhibition by sesquiter-pene lactonesrdquoCellular Physiology and Biochemistry vol 32 no6 pp 1867ndash1877 2013

[32] S J Shankland JW Pippin J Reiser andPMundel ldquoPodocytesin culture past present and futurerdquo Kidney International vol72 no 1 pp 26ndash36 2007

[33] E Tarabra S Giunti F Barutta et al ldquoEffect of the monocytechemoattractant protein-1CC chemokine receptor 2 system onnephrin expression in streptozotocin-treated mice and humancultured podocytesrdquoDiabetes vol 58 no 9 pp 2109ndash2118 2009

[34] Q Zhang Y Lu Y Ding et al ldquoGuaianolide sesquiterpenelactones a source to discover agents that selectively inhibit acutemyelogenous leukemia stem and progenitor cellsrdquo Journal ofMedicinal Chemistry vol 55 no 20 pp 8757ndash8769 2012

[35] X F Wei Q G Zhou F F Hou B Y Liu and M LiangldquoAdvanced oxidation protein products induce mesangial cellperturbation through PKC-dependent activation of NADPHoxidaserdquo American Journal of Physiology Renal Physiology vol296 no 2 pp F427ndashF437 2009

[36] W Cao J Xu Z M Zhou G B Wang F F Hou and J NieldquoAdvanced oxidation protein products activate intrarenal renin-angiotensin system via a CD36-mediated redox-dependentpathwayrdquo Antioxidants and Redox Signaling vol 18 no 1 pp19ndash35 2013

[37] Z J Guo H X Niu F F Hou et al ldquoAdvanced oxidationprotein products activate vascular endothelial cells via a RAGE-mediated signaling pathwayrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1699ndash1712 2008

[38] Y Iwao K Nakajou R Nagai et al ldquoCD36 is one of importantreceptors promoting renal tubular injury by advanced oxidationprotein productsrdquo American Journal of Physiology Renal Physi-ology vol 295 no 6 pp F1871ndashF1880 2008

[39] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[40] R G Baker M S Hayden and S Ghosh ldquoNF-120581B inflamma-tion and metabolic diseaserdquo Cell Metabolism vol 13 no 1 pp11ndash22 2011

[41] S Kiechl J Wittmann A Giaccari et al ldquoBlockade of receptoractivator of nuclear factor-120581B (RANKL) signaling improveshepatic insulin resistance and prevents development of diabetesmellitusrdquo Nature Medicine vol 19 no 3 pp 358ndash363 2013

[42] Q G Zhou M Zhou A J Lou D Xie and F F HouldquoAdvanced oxidation protein products induce inflammatoryresponse and insulin resistance in cultured adipocytes viainduction of endoplasmic reticulum stressrdquo Cellular Physiologyand Biochemistry vol 26 no 4-5 pp 775ndash786 2010

[43] S Crunkhorn ldquoMetabolic disorders breaking the links betweeninflammation and diabetesrdquo Nature Reviews Drug Discoveryvol 12 no 4 article 261 2013

[44] A K H Lim and G H Tesch ldquoInflammation in diabeticnephropathyrdquo Mediators of Inflammation vol 2012 Article ID146154 12 pages 2012

[45] Y H Ding H X Fan J Long Q Zhang and Y Chen ldquoTheapplication of Heck reaction in the synthesis of guaianolide

sesquiterpene lactones derivatives selectively inhibiting resis-tant acute leukemic cellsrdquo Bioorganic and Medicinal ChemistryLetters vol 23 no 22 pp 6087ndash6092 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom