research article red ginseng extract ameliorates autoimmune arthritis via regulation...

Research ArticleRed Ginseng Extract Ameliorates AutoimmuneArthritis via Regulation of STAT3 Pathway Th17TregBalance and Osteoclastogenesis in Mice and Human

JooYeon Jhun1 Jennifer Lee12 Jae-Kyeong Byun1 Eun-Kyung Kim1

Jung-Won Woo1 Jae-Ho Lee12 Seung-Ki Kwok12 Ji-Hyeon Ju12 Kyung-Su Park12

Ho-Youn Kim12 Sung Hwan Park12 and Mi-La Cho123

1 The Rheumatism Research Center Catholic Research Institute of Medical Science The Catholic University of KoreaSeoul Republic of Korea

2Divison of Rheumatology Department of Internal Medicine School of Medicine The Catholic University of KoreaSeoul St Maryrsquos Hospital 505 Banpo-dong Seocho-gu Seoul 137-701 Republic of Korea

3 Conversant Research Consortium in Immunologic Disease College of Medicine The Catholic University of KoreaKorea 505 Banpo-Dong Seocho-Ku Seoul 137-040 Republic of Korea

Correspondence should be addressed to Sung Hwan Park raparkcatholicackr and Mi-La Cho iammilacatholicackr

Received 28 February 2014 Revised 5 June 2014 Accepted 18 June 2014 Published 23 July 2014

Academic Editor Marilia Seelaender

Copyright copy 2014 JooYeon Jhun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation Red ginseng is asteamed and dried Panax ginseng CA Meyer which has been used as alternative medicine for thousands of years This studywas undertaken to investigate the effects of red ginseng extracts (RGE) on autoimmune arthritis in mice and humans and todelineate the underlying mechanism RGE was orally administered three times a week to mice with arthritis Oral administrationof RGEmarkedly ameliorated clinical arthritis score and histologically assessed joint inflammation in mice with CIA A significantreduction in STAT3 phosphorylation and a decrease in the number of Th17 cells were observed with RGE treatment There wasalso a marked reduction in RANKL-induced osteoclastogenesis with treatment of RGE The inhibitory effect of RGE on Th17differentiation and osteoclastogenesis observed inmice was also confirmed in the subsequent experiments performed using humanperipheral blood mononuclear cells Our findings provide the first evidence that RGE can regulate Th17 and reciprocally promoteTreg cells by inhibiting the phosphorylation of STAT3 Therefore RGE can ameliorate arthritis in mice with CIA by targetingpathogenic Th17 and osteoclast differentiation suggesting a novel therapy for treatment of RA

1 Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune diseasecharacterized by chronic joint inflammation that can lead tojoint destruction and disability Although the exact molec-ular mechanism of the RA pathogenesis remains elusiveproinflammatory cytokines including tumor necrosis factor-(TNF-) 120572 and interleukin- (IL-) 1120573 and autoreactive T cellsare known to play central roles in the development andprogression of RA [1] Although RA was conventionallyconceived asTh1-driven inflammatory disease it is becomingclear that Th17 rather than Th1 is a major pathogeniccell that orchestrates the complex network of the sustained

inflammation and disease progression [2ndash4] Th17 producesIL-17 which is abundantly expressed in the arthritic joints andneutralization of IL-17 results in attenuation of arthritis [4]IL-17 not only induces proinflammatory cytokines but alsodirectly enhances osteoclastogenesis by upregulating receptoractivator nuclear kappa ligand (RANKL) on osteoblasts [5]contributing to bone erosion in RA Recently Th17 wasreported to express RANKL itself as well as directly inducingmature osteoclasts via cell-to-cell contact substantiating itsrole in inflammatory bone destruction [6]

Thedevelopment ofTh17 is largely dependent on cytokinemilieu Transforming growth factor- (TGF-) 120573 and IL-6

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 351856 13 pageshttpdxdoiorg1011552014351856

2 Mediators of Inflammation

primes initial differentiation and IL-23 promotes functionalmaturation of Th17 cells [7] As signal transducer and acti-vator of transcription (STAT) 3 phosphorylation directly reg-ulates retinoic acid receptor-related orphan receptor (ROR)120574t which is the master molecule of Th17 the Janus kinase(JAK)2-STAT3 pathway initiated by IL-6 is essential in theTh17 development With recent advances in understandingthe central role of Th17 in the pathogenesis of RA noveltherapeutics targeting Th17 are being utilized or are stillunder development The blockade of IL-6 (Tocilizumab) iscurrently available on the market while the JAK2 inhibitorandmonoclonal antibody against IL-17 are still under clinicaltrials among RA patients

Ginseng the root of Panax ginseng CA Meyer (Arali-aceae) is a perennial herb which has been used as herbalmedicine in Eastern Asia for thousands of years [8 9]Ginseng has two types of preparation one is air-dried whiteginseng and the other is steamed and sun-dried red ginsengIt contains various active components including ginseno-sides polysaccharides peptides polyacetylenic alcohols andfatty acids [10] Ginsenosides in particular are also calledsaponin extracts and are known to exert the majority ofthe pharmacologic effects seen from ginseng Red ginsengis made from a ginseng plant going through an intensiveprocess of cleaning steaming and drying Heat treatmentof ginseng leads to chemical changes of ginsenosides givingthem distinct physical characteristics Major ginsenosidesof red ginseng saponin extract were shown to include lesspolar red ginseng-unique saponins Rg3 Rk1 and Rg5 in ahigh-performance liquid chromatographic analysis [11] Assupplementation with red ginseng is believed to improvehealth numerous studies have been conducted to validateits beneficial effects They demonstrated that RGEs had anti-inflammatory [12 13] antidiabetic [14 15] and anticancer[16] effects Anti-inflammatory property of RGE leads to theunderstanding that it may have antiarthritic effect via mod-ulating inflammation Indeed accumulating evidence hassuggested that ginseng extracts can ameliorate autoimmunearthritis It was reported that treatmentwith ginsenoside Rb-1significantly attenuated arthritis inmice with established CIA[17] Orally administered RGEs including Rg3 Rk1 and Rg5asmajor components also successfully suppressedCIA shownby decreased production of proinflammatory cytokinesmatrix metalloproteinase (MMP) and nitrotyrosine in thearthritic joints [11] More recently compound K an activeginsenoside metabolite with a high intestinal absorption ratewas shown to decrease TNF-120572-induced MMP production inRA-fibroblast-like-synoviocyte (FLS) and suppress osteoclas-togenesis by inhibiting the expression of nuclear factor ofactivated T cells (NFATc1) [18] However it has not beensuggested whether or not RGE has a regulatory effect onTh17 which is a central pathogenic cell in RA Given thatginseng was reported to repress STAT3 activation in cancercells [19] it is plausible to assume that RGE can suppressTh17by inhibiting STAT3 phosphorylation enhancing enhancedTreg cells by reciprocal regulation [20]

In the present study we verified that oral administrationof RGE could suppress arthritis in CIA model To delineatethe mechanism underlying the antiarthritic effect in terms of

Th17 the effect of RGE on pathogenicTh17 cell differentiationboth in vivo and in vitro was investigated In addition theeffect of RGE osteoclast formation which is implicated inbone erosion in RA was examined

2 Methods and Materials

21 Animals Six week oldmale DBA1J mice were purchasedfrom SLC Inc (Shizouka Japan) and IL-10 knockout (KO)mice in the DBA1J background were kindly provided byLinda K Myers (University of Tennessee) Mice were main-tained under specific pathogen-free conditions at the instituteof Medical Science at the Catholic University of Korea andwere provided standard mouse chow (Ralston Purina StLouis MO USA) and water ad libitum All experimentalprocedures were examined and approved by the AnimalResearch Ethics Committee of the Catholic University ofKorea (permit number CUMC20) which conforms to allNational Institutes of Health of the USA guidelines

22 Preparation of Red Ginseng Extract Red ginseng extract(RGE) was kindly provided by the Korea Ginseng Cooper-ation Daejeon Cooperation (Daejeon Republic of Korea)RGEyields 437 saponins themain components of ginseno-sides were Rb1 (126) Rb2 (62) Rc (69) Rd (34) Re(64) Rf (21) Rg1 (158) and Rg3 (14)The identifiedconstituents are well standardized and qualified by the KoreaGinseng Cooperation Other constituents in RGE are starchsugars fat fiber proteins vitamins minerals and so forth

23 Induction of CIA and Treatment with Red Ginseng Toinduce CIA in mice 100 120583L of an emulsion containing 100120583gbovine type II collagen (CII) and complete Freundrsquos adjuvant(Chondrex RedmondWA USA) was injected intradermallyinto the base of the tail as the primary immunization Twoweeks later 100 120583g CII dissolved and emulsified 1 1 withincomplete Freundrsquos adjuvant (Difco Detroit MI USA) wasadministered into the footpad as a booster injection To assessthe influence of RGE on symptom severity in the CIAmodelmice were orally treated with 10mgkg RGE in saline or withvehicle alone three times a week after booster immunizationover the course of 6 weeks The arthritis index in these micewas scored twice weekly and expressed as the sum of thescores of four limbs

24 Measurement of CII-Specific IgG and IgG Serum levels ofCII-specific IgG (Total IgG IgG1 IgG2a) and IgG (Total IgGIgG1 IgG2) measurement antibodies were measured usinga commercially available ELISA kit (Beathyl LaboratoriesMontgomery TX USA)

25 Immunohistochemistry Mouse joint tissue was fixed in4 paraformaldehyde decalcification EDTA bone decalcifierand embedded in paraffin The section (7 120583m) was stainedwith hematoxylin and eosin Safranin O and toluidine blueto detect proteoglycans

Immunohistochemistry was performed using the Vec-tastatin ABC kit (Vector Laboratories Burlingame CAUSA) Tissue was first incubated with primary antibodiesto IL-17 IL-6 IL-1b TNF-120572 Nitrotyrosine NRF2 (Santa

Mediators of Inflammation 3

Cruz Biotechnology Santa Cruz CA USA) and HO-1Inos (Abcam Cambridge Science Park Cambridge UK)overnight at 4∘C The sections were counterstained withhematoxylin Samples were photographed with an Olympusphotomicroscope (Tokyo Japan)

26 Mouse In Vitro Osteoclastogenesis Isolation of bone-marrow-derived monocytemacrophage (BMM) cells anddifferentiation of osteoclast precursor cells (preosteoclasts)was performed as described [21]Three days later the nonad-herent cells werewashed out and preosteoclasts were culturedfurther in the presence of 10 ngmL M-CSF 100 ngmLRANKL (Peprotech London UK) and various concentra-tions of RGE for four days to generate osteoclasts On day2 the medium was replaced with fresh medium containingM-CSF RANKL and RGE

27 Human in CD4 T Cell Isolation and Differentiation CD4T cells were isolated fromperipheral bloodmononuclear cells(PBMCs) using a CD4 T cell isolation kit (Miltenyi Biotec)according to themanufacturerrsquos instruction To establishTh17cell-polarizing conditions the CD4+ T cells were stimulatedwith plate-bound anti-CD3 anti-CD28 anti-IFN-r anti-IL-4 IL-1120573 (20 ngmL) and IL-6 (20 ngmL) for 3 days Allcytokines were from RampD Systems with the exception ofTGF-120573

28 Intracellular Staining and Flow Cytometry The followingantibodies were used for mouse cells Th17 cells PerCP-Cy55-conjugated anti-CD4 (eBioscience) and FITC conju-gated anti-IL-17A (eBioscience) was used for intracellularstainingThe following antibodies were used for human cellsTh17 and Treg cells were from PE-Cy7-conjugated anti-CD4APC- conjugated anti-CD25 (both from BD Pharmingen)and FITC-conjugated anti-Foxp3 PE-conjugated anti-IL-17(both from eBioscience)

29 Enzyme-Linked Immunosorbent Assay (ELISA) Theamount of IL-17 IL-21 IL-22 and IL-10 were measured usinga sandwich ELISA (RampD Systems) Absorbance at 405 nmwas measured using an ELISA microplate reader (MolecularDevices)

210 Human In Vitro Osteoclastogenesis The generation ofhuman preosteoclasts was performed as described [21] Afterthree days these preosteoclasts were cultured further in thepresence of 25 ngmLM-CSF 30 ngmL RANKL and variousconcentrations of RGE for nine days to generate osteoclastsOn day 3 the medium was replaced with fresh mediumcontaining M-CSF RANKL and RGE TRAP stain wasperformed as described [21] All the subjects gave informedconsent before the study The study received the approval ofthe institutional review board of Seoul St Maryrsquos Hospitalfrom all healthy volunteers

211 TRAP Staining A commercial TRAP kit (Sigma StLouis ML USA) was used according to the manufacturerrsquosinstructions however counterstaining with hematoxylin wasomitted TRAP-positive multinuclear cells (MNCs) contain-ing three or more nuclei were counted as osteoclasts

212 Gene Expression Analysis Using Real-Time PCR PCRamplification and analysis were performed on a Light Cycler20 instrument (Roche Diagnostic Mannheim Germany)with software version 40 All reactions were performed usingLightCycler FastStart DNA master SYBR green I (TakaraShiga Japan) according to the manufacturerrsquos instructionThe following primers for mouse samples were used IL-1751015840-CCT CAA AGC TCA GCG TGT CC-31015840(sense) and 51015840-GAG CTC ACT TTT GCG CCA AG-31015840(anti-sense) RORrt51015840-TGT CCT GGG CTA CCC TAC TG-31015840(sense) and 51015840-GTCCAGGAGTAGGCCACATT-31015840(antisense) CCR6 51015840-CCA TGA CTG ACG TCT ACC TGT TGA ACA-31015840(sense)and 51015840-GAA CAG CTC CAG TCC CAT ACC CAG CAG-31015840(antisense) Foxp3 51015840-GGC CCTT CTC CAG GAC AGA-31015840(sense) and 51015840-GCT GAT CAT GGC TGG GTT GT-31015840(antisense) SOCS3 51015840-CCT TTG ACA AGC GGA CTCTC-31015840(sense) and 51015840-GCC AGC ATA AAA ACC CTT CA-31015840(antisense) RANK 51015840-TGT ACT TTC GAG CGC AGATG-31015840(sense) and 51015840-CCA CAA TGT GTT GCA GTT CC-31015840(antisense) MMP9 51015840-CTG TCC AGA CCA AGG GTACAGCCT-31015840(sense) and 51015840-GAGGTA TAGTGGGACACATAG TGG-31015840(antisense) cathepsin K 51015840-CAG CAG AGGTGT GTA CTA TG-31015840(sense) and 51015840-GCG TTG TTC TTACGAGC-31015840(antisense) TRAP 51015840-TCCTGGCTCAAAAAGCAG TT-31015840(sense) and 51015840-ACA TAG CCC ACA CCG TTCTC-31015840(antisense) The following primers for human sampleswere used RANK 51015840-GCT CTA ACA AAT GTG AAC CAGGA-31015840(sense) and 51015840-GCC TTG CCT GTA TCA CAA ACT-31015840(antisense) MMP9 51015840-CGC AGA CAT CGT CAT CCAGT-31015840(sense) and 51015840-GGA TTG GCC TTG GAA GAT GA-31015840(antisense) cathepsin K 51015840-TGA GGC TTC TCT TGGTGT CCA TAC-31015840(sense) and 51015840-AAA GGG TGT CAT TACTGC GGG-31015840(antisense) CTR 51015840-TGG TGC CAA CCACTA TCC CTG A-31015840(sense) and 51015840-CAC AAG TGC CGCCAT GAC AG-31015840(antisense) The level of mRNA expressionwas normalized to that of 120573-actin

213 Immunoblot Analysis PBMC were cultured with anti-CD3 and anti-CD28 in the presence or absence of RDEfor 72 h Mice splenocytes were cultured with the Th17condition in the presence or absence of RDE for 72 hBoth cells were then harvested and lysed with lysis bufferProtein concentration was measured using the Bradfordmethod (Bio-Rad Herculed CA USA) Protein sampleswere separated using 12 SDS-PAGE and transferred ontonitrocellulose membranes (AmersharmPharmacia BiotechPiscataway NJ USA) For Western blot hybridization themembrane was preincubated with blocking buffer for 2 hand then incubated with primary antibodies against TotalIkBa p-IkBa Total ERK p-ERK Total STAT5 pSTAT5 TotalSTAT3 pSTAT3(727) pSTAT3(705) (all from Cell SignalingDanvers ma) and 120573-actin for 1 h After washing horseradishperoxidase-conjugated secondary antibodies were addedand the membranes were incubated for 1 h at room tem-perature After washing the hybridized bands were detectedusing an ECL detection kit (Pierce Rockford IL USA) andHyperfilm (Agfa Belgium)


214 Statistical Analysis Experimental values are presentedas mean plusmn SD of triplicate cultures and representative ofexperiments performed on three occasions Statistical signif-icance was determined by Mann-Whitney 119880 test or ANOVAwith Bonferronirsquos post-hoc test using the Graphpad Prism(v501) Values of 119875 lt 005 were considered statisticallysignificant lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

3 Results

31 Red Ginseng Extract Suppresses Collagen-Induced Arthri-tis Results showed that administration of oral RGE threetimes a week (10mgkg) reduced the arthritic score andarthritis incidence almost completely compared to the oraladministration of the vehicle (Figure 1(a)) When CIA wasinduced among the IL-10 KO mice there were higher ratesof clinical signs and more severe knee and paw injury whencompared to the wild type mice As shown in Figure 1(b)RGE successfully suppressed the arthritic score and arthritisincidence in the CIAmice of the IL-10 knockout backgroundHistological examination of the joints demonstrated that thepaws and ankles of the red ginseng extract-treated mice hada lower degree of inflammation and cartilage damage com-pared with those of the vehicle-treated mice as determinedon day 49 after immunization (Figure 1(c)) In addition thered ginseng extract-treated mice expressed markedly lowerlevels of not only proinflammatory cytokines such as TNF-120572IL-1b IL-6 and IL-17 but also oxidative stress markers suchas nitrotyrosine and iNOS as demonstrated by immunohis-tochemical analysis (Figure 1(d)) The results implicated thatantioxidant activity of RGE might contribute to attenuatingoxidative stresses in CIA mice We next examined whethertreatment with red ginseng extract would modulate humoralimmune responses by assessing Ab production in CIA miceFigure 1(e) illustrates that treatment with RGE efficientlyattenuated the production of total IgG and IgG2a the Th1-type Ab in the sera of CIA mice The effect of red ginsengextract on the Ag-specific humoral immune responses invivo was also assessed The serum levels of the CII-specificIgG2a and total IgG were significantly lower in the micetreated with red ginseng extract (Figure 1(e)) Interestinglythe CII-specific IgG1a the Th2-type Ab was considerablyincreased among the red ginseng extract-treated CIA mice(Figure 1(e))

32 RedGinseng Extract ReciprocallyModulates Populations ofRegulatory T Cells and Th17 Cells in CIA Mice mRNA fromsplenocytes of either red ginseng extract-treated CIAmice orvehicle-treated CIA mice was isolated and the expression ofTh17 cell- and Treg cell-related markers by RT-PCR was thenanalyzed The results showed that the mRNA levels of Th17cell-related molecules such as IL-17 RORC and CCR6 weredownregulated whereas the mRNA levels of Treg cell-relatedmolecules such as Foxp3 and SOCS3 were upregulated inthe red ginseng-treatedCIAmice (Figure 2(a)) Furthermorethe red ginseng treatment reduced the number of IL-17-producing CD4+ T cells in the splenocytes of CIA mice asanalyzed by flow cytometry (Figure 2(b)) Additionally we

measured the numbers of CD4+CD25+Foxp3+ regulatory Tcells and CD4+IL-17+ T cells (Th17 cells) in tissues of spleensand drain lymph nodes by immunofluorescence confocalmicroscopyThe spleen tissues from themice treated with redginseng extract showed an increased number of Foxp3+ regu-latory T cells and a decreased number ofTh17 cells comparedwith those of the vehicle-treated mice (Figure 2(c)) Thenumber of Th17 cells was also significantly decreased in thetissues of the drain lymph nodes (data not shown) althoughthe number of Treg cells remained unchanged (Figure 2(c))

33 Red Ginseng Extract Reduces STAT3 Phosphorylation inthe CD4+ TCells inMice Thenumber of pSTAT3-expressingCD4+ T cells was indeed decreased in the red ginseng-treated CIA mice compared with those of the vehicle-treated CIA mice (Figure 3(a)) To evaluate whether RGEpromotes phosphorylation of STAT3 in vitro we performedimmunoblot analysis of protein extracts prepared fromCD4+T cells isolated from spleens cultured in a Th17-polarizingcondition with various concentrations of RGE RGE dose-dependently decreased the amount of phosphorylated STAT3at tyrosine 705 under the Th17-generating condition whilethe total amount of STAT3 remained the same (Figure 3(b))Phosphorylation of IkB and ERK was also reduced with RGEtreatment (Figure 3(b)) After stimulation under conditionsfavoring the development of Th17 cells we found thatphosphorylation of STAT3 at 705 and 727 was significantlydecreased by the addition of red ginseng extract althoughphosphorylation of STAT3 at 727 was reduced in a less degree(Figure 3(b))

34 Red Ginseng Extract Inhibits Osteoclastogenesis in CIAMice The number of TRAP positive cells was markedlyreduced in the joint tissues of RGE-treated mice comparedwith those of vehicle-treated mice (Figure 4(a)) We nextinvestigated whether RGE would directly inhibit osteoclastformation in vitro The BMM cells were prepared from WTmice and stimulated with M-CSF andor RANKL to induceosteoclast differentiation The addition of various concentra-tions of RGE during the induction of osteoclastogenesis sig-nificantly inhibited osteoclast formation in a dose-dependentmanner (Figure 4(b)) Transcripts of various osteoclastogenicmarkers such as RANK MMP9 cathepsin K and TRAPwere also considerably decreased by the addition of RGE(Figure 4(c))

35 Red Ginseng Extract Increased Foxp3-Expressing Regu-latory T Cells and Decreased IL-17-Expressing Th17 Cells inHuman PBMCs Concentrations of RGE used in these invitro experiments did not affect cell viability as demon-strated by the MTT assay (Figure 5(a)) Transcripts of Treg-related molecules such as Foxp3 Socs3 and IL-10 signif-icantly increased whereas mRNAs of IL-17 and IL-6 weremarkedly reduced by red ginseng extract in CD4+ T cells ofhuman PBMCs (Figure 5(c)) Additionally Th17-associatedcytokines like IL-26 and IL-21 were also decreased althoughIL-22 did not change significantly (Figure 5(c)) at the mRNAlevel Subsequent flow cytometry analysis confirmed that the


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Figure 1 Red ginseng extract suppresses collagen-induced arthritis Collagen-induced arthritis (CIA) was induced Mice were orally treatedwith 10mgmL of red ginseng extract (RGE) or vehicle only (a) Clinical scores and incidence of arthritis in CIA-induced DBA1J mice (b)Clinical scores and incidence of arthritis in IL-10 KOmice (c) Representative histological features of the joint of RGE or vehicle-treated micewith CIA HampE safranin O and toluidine blue staining are shown (d) Representative immunohistochemical staining of the joint of RGEor vehicle-treated mice with CIA (e) Concentrations of CII-specific IgG and IgG2a in sera of RGE or vehicle-treated mice with CIA weredetermined by ELISA Data are presented as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001compared to control mice

populations of Treg and Th17 cells were indeed reciprocallyregulated in which Treg cells were increased and Th17cells were decreased by RGE in a dose-dependent manner(Figure 5(b))

CD4+ T cells either when activated with anti-CD328antibodies alone (data not shown) orwhen cultured in aTh17-polarizing condition produced considerably lower levels ofIL-17 IL-21 and IL-22 after the treatment with RGE Inter-estingly Treg-associated cytokines IL-10 was significantlyincreased by the addition of RGE (Figure 5(d)) Phospho-rylation of STAT3 at tyrosine 705 and 727 was markedlyreduced with the treatment of 1000 120583gmL red-ginsengextract (Figure 5(e)) However the amount of pSTAT5 wasnot affected by RGE treatment

36 Red Ginseng Extract Inhibits Osteoclastogenesis inHumans Figure 6(a) displays that RGE treatment effectivelyprevented human monocytes from differentiating intomature osteoclasts which was determined by TRAPstaining It also reduced the expressions of osteoclastogenicmarkers such as the calcitonin receptor (CTR) cathepsin Kand MMP-9 (Figure 6(b))

4 Discussion

In this study oral administration of RGE ameliorated theclinical arthritis score and the histological severity of jointinflammation in mice with CIA RGE inhibited differenti-ation of Th17 which is a main pathogenic T cell in RAby suppressing phosphorylation of STAT3 and reciprocallyincreasedTreg population Furthermore treatmentwithRGEsubstantially suppressed osteoclastogenesis which mightcontribute to less bone erosion inCIAmice treatedwith RGE

A significant reduction in proinflammatory cytokinesincluding IL-17 IL-6 TNF-120572 and IL-1120573 was observed in thejoints of RGE-treated CIA mice As in vitro studies havedemonstrated that IL-17 is a powerful inducer of IL-6 TNF-120572 and IL-1120573 [3] a decrease in Th17 which in turn reducedIL-17 in the arthritic joints seemed to cause the decrease inproduction of those cytokines Moreover decreased produc-tion of IL-6 and IL-1b might have contribution to suppressedTh17-IL-17 activation

As oxidative stress is known to play an important rolein RA pathogenesis [22ndash24] oxidative stress representedby iNOS and nitrotyrosine expression was also measuredwhich diminished with RGE treatment in CIA Nrf2 is atranscription factor activating upon the exposure to ROSExpression of Nrf2 and HO-1 suppressed regulation ofoxidative stress [25] Activated Nrf2 binds to antioxidantresponse element (ARE) located in the regulatory regionsof the genes coding for antioxidative enzymes such as HOand enhances transcription of the antioxidative enzymes [26]Thus decreased expression of Nrf2 and HO in the jointsof RGE-treated mice indicates that the oxidative stress waslower than the vehicle-treated mice Our data corroboratethe previously reported antioxidative effect of RGE [27 28]In addition to inhibitory effect on Th17 the antioxidativeproperty of RGE seems to exert an additional effect insuppressing CIA

The main focus of this study was to investigate whetherthe antiarthritic effect of RGE was mediated by the regu-lation of pathogenic Th17 cells As expected the numberof Th17 cells was significantly reduced among the RGE-treated mice with CIA indicating that RGE suppressedTh17 differentiation in arthritic condition As mentionedDevelopment of Th17 cell was caused by activation of STAT3signal pathway [7] Therefore we investigated whether RGEcould suppress STAT3 phosphorylation and discovered thatphosphorylation of STAT3 was decreased in RGE-treatedmice as demonstrated by immunofluorescence confocalmicroscopy and inRGE-treatedCD4+ Tcells cultured inTh17polarizing conditions measured by immunoblotting Thesedata suggest that RGE might block the signal transductionpathway initiated by binding of IL-6 to its receptor whichactivates downstream kinases and subsequently allows thephosphorylation of STAT3 This was consistent with pre-vious report that American ginseng albeit different genusdramatically suppressed JAK2-STAT3 activation in aorticsmooth muscle cell in rats [29] Diminished expression ofIL-6 also seems to have contributed to the Th17 regulationTo elucidate whether RGE directly regulates IL-6 expressionrequires future research

The effect of RGE on Treg as well as Th17 in pathologicinflammatory condition in mice with CIA or in CD4+ T cells


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Figure 2 Red ginseng extract reciprocally modulates populations of regulatory T cells and Th17 cells in CIA mice (a) Messenger RNAwas isolated from splenocytes of either red ginseng extract (RGE) or vehicle-treated CIA mice The mRNA expressions of Th17 cell-relatedmolecules weremeasured by RT-PCR (b)The proportion of IL-17-producing CD4+ T cells in the splenocytes was analyzed by flow cytometry(c) Spleens were subjected to immunostaining for CD4+CD25+Foxp3+ or CD4+IL-17+ cells The number of cells was counted in fourindependent quadrants Data are presented as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001compared to control mice

cultured in Th17 polarizing condition was investigated Ourdata showed that the number of Treg cells increased withRGE treatment while Th17 differentiation was suppressedThis was in line with the previous finding that ginsenosideRp-1 activated Treg cells [30] However this simultaneousregulation on Th17 and Treg by red ginseng has neverbeen reported The notion that Treg is reciprocally regulatedwith Th17 originates from the report that advocated theplasticity of T cell subsets [31] Th17 and Treg are thoughtto have common precursors cell before they are destined tocertain effecter cells As transcription of IL-17 is regulatedby competitive binding of pSTAT3 and pSTAT5 [20] and

pSTAT5 is a critical transcriptional factor for Foxp3 themaster molecule of Treg the ratio of pSTAT3 and pSTAT5is expected to be one of determinants for final effector celltype While RGE significantly reduced pSTAT3 the levelof STAT5 phosphorylation was not increased with RGEtreatment Although the RGE failed to increase the amountof pSTAT5 the decreased ratio of pSTAT3pSTAT5 seemed tocontribute to theTh17 suppression and the reciprocal increasein Treg Namely the relative reduction in pSTAT3 may haveresulted in pSTAT5 binding on promoter site of IL-17 andsuppressed IL-17 expression With the suppression of Th17differentiation a shift toward Treg differentiation seemed to


Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)

Th17 (RGE 120583gmL)

Th17 (RGE 120583gmL) Th17 (RGE 120583gmL)

Th17 (RGE 120583gmL) Th17 (RGE 120583gmL) Th17 (RGE 120583gmL)

mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)

Figure 3 Red ginseng extract reduces STAT3 phosphorylation in the CD4+ T cells in mice (a) Spleens of red ginseng extract or vehicle-treated CIA mice were subjected to immunostaining for CD4+pSTAT3y705+ or CD4+pSTAT3s727+ cells The number of cells was countedin four independent quadrants (b) Isolated CD4+ T cells were cultured in a Th17-polarizing condition for 3 days in the absence or presenceof various concentrations of red ginseng extract (RGE) The expression of various signaling molecules was determined by western blottingData are presented as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to control mice


TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash

RGE 001120583gmL RGE 1120583gmLRGE 01120583gmL

RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP

MCSF + RANKL (RGE 120583gmL)

+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA

lowastlowast lowastlowast

lowastlowastlowast00

05

10

15

1 10 100mdash

lowastlowastlowast lowast

00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA

MCSF + RANKL (RGE 120583gmL) MCSF + RANKL (RGE 120583gmL) MCSF + RANKL (RGE 120583gmL)

(c)

Figure 4 Red ginseng extract inhibits osteoclastogenesis in CIA mice (a) The number of TRAP+ cells in the arthritic joint of red ginsengextract (RGE) or CIA was counted using light microscopy The BMM cells from the DBA1J mice were cultured with M-CSF (10 ngmL)and RANKL (50 ngmL) in the presence or absence of various concentrations of RGE (b) Cells were fixed and stained for TRAP (originalmagnification times100) The number of TRAP+ cells was counted using light microscopy The representative photographs from each group areshown (b)The mRNA expressions of various osteoclastogenic markers such as RANK MMP9 Cathepsin K and TRAP were analyzed usingreal time PCR BMM bone-marrow-derived monocytemacrophage MMP9 matrix metalloproteinase 9 Data are presented as the mean plusmnSD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to control mice

be augmented Although the differentiation into a specificT cell subset is mainly determined by master transcrip-tional regulator (eg ROR120574t Foxp3) and phosphorylationof STATs recent data indicate that other signaling pathwaysare also involved in this fine-tuning process ERK signalingwas suggested to be one of those pathways [32] where aninhibition of ERK decreases IL-6-induced ROR120574t expressionand reciprocally enhances TGF-120573-induced Foxp3 expressionresulting in decreased Th17Treg ratio Hence the ability ofRGE to suppress phosphorylation of ERK as well as STAT3may be another efficient mechanism by which RGE regulatedTh17Treg balance

The suppression of osteoclastogenesis by RGE wasdemonstrated by both in vivo and in vitro In CIA joints

the decrease in number ofTh17 cells seems to have resulted ina reduction in TRAP(+) osteoclasts The inhibitory effect ofRGE on osteoclastogenesis in vitro was previously reportedby He et al [33] They demonstrated that ginsenoside Rh2a component of red ginseng suppressed osteoclast differ-entiation by inhibiting RANKL-induced c-fos and NFATc1expression Ginsenoside Rh2 also inhibited phosphorylationof ERK and I120581B120572 thereby suppressing NF-120581B which wasconsistent with our findings observed in CD4+ T cells Inaddition these results showed that RGE directly suppressedRANK expression during osteoclastogenesis in vitro ham-pering RANKL-induced signaling

The limitation of this study is that the exact chemicalcomposition of RGE used was not analyzed Therefore it


000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

FL1-HFL1-FoxP3- FITC FL1-HFL1-FoxP3- FITCFL1-HFL1-FoxP3- FITC FL1-HFL1-FoxP3- FITCFL1-HFL1-FoxP3- FITC

FL2

-HF

L2-I

L-17

PE

521594

IL-17

Foxp3 Foxp3 Foxp3 Foxp3 Foxp3

861 598660

1359

420

686

664

Th17 RGE 10120583gmL RGE 100120583gmL RGE 300120583gmL RGE 1000 120583gmL

(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus

RGE1000 + +minus+ + ++ + minus+ +minus

+ + ++ + minus+ +minus




Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm

L)lowastlowastlowast

lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash

120573-Actin Th0 (RGE 120583gmL)

Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)

Figure 5 Red ginseng extract increased Foxp3-expressing regulatory T cells and decreased IL-17-expressing Th17 cells in human PBMCsCD4+ T cells were isolated from human peripheral blood and cultured with various concentrations of red ginseng extract (RGE) (a) MTTassay (b)The proportion of IL-17 positive cells and Foxp3 positive cells was analyzed by flow cytometry (c) The mRNA expressions ofTh17 -and Treg-associatedmolecules weremeasured by RT-PCR (d)The concentrations of IL-17 IL-21 and IL-22 in the supernatant weremeasuredby ELISA (e) The level of total and phosphorylated STAT3 (at Y705 and S727 resp) and STAT5 was assessed by western blotting Data arepresented as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to control



MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL

RGE 30120583gmLRGE 10120583gmL

mdash

lowastlowast lowastlowast lowastlowast

lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)

MCSF + RANKL (RGE 120583gmL) MCSF + RANKL (RGE 120583gmL) MCSF + RANKL (RGE 120583gmL) MCSF + RANKL (RGE 120583gmL)

RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15


lowastlowastlowastlowast


lowastlowast lowastlowastlowast lowast

100 300 1000mdash 100 300 1000mdash 100 300 1000mdash 100 300 1000mdash

CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)

Figure 6 Red ginseng extract inhibits osteoclastogenesis in humans Human peripheral blood mononuclear cells were cultured with M-CSF(10 ngmL) and RANKL (50 ngmL) in the presence or absence of various concentrations of RGE (a) Cells were fixed and stained for TRAP(original magnification times100) The number of TRAP+ cells was counted using light microscopy The representative photographs from eachgroup are shown (b)ThemRNAexpressions of various osteoclastogenicmarkers such as RANKMMP9 CathepsinK andCTRwere analyzedusing real time PCR BMM bone-marrow-derived monocytemacrophage CTR calcitonin receptor MMP9 matrix metalloproteinase 9Data are presented as the mean plusmn SD of three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared to control

is unclear which component of RGE is involved in specificmolecular event For instance one ginsenoside may inhibitSTAT3 phosphorylation in Th17 polarizing condition andanother ginsenoside may suppress RANK expression duringosteoclastogenesis or both effects can be mediated by oneginsenoside Namely the antiarthritic effect observed inthe present study may be a constellate effect of severalginsenosides However red ginseng is generally ingested asa whole rather than any extracted form of specific ingredientHence our data suggest red ginseng as a whole can beutilized as a novel therapeutic for RA

Thepresent study provides the first evidence that RGE canregulate Th17 differentiation By inhibiting STAT3 phospho-rylation RGE concurrently suppresses Th17 and enhancesTreg This novel mechanism will provide the solid basis thatred ginseng conceiving as an alternative medicine can be apromising therapeutic with scientific evidence Furthermoreit can be expected that RGE may have therapeutic potentialin other diseases whereTh17 plays major role in pathogenesissuch as inflammatory bowel disease or graft-versus-hostdisease Future research on this topic will be promising

5 Conclusion

In conclusion our data demonstrate that RGE has a profoundinhibitory effect on CIA development as well as its severity

The inhibitory effect appeared to be mediated through recip-rocal regulation of Th17 and Treg by suppressive effect onSTAT3 phosphorylation of RGE RGE also directly inhibitedosteoclastogenesis which is critical for bone erosion in RAThe regulatory effect was consistently found in experimentsusing human PBMCs suggesting that antiarthritic effect ofRGE could be applicable to human RA In addition RGE hasbeen used widely in alternative medicine with relatively goodsafety profile Therefore RGE may be a novel therapeuticagent for the treatment of RA

Conflict of Interests

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

Authorsrsquo Contribution

Jennifer Lee and Joo Yeon Jhun have equally contributed tothis work Sung Hwan Park and Mi-La Cho have equallycontributed to this work

Acknowledgments

This work was supported by the Basic Science ResearchProgram through the National Research Foundation of


Korea (NRF) funded by the Ministry of Education Sci-ence and Technology (NRF-2013R1A2A2A01007720) andby a Grant of the Korea Health Technology R amp DProject Ministry for Health amp Welfare Republic of Korea(Grant no HI09C1555) The English in this document hasbeen checked by at least two professional editors bothnative speakers of English For a certificate please seehttpwwwtextcheckcomcertificateLVMKBG

References

[1] I B McInnes and G Schett ldquoThe pathogenesis of rheumatoidarthritisrdquoTheNew England Journal of Medicine vol 365 no 23pp 2205ndash2219 2011

[2] S Nakae A Nambu K Sudo and Y Iwakura ldquoSuppressionof immune induction of collagen-induced arthritis in IL-17-deficient micerdquo Journal of Immunology vol 171 no 11 pp 6173ndash6177 2003

[3] M I Koenders L A B Joosten and W B Van Den BergldquoPotential new targets in arthritis therapy Interleukin (IL)-17 and its relation to tumour necrosis factor and IL-1 inexperimental arthritisrdquo Annals of the Rheumatic Diseases vol65 no 3 pp iii29ndashiii33 2006

[4] P J Egan A vanNieuwenhuijze I K Campbell and I PWicksldquoPromotion of the local differentiation of murine Th17 cellsby synovial macrophages during acute inflammatory arthritisrdquoArthritis amp Rheumatism vol 58 no 12 pp 3720ndash3729 2008

[5] E Lubberts L van den Bersselaar B Oppers-Walgreen etal ldquoIL-17 promotes bone erosion in murine collagen-inducedarthritis through loss of the receptor activator of NF-120581B lig-andosteoprotegerin balance1rdquo Journal of Immunology vol 170no 5 pp 2655ndash2662 2003

[6] J Kikuta Y Wada T Kowada et al ldquoDynamic visualization ofRANKL andTh17-mediated osteoclast functionrdquoThe Journal ofClinical Investigation vol 123 no 2 pp 866ndash873 2013

[7] L A Zuniga R Jain C Haines and D J Cua ldquoTh17 celldevelopment from the cradle to the graverdquo ImmunologicalReviews vol 252 no 1 pp 78ndash88 2013

[8] J T Coon and E Ernst ldquoPanax ginseng a systematic review ofadverse effects and drug interactionsrdquo Drug Safety vol 25 no5 pp 323ndash344 2002

[9] E Ernst ldquoPanax ginseng an overview of the clinical evidencerdquoJournal of Ginseng Research vol 34 no 4 pp 259ndash263 2010

[10] Y Z Xiang H C Shang X M Gao and B L ZhangldquoA comparison of the ancient use of ginseng in traditionalChinese medicine with modern pharmacological experimentsand clinical trialsrdquo Phytotherapy Research vol 22 no 7 pp 851ndash858 2008

[11] K R Kim T Y Chung H Shin et al ldquoRed ginseng saponinextract attenuates murine collagen-induced arthritis by reduc-ing pro-inflammatory responses andmatrix metalloproteinase-3 expressionrdquoBiological and Pharmaceutical Bulletin vol 33 no4 pp 604ndash610 2010

[12] H Jung H Choi H Lim et al ldquoEnhancement of anti-inflammatory and antinociceptive actions of red ginseng extractby fermentationrdquo Journal of Pharmacy and Pharmacology vol64 no 5 pp 756ndash762 2012

[13] E Cho and S H Cho ldquoEffects of Korean red ginseng extract onthe prevention of atopic dermatitis and its mechanism on earlylesions in a murine modelrdquo Journal of Ethnopharmacology vol145 no 1 pp 294ndash302 2013

[14] Y J Hong N Kim K Lee et al ldquoKorean red ginseng (Panaxginseng) ameliorates type 1 diabetes and restores immune cellcompartmentsrdquo Journal of Ethnopharmacology vol 144 no 2pp 225ndash233 2012

[15] H J Kim S G Lee I G Chae et al ldquoAntioxidant effectsof fermented red ginseng extracts in streptozotocin-induceddiabetic ratsrdquo Journal of Ginseng Research vol 35 no 2 pp 129ndash137 2011

[16] Y J Surh H K Na J Y Lee and Y S Keum ldquoMolecularmechanisms underlying anti-tumor promoting activities ofheat-processed Panax ginseng CA Meyerrdquo Journal of KoreanMedical Science vol 16 supplement pp S38ndashS41 2001

[17] H A Kim S Kim S H Chang H J Hwang andY Choi ldquoAnti-arthritic effect of ginsenoside Rb1 on collagen induced arthritisin micerdquo International Immunopharmacology vol 7 no 10 pp1286ndash1291 2007

[18] Y S Choi E H Kang E Y Lee et al ldquoJoint-protective effectsof compound K amajor ginsenosidemetabolite in rheumatoidarthritis in vitro evidencerdquo Rheumatology International vol 33no 8 pp 1981ndash1990 2013

[19] S Park H Lee S Jeong et al ldquoInhibition of JAK1STAT3signaling mediates compound K-induced apoptosis in humanmultiple myeloma U266 cellsrdquo Food and Chemical Toxicologyvol 49 no 6 pp 1367ndash1372 2011

[20] X-P Yang K Ghoreschi S M Steward-Tharp et al ldquoOpposingregulation of the locus encoding IL-17 through direct reciprocalactions of STAT3 and STAT5rdquo Nature Immunology vol 12 no3 pp 247ndash254 2011

[21] J S ParkMK ParkH JOh et al ldquoGrape-seed proanthocyani-din extract as suppressors of bone destruction in inflammatoryautoimmune arthritisrdquo PLoS ONE vol 7 no 12 Article IDe51377 2012

[22] C AHitchon andH S El-Gabalawy ldquoOxidation in rheumatoidarthritisrdquo Arthritis Research and Therapy vol 6 no 6 pp 265ndash278 2004

[23] J Fay D Varoga C J Wruck B Kurz M B Goldring and TPufe ldquoReactive oxygen species induce expression of vascularendothelial growth factor in chondrocytes and human articularcartilage explantsrdquoArthritis Research andTherapy vol 8 articleR189 2006

[24] L K Stamp I Khalilova J M Tarr et al ldquoMyeloperoxidase andoxidative stress in rheumatoid arthritisrdquo Rheumatology vol 51no 10 pp 1796ndash1803 2012

[25] C J Wruck A Fragoulis A Gurzynski et al ldquoRole of oxidativestress in rheumatoid arthritis insights from the Nrf2-knockoutmicerdquo Annals of the Rheumatic Diseases vol 70 no 5 pp 844ndash850 2011

[26] J-M Lee and J A Johnson ldquoAn important role of Nrf2-ARE pathway in the cellular defense mechanismrdquo Journal ofBiochemistry and Molecular Biology vol 37 no 2 pp 139ndash1432004

[27] T Shen J LeeMH Park et al ldquoGinsenoside Rp1 a ginsenosidederivative blocks promoter activation of iNOS and Cox-2genes by suppression of an IKK120573-mediated NF-120581B pathway inHEK293 cellsrdquo Journal of Ginseng Research vol 35 no 2 pp200ndash208 2011

[28] T Ramesh S Kim S Hwang S Sohn S Yoo and S KimldquoPanax ginseng reduces oxidative stress and restores antioxidantcapacity in aged ratsrdquoNutrition Research vol 32 no 9 pp 718ndash726 2012


[29] QWuWWang S Li et al ldquoAmerican ginseng inhibits vascularsmooth muscle cell proliferation via suppressing JakStat path-wayrdquo Journal of Ethnopharmacology vol 144 no 3 pp 782ndash7852012

[30] J Bae J Koo S Kim T Y Park and M Y Kim ldquoGinsenosideRp1 exerts anti-inflammatory effects via activation of dendriticcells and regulatory T cellsrdquo Journal of Ginseng Research vol 36no 4 pp 375ndash382 2012

[31] L Zhou M M W Chong and D R Littman ldquoPlasticity ofCD4+ T Cell Lineage Differentiationrdquo Immunity vol 30 no 5pp 646ndash655 2009

[32] H Liu S Yao S M Dann H Qin C O Elson and Y CongldquoERK differentially regulates Th17- and Treg-cell developmentand contributes to the pathogenesis of colitisrdquo European Journalof Immunology vol 43 no 7 pp 1716ndash1726 2013

[33] L He J Lee J H Jang et al ldquoGinsenoside Rh2 inhibits osteo-clastogenesis through down-regulation of NF-120581B NFATc1 andc-Fosrdquo Bone vol 50 no 6 pp 1207ndash1213 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


primes initial differentiation and IL-23 promotes functionalmaturation of Th17 cells [7] As signal transducer and acti-vator of transcription (STAT) 3 phosphorylation directly reg-ulates retinoic acid receptor-related orphan receptor (ROR)120574t which is the master molecule of Th17 the Janus kinase(JAK)2-STAT3 pathway initiated by IL-6 is essential in theTh17 development With recent advances in understandingthe central role of Th17 in the pathogenesis of RA noveltherapeutics targeting Th17 are being utilized or are stillunder development The blockade of IL-6 (Tocilizumab) iscurrently available on the market while the JAK2 inhibitorandmonoclonal antibody against IL-17 are still under clinicaltrials among RA patients

Ginseng the root of Panax ginseng CA Meyer (Arali-aceae) is a perennial herb which has been used as herbalmedicine in Eastern Asia for thousands of years [8 9]Ginseng has two types of preparation one is air-dried whiteginseng and the other is steamed and sun-dried red ginsengIt contains various active components including ginseno-sides polysaccharides peptides polyacetylenic alcohols andfatty acids [10] Ginsenosides in particular are also calledsaponin extracts and are known to exert the majority ofthe pharmacologic effects seen from ginseng Red ginsengis made from a ginseng plant going through an intensiveprocess of cleaning steaming and drying Heat treatmentof ginseng leads to chemical changes of ginsenosides givingthem distinct physical characteristics Major ginsenosidesof red ginseng saponin extract were shown to include lesspolar red ginseng-unique saponins Rg3 Rk1 and Rg5 in ahigh-performance liquid chromatographic analysis [11] Assupplementation with red ginseng is believed to improvehealth numerous studies have been conducted to validateits beneficial effects They demonstrated that RGEs had anti-inflammatory [12 13] antidiabetic [14 15] and anticancer[16] effects Anti-inflammatory property of RGE leads to theunderstanding that it may have antiarthritic effect via mod-ulating inflammation Indeed accumulating evidence hassuggested that ginseng extracts can ameliorate autoimmunearthritis It was reported that treatmentwith ginsenoside Rb-1significantly attenuated arthritis inmice with established CIA[17] Orally administered RGEs including Rg3 Rk1 and Rg5asmajor components also successfully suppressedCIA shownby decreased production of proinflammatory cytokinesmatrix metalloproteinase (MMP) and nitrotyrosine in thearthritic joints [11] More recently compound K an activeginsenoside metabolite with a high intestinal absorption ratewas shown to decrease TNF-120572-induced MMP production inRA-fibroblast-like-synoviocyte (FLS) and suppress osteoclas-togenesis by inhibiting the expression of nuclear factor ofactivated T cells (NFATc1) [18] However it has not beensuggested whether or not RGE has a regulatory effect onTh17 which is a central pathogenic cell in RA Given thatginseng was reported to repress STAT3 activation in cancercells [19] it is plausible to assume that RGE can suppressTh17by inhibiting STAT3 phosphorylation enhancing enhancedTreg cells by reciprocal regulation [20]

In the present study we verified that oral administrationof RGE could suppress arthritis in CIA model To delineatethe mechanism underlying the antiarthritic effect in terms of

Th17 the effect of RGE on pathogenicTh17 cell differentiationboth in vivo and in vitro was investigated In addition theeffect of RGE osteoclast formation which is implicated inbone erosion in RA was examined

2 Methods and Materials

21 Animals Six week oldmale DBA1J mice were purchasedfrom SLC Inc (Shizouka Japan) and IL-10 knockout (KO)mice in the DBA1J background were kindly provided byLinda K Myers (University of Tennessee) Mice were main-tained under specific pathogen-free conditions at the instituteof Medical Science at the Catholic University of Korea andwere provided standard mouse chow (Ralston Purina StLouis MO USA) and water ad libitum All experimentalprocedures were examined and approved by the AnimalResearch Ethics Committee of the Catholic University ofKorea (permit number CUMC20) which conforms to allNational Institutes of Health of the USA guidelines

22 Preparation of Red Ginseng Extract Red ginseng extract(RGE) was kindly provided by the Korea Ginseng Cooper-ation Daejeon Cooperation (Daejeon Republic of Korea)RGEyields 437 saponins themain components of ginseno-sides were Rb1 (126) Rb2 (62) Rc (69) Rd (34) Re(64) Rf (21) Rg1 (158) and Rg3 (14)The identifiedconstituents are well standardized and qualified by the KoreaGinseng Cooperation Other constituents in RGE are starchsugars fat fiber proteins vitamins minerals and so forth

23 Induction of CIA and Treatment with Red Ginseng Toinduce CIA in mice 100 120583L of an emulsion containing 100120583gbovine type II collagen (CII) and complete Freundrsquos adjuvant(Chondrex RedmondWA USA) was injected intradermallyinto the base of the tail as the primary immunization Twoweeks later 100 120583g CII dissolved and emulsified 1 1 withincomplete Freundrsquos adjuvant (Difco Detroit MI USA) wasadministered into the footpad as a booster injection To assessthe influence of RGE on symptom severity in the CIAmodelmice were orally treated with 10mgkg RGE in saline or withvehicle alone three times a week after booster immunizationover the course of 6 weeks The arthritis index in these micewas scored twice weekly and expressed as the sum of thescores of four limbs

24 Measurement of CII-Specific IgG and IgG Serum levels ofCII-specific IgG (Total IgG IgG1 IgG2a) and IgG (Total IgGIgG1 IgG2) measurement antibodies were measured usinga commercially available ELISA kit (Beathyl LaboratoriesMontgomery TX USA)

25 Immunohistochemistry Mouse joint tissue was fixed in4 paraformaldehyde decalcification EDTA bone decalcifierand embedded in paraffin The section (7 120583m) was stainedwith hematoxylin and eosin Safranin O and toluidine blueto detect proteoglycans

Immunohistochemistry was performed using the Vec-tastatin ABC kit (Vector Laboratories Burlingame CAUSA) Tissue was first incubated with primary antibodiesto IL-17 IL-6 IL-1b TNF-120572 Nitrotyrosine NRF2 (Santa













3 Results








0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1

6w2

0

4

8

12

CIARGE


Art

hriti

s sco

re


(a)

CIA(IL-10KO)RGE

0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1


0

4

8

12

16

Art

hriti

s sco

re

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

(b)

lowastlowast

lowastlowast

CIA

RGE


CIA RGE

CIA RGE

01234

01234

Infla

mm

atio

nCa

rtila

ge d

amag

e

(c)

CIA

RGE

CIA

RGE



(d)

Figure 1 Continued


IgG2

a (ng

mL)

IgG1

(ng

mL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowast


10152025

Tota

l IgG

(ng

mL)

050

100150200

01020304050

000002004006008010

CII-

spec

ific t

otal

IgG

(a

u)

0001020304

000

005

010

015

CII-

spec

ific I

gG1

(au

)

CII-

spec

ific I

gG2

a(a

u)

(e)





4 Discussion







CIA RGE00

05

10

15

IL-1

7 m

RNA

lowastlowastlowast

CIA RGE00

05

10

15

lowast

lowastlowast

RORC

mRN

A


05

10

15

lowastlowast

CCR6

mRN

A

00

05

10

15

20

SOCS

3 m

RNA

00

05

10

15

20

Foxp

3 m

RNA

(a)

10 k 10 k

419

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

266

CIA RGE

CIA RGE

lowastlowast

012345

IL-1

7in

CD

4T

cells

()

IL-17 IL-17

(b)

CIA RGE

CIA RGE

Sple

enSp

leen

Foxp

3CD

4CD

25CD

4IL

-17

CIA RGE

0

10

20

30

0

10

20

30

Spleen

Spleen

lowastlowastlowast

lowastlowastlowast

Num

ber o

fCD

4+CD

25+

Foxp

3+ce

llsN

umbe

r of

CD4+

IL-17+

cells

(c)





Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3 Results








0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1

6w2

0

4

8

12

CIARGE


Art

hriti

s sco

re


(a)

CIA(IL-10KO)RGE

0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1


0

4

8

12

16

Art

hriti

s sco

re

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

(b)

lowastlowast

lowastlowast

CIA

RGE


CIA RGE

CIA RGE

01234

01234

Infla

mm

atio

nCa

rtila

ge d

amag

e

(c)

CIA

RGE

CIA

RGE



(d)

Figure 1 Continued


IgG2

a (ng

mL)

IgG1

(ng

mL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowast


10152025

Tota

l IgG

(ng

mL)

050

100150200

01020304050

000002004006008010

CII-

spec

ific t

otal

IgG

(a

u)

0001020304

000

005

010

015

CII-

spec

ific I

gG1

(au

)

CII-

spec

ific I

gG2

a(a

u)

(e)





4 Discussion







CIA RGE00

05

10

15

IL-1

7 m

RNA

lowastlowastlowast

CIA RGE00

05

10

15

lowast

lowastlowast

RORC

mRN

A


05

10

15

lowastlowast

CCR6

mRN

A

00

05

10

15

20

SOCS

3 m

RNA

00

05

10

15

20

Foxp

3 m

RNA

(a)

10 k 10 k

419

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

266

CIA RGE

CIA RGE

lowastlowast

012345

IL-1

7in

CD

4T

cells

()

IL-17 IL-17

(b)

CIA RGE

CIA RGE

Sple

enSp

leen

Foxp

3CD

4CD

25CD

4IL

-17

CIA RGE

0

10

20

30

0

10

20

30

Spleen

Spleen

lowastlowastlowast

lowastlowastlowast

Num

ber o

fCD

4+CD

25+

Foxp

3+ce

llsN

umbe

r of

CD4+

IL-17+

cells

(c)





Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1

6w2

0

4

8

12

CIARGE


Art

hriti

s sco

re


(a)

CIA(IL-10KO)RGE

0d 1w1

1w2

2w1

2w2

3w1

3w2

4w1

4w2

5w1

5w2

6w1


0

4

8

12

16

Art

hriti

s sco

re

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

(b)

lowastlowast

lowastlowast

CIA

RGE


CIA RGE

CIA RGE

01234

01234

Infla

mm

atio

nCa

rtila

ge d

amag

e

(c)

CIA

RGE

CIA

RGE



(d)

Figure 1 Continued


IgG2

a (ng

mL)

IgG1

(ng

mL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowast


10152025

Tota

l IgG

(ng

mL)

050

100150200

01020304050

000002004006008010

CII-

spec

ific t

otal

IgG

(a

u)

0001020304

000

005

010

015

CII-

spec

ific I

gG1

(au

)

CII-

spec

ific I

gG2

a(a

u)

(e)





4 Discussion







CIA RGE00

05

10

15

IL-1

7 m

RNA

lowastlowastlowast

CIA RGE00

05

10

15

lowast

lowastlowast

RORC

mRN

A


05

10

15

lowastlowast

CCR6

mRN

A

00

05

10

15

20

SOCS

3 m

RNA

00

05

10

15

20

Foxp

3 m

RNA

(a)

10 k 10 k

419

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

266

CIA RGE

CIA RGE

lowastlowast

012345

IL-1

7in

CD

4T

cells

()

IL-17 IL-17

(b)

CIA RGE

CIA RGE

Sple

enSp

leen

Foxp

3CD

4CD

25CD

4IL

-17

CIA RGE

0

10

20

30

0

10

20

30

Spleen

Spleen

lowastlowastlowast

lowastlowastlowast

Num

ber o

fCD

4+CD

25+

Foxp

3+ce

llsN

umbe

r of

CD4+

IL-17+

cells

(c)





Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IgG2

a (ng

mL)

IgG1

(ng

mL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowast


10152025

Tota

l IgG

(ng

mL)

050

100150200

01020304050

000002004006008010

CII-

spec

ific t

otal

IgG

(a

u)

0001020304

000

005

010

015

CII-

spec

ific I

gG1

(au

)

CII-

spec

ific I

gG2

a(a

u)

(e)





4 Discussion







CIA RGE00

05

10

15

IL-1

7 m

RNA

lowastlowastlowast

CIA RGE00

05

10

15

lowast

lowastlowast

RORC

mRN

A


05

10

15

lowastlowast

CCR6

mRN

A

00

05

10

15

20

SOCS

3 m

RNA

00

05

10

15

20

Foxp

3 m

RNA

(a)

10 k 10 k

419

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

266

CIA RGE

CIA RGE

lowastlowast

012345

IL-1

7in

CD

4T

cells

()

IL-17 IL-17

(b)

CIA RGE

CIA RGE

Sple

enSp

leen

Foxp

3CD

4CD

25CD

4IL

-17

CIA RGE

0

10

20

30

0

10

20

30

Spleen

Spleen

lowastlowastlowast

lowastlowastlowast

Num

ber o

fCD

4+CD

25+

Foxp

3+ce

llsN

umbe

r of

CD4+

IL-17+

cells

(c)





Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CIA RGE00

05

10

15

IL-1

7 m

RNA

lowastlowastlowast

CIA RGE00

05

10

15

lowast

lowastlowast

RORC

mRN

A


05

10

15

lowastlowast

CCR6

mRN

A

00

05

10

15

20

SOCS

3 m

RNA

00

05

10

15

20

Foxp

3 m

RNA

(a)

10 k 10 k

419

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

100 101 102 103 104

FL1-HIL-17 FITC

0200400600800

SSC-

H

266

CIA RGE

CIA RGE

lowastlowast

012345

IL-1

7in

CD

4T

cells

()

IL-17 IL-17

(b)

CIA RGE

CIA RGE

Sple

enSp

leen

Foxp

3CD

4CD

25CD

4IL

-17

CIA RGE

0

10

20

30

0

10

20

30

Spleen

Spleen

lowastlowastlowast

lowastlowastlowast

Num

ber o

fCD

4+CD

25+

Foxp

3+ce

llsN

umbe

r of

CD4+

IL-17+

cells

(c)





Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sple

enSp

leen

Sple

en

CD4

s727

DA

PICD

4N

rf2CD

4y7

05

CIA RGE

CIA RGE

CIA RGE

CIA RGE

0

10

20

30

40

0

10

20

30

40

lowastlowastlowast

lowastlowast

lowastlowast

Num

ber o

fN

umbe

r of

0

20

40

60

Num

ber o

fCD

4+72

7+ce

llsCD

4+y7

05+

cells

DA

PI+

CD4+

Nrf2

+ce

lls(a)

p-I120581

Ba

p-I120581Ba

Total I120581Ba

p-ST

AT3(

tyr7

05)

120573-Actin

p-ERK

Total ERK

p-STAT5

Total STAT5

Total STAT3

p-STAT3(705)

p-STAT3(727)




mdash 100 300 1000

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT3(

ser7

27)

00

02

04

06

08

10

100 500 1000mdash

p-ST

AT5

00

02

04

06

08

10

100 500 1000mdash

00

02

04

06

08

10

100 500 1000mdash

p-ER

K

(b)



TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TRA

P

CIA RGE 10 mgkg

CIA RGE0

2

4

6

8

lowastlowast

Num

ber o

fTR

AP+

cells

(a)

0

100

200

300

400

lowast lowast

lowast

001 01 1 10 100mdash


RGE 10120583gmL RGE 100120583gmL

MCSF + RANKL

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP


+

(b)


00

05

10

15

1 10 100mdash

RAN

K m

RNA



05

10

15

1 10 100mdash


00

05

10

15

1 10 100mdashTR

AP

mRN

A00

05

10

15

1 10 100mdash

MM

P9m

RNA

lowastlowast

lowastlowast

Cath

epsin

K m

RNA


(c)







000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000102030405

(RGE 120583gmL)10 30 100 300 1000mdash

OD

(a)

771

100

101

102

103

104

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104


FL2

-HF

L2-I

L-17

PE

521594

IL-17


861 598660

1359

420

686

664


(b)

IL-1

7 m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15

000510152025

000510152025

Th17 + + +RGE300 + + minus






Foxp

3 m

RNA

IL-6

mRN

A

IL-2

1 m

RNA

IL-2

6 m

RNA

IL-1

0 m

RNA


lowastlowastlowast

lowast

lowast

lowastlowast

(c)

Th17 + + +RGE300 + + minus

RGE1000 + +minus




0

5000

10000

15000

0

5000

10000

15000

IL-1

7(p

gm

L)

IL-2

2(p

gm

L)

0

50

100

150

IL-2

1(p

gm

L)

0200400600800

1000

IL-1

0(p

gm


lowastlowastlowast

lowast


lowastlowastlowast

(d)

mdash

mdash


Th0 (RGE 120583gmL)

00

05

10

15

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000 mdashTh0 (RGE 120583gmL)

100 300 1000

p-ST

AT3(

ser7

27)

00

05

10

15

p-ST

AT3(

tyr7

05)

00

05

10

15

p-ST

AT5

p-STAT3(705)

p-STAT3(727)

Total STAT3

p-STAT5

Total STAT5

100 300 1000

(e)




MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















MCSF + RANKL

0

100

200

300

10 30 100 3001000

RGE 100120583gmL

RGE 300120583gmL RGE 1000120583gmL


mdash


lowast

cells

wel

l (nu

cleigt

3)N

umbe

r of T

RAP+

(a)


RAN

K m

RNA

00

05

10

15

00

05

10

15

00

05

10

15

00

05

10

15






CTR

mRN

A

MM

P9 m

RNA

Cath

epsin

K m

RNA

(b)




5 Conclusion







Acknowledgments




References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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