research article effects of the pinggan qianyang recipe on...

Research ArticleEffects of the Pinggan Qianyang Recipe onMicroRNA Gene Expression in the Aortic Tissue ofSpontaneously Hypertensive Rats

Guangwei Zhong1 Xia Fang2 Dongsheng Wang1 Qiong Chen2 and Tao Tang2

1 Institute of Integrated Traditional Chinese and Western Medicine Xiangya Hospital Central South UniversityChangsha 410008 China2Department of Geriatrics Xiangya Hospital Central South University Changsha 410008 China

Correspondence should be addressed to Qiong Chen qiongch163com

Received 9 September 2014 Revised 24 January 2015 Accepted 28 January 2015

Academic Editor Joen-Rong Sheu

Copyright copy 2015 Guangwei Zhong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present study aimed to investigate the relationship between miRNAs and in spontaneously hypertensive rats (SHR) vascularremodeling and analyze the impact of the Pinggan Qianyang recipe (PQR) on miRNAs Mammalian miRNA microarrayscontaining 509 miRNA genes were employed to analyze the differentially expressed miRNAs in the three groups MiRNAs wereconsidered to be up- or downregulated when the fluorescent intensity ratio between the two groups was over 4-fold Validationof those miRNAs changed in SHR after PQR treatment was used by real-time quantitative RT-PCR (qRT-PCR) Compared withthe normal group a total of 32 miRNAs were differentially expressed by more than twofold among these 18 were upregulated and14 were downregulated in the model group Compared with the normal group there were a number of 17 miRNAs which weresignificantly expressed by more than twofold in the different expressions of 32 miRNAs among these 10 were downregulated and 7were upregulated in the PQR group qRT-PCR verified that miR-20a miR-145 miR-30 and miR-98 were significantly expressed inthe three groupsThese data show that PQR could exert its antihypertensive effect through deterioration of the vascular remodelingprocess The mechanism might be associated with regulating differentially expressed miRNAs in aorta tissue

1 Introduction

Hypertension a lifelong condition is one of the most com-mon cardiovascular diseases Among patients treated by theauthors the prevalence of hypertension in 15 to 69-year-oldpatients is 234 greater than the current estimate of patientswith hypertension in China [1] Because hypertension is animportant risk factor for coronary heart disease and strokedamage to the vital organs such as the heart brain andkidneys can be avoided or minimized by preventing and con-trolling high blood pressure [2] A Chinese medicine scholarhas successfully explored the pathogenesis of spontaneoushypertension and various therapy approaches including thePinggan Qianyang recipe (PQR) a Chinese medicine recipefor calming the liver and suppressing yang [3] PQR whichoriginated from the use of Tianma Guoteng beverages has

been used to treat essential hypertension with satisfactoryresults [4] Recent research has found that Chinese herbalmedicines that involve PQR have a beneficial effect on reduc-ing blood pressure and recovering circadian rhythm in essen-tial hypertension patients [5 6] However the underlyingmechanism of these therapeutic effects remains unknown

miRNAs are a class of highly conserved noncodingsmall-molecule RNAs consisting of about 22 nucleotideseach They adjust protein levels by promoting mRNA degra-dation or inhibiting mRNA translation miRNAs thus partic-ipate in many important biological processes throughout thebody [7 8] miRNAs are involved in cell proliferation differ-entiationmigration and apoptosis [9 10] Cordes et al foundthat reducing miRNA-143 levels could inhibit adipocytedifferentiation in vitro suggesting that miRNAs may play

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 154691 10 pageshttpdxdoiorg1011552015154691

2 Evidence-Based Complementary and Alternative Medicine

a significant role in the renin-angiotensin system (RAAS)mdashan important modulator of systemic blood pressure [11]Some miRNAs including miR-1 miR-145 miR-122 miR-221 and miR-222 have been linked to vascular endothelialdysfunction [12] Others have been linked to the regulationof vascular smooth muscle cells these include miR-145 let-7d miR-24 miR-26a and miR-146 [13] The miRNAs miR-1miR-155 and miR-208 have significant effects on the RAAS[14] Therefore a new strategy for hypertension treatmentmight involve maintenance and restoration of stability bytargeting corresponding miRNA expression in the organ ofinterest

To elucidate the association between miRNA expressionand PQR treatment for essential hypertension we carried outanalysis of miRNA gene expression in aortic tissue from SHRthat had received PQR interventionWe tested the hypothesisthat PQRplays an antihypertensive role by regulatingmiRNAexpression in rat aortic tissueThis research may also providenew insights into potential therapeutic targets to prevent andtreat hypertension

2 Materials and Methods

21 Animals and Drugs Forty 16-week-old male sponta-neously hypertensive rats (SHR) and 20 male Wistar (WKY)rats (Vital River Laboratory Animal Technology Co LtdBeijing China) of the same age were housed in a sterileenvironment at a temperature of 21 plusmn 1∘C and a relativehumidity of 50 plusmn 10 in a 12-hour day-night cycle Bothgroups of rats had been fed standard rat chow and wateruntil they were 16 weeks old All animal study protocolswere approved by the Animal Care and Use Committee ofCentral SouthUniversity (201303117) and followed the animalmanagement rules set out by the Ministry of Health Chinaand the US National Institutes of Health Guide for the Careand Use of Laboratory Animals The PQR medication recipewas composed of Rhizoma Gastrodiae Ramulus Uncariaecum Uncis Concha Haliotidia Concha Ostreae and RadixAchyranthis Bidentatae all componentswere purchased fromthe Department of Pharmacy Xiangya Hospital CentralSouth University One gram of extract was equal to 425 g ofcrude material

22 Animal Groupings and Treatments The WKY rats andSHR were arbitrarily separated into three groups the normalgroup (119899 = 20) the model group (119899 = 20) and the PQRgroup (119899 = 20) Rats in the PQR group were administeredPQR at a dose of 50mgsdotkgminus1sdotdminus1 by gastrogavageThe otherswere given an equal volume of distilled water For all groupsthe administration course lasted 4 weeks All animals wereused for the miRNA analysis and verification study FortySHR were randomly divided into two groups and were given50mgkg of PQR by gastrogavage once daily for 4 weeksnormal saline was given as the negative control

23 Blood Pressure Detection Systolic blood pressure (SBP)was measured in all rats as previously described [15] Tail-cuff plethysmography (TCP) with a rat tail blood pressuremonitor was used The SBP of each rat was measured five

timesmdashonce before treatment and 1 2 3 and 4 weeks aftertreatment At every time point the mean of the lowest threevalues within 5mmHg was regarded as the SBP value

24 Histological and Morphological Assay Rats were anes-thetizedwith 10 chloral hydrate (400mgkg intraperitonealinjection) at the end of each week of whole-day drug admin-istration The thoracic aorta below the aortic arch of each ratwas stripped and clipped A portion was fixed in 8 neutralformaldehyde embedded in paraffin sectioned at 5 120583mand stained with the hematoxylin-eosin (HE) and Massonmethods [16] Light microscopy was used to image eachcross-sectional slice of which there were five per rat Eachvascular ring in the perpendicular position and the vesselmedia wall were observed The images were observed undera Leica imaging system (LeicaMicrosystems GmbHWetzlarGermany) The media thickness (MT) and inner diameter(LD)weremeasured and the ratio ofmedia thickness to innerdiameter (MTLD)was calculatedOther parts of the thoracicaorta were removed from the adventitia and were promptlyrefrigerated at minus80∘C for miRNA assay

25 RNA Microarray and Hybridization

RNA Extraction Total RNA was extracted by a one-stepmethod using TRIzol (Invitrogen USA) following the manu-facturer protocol concentrated using isopropanol precipita-tion and quantified using a spectrophotometer and agarosegel electrophoresis The polyethylene glycol (PEG) methodwas used to isolate and purify 50 120583g of total RNA

Fluorescently Labeled miRNA miRCURY LNA array labelingkit (Exiqon Denmark) was used Total RNA (10 120583g) wasadded to 2 120583L of Hy

3fluorescent label solution and 2 120583L of

labeling enzyme mixed by pipetting and then incubated at65∘C for 15min to terminate the labeling process

miRNA Microarray Hybridization A miRCURY LNA arraylabeling kit using Macro Kit (ID 208000V71) and hybridbox II (ID 40080) was purchased from Exiqon Biochipslides and cover slips were purchased from Ambion Inc(USA) miRNA microarray hybridization was performedaccording to the miRCURY LNA array kit instructions10 120583L of total RNA was added to 10 120583L of 2x hybridizationbuffer and incubated for 3ndash5min at 95∘C Then 20120583L of thehybridization solution was placed on a microarray slide andcompletely covered with a Bioarray Lifter Slip coverslip Themicroarray slide was placed into the Hybridization ChamberII in a horizontal orientation and bathed at 60∘C for 16 hFollowing incubation hybridization samples were removedfrom the microarray slides with a wash solution Each of 509miRNAs was detected by three replicate probe spots on eachmicroarray slide for a total of six measurements per miRNAper sample after repeated fluorescence exchange

Image Acquisition andQuantification Eachmicroarray (chip)was rinsed and immediately dried then illuminated by a sin-gle 635 nm beam and scanned by a GenePix 4000B dual laserscanner (Molecular Devices LLC USA) Image files were

Evidence-Based Complementary and Alternative Medicine 3

saved in TIFF format The data were analyzed by GenePixPro 60 software (Molecular Devices LLC USA) After pre-processing the data were normalized to the same interchipglobal mean Finally the differentially expressed genes wereanalyzed by SAM (Significance Analysis of Microarraysversion 21)We used the following screening conditions falsediscovery rate of lt5 and expression differences of ge2-fold

26 Target Prediction Methods Predicted miRNA targetgenes were determined by four software programs miRanda(httpwwwmicrornaorg)miRBaseTargetDatabase (httpmicrornasangeracuk) and Target Scan (httpwwwtarget-scanorg) [17] Outputs varied among the programs Genespredicted by at least two programs were selected as predictedmiRNA target genes

27 Quantitative RT-PCR Differentially expressed miRNAsselected according to ge2-fold upregulation or downregula-tion by microarray analysis were measured by qRT-PCRusing RNA-tailing and primer extension Briefly 2120583g ofRNA was added to 25U120583L of poly (A) polymerase and1mmolL of ATP and incubated in water for 30min at 37∘CPCR primers were designed according to miRNA sequencesindicated by the aforementioned online software programs(26)U6 small nuclear RNA in the ratswas used as an internalcontrol gene Real-time PCR reactions were amplified ina 96-well PCR fluorescence analyzer (MJ real-time PCRinstrument Bio-Rad Laboratories Inc USA) Samples werepredenatured for 5min at 95∘C denatured for 20 s at 94∘Cannealed for 20 s at 58∘C and extended for 30 s at 72∘C fora total of 40 cycles with each sample analyzed in triplicateThe specific product in each PCR reaction was confirmedby the amplification curve Quantification of relative geneexpression was determined by the standard 2minusΔΔCt methodrelative gene expression = 2minus(ΔCtsampleminusΔCtcontrol)

28 StatisticalAnalysis Allresultsarepresented as themeanplusmnstandard deviation All experiments were repeated threetimes An independent sample 119905-test was applied when onlytwo groups were compared whereas comparisons betweenmore than two groups were made by analysis of variance(ANOVA) followed by a Bonferroni posttest Differenceswere considered significant at the level of 119875 lt 005

3 Results

31 PQR Significantly Decreased SBP At the beginning oftreatment SBP was 126 plusmn 11mmHg in the normal groupand 208 plusmn 14mmHg in the model and PQR groups (119875 lt001) However a decrease in SBP was observed in the PQRgroup after 2 weeks of treatment (119875 lt 005) After 4 weeksof treatment the SBP of the PQR group was approximately45mmHg lower than at the beginning of treatment (Figure 1)

32 Morphology and Histology of Vascular Tissue ChangesMasson and HE staining showed that the aortic tunica mediaof the model group was thicker than that of normal groupand the aortic tunica media of PQR-treated rats was thinner

0 1 2 3 4

0

100

200

300

Normal groupModel groupPQR group

SBP

(mm

Hg)

998771

Week

Figure 1 SBP changes inWKY rats or SHR receiving an ia of PQRor distilled water at various times Data are shown as the mean plusmnSD for twenty rats of each group 119875 values for statistical significancewere as 995333119875 lt 001 compared with the model group 998771119875 lt 005 andX119875 lt 001 compared with the PQR group respectively

Table 1 A260 A280 and A260A280 ratios and miRNA concen-trations

Group A260 A280 Ratio ofA260A280

Concentration(120583guL)

Normal group 057 028 196 0183Model group 106 051 208 0295PQR group 092 047 195 0266

than that of control rats in the model group (Figures 2(a)and 2(b)) As shown in Figures 2(c) and 2(d) both MT andMTLD were higher in the model group than in the normalgroup (MT 1267 plusmn 116 120583m versus 843 plusmn 83 120583m resp 119875 =002 MTLD 192 plusmn 019 versus 123 plusmn 021 resp 119875 = 0009)However both MT and MTLD were significantly lower inthe PQR group than in the model group (MT 1024 plusmn 94 120583mversus 1267 plusmn 116 120583m resp 119875 = 004 MTLD 145 plusmn 022versus 192 plusmn 019 resp 119875 = 003)

33 Quality Assessment of Total RNA We extracted totalRNA from the aortic tissues of all rats The purity of the totalRNA was high as indicated by the A260A280 ratio beinggreater than 190 Quality assessment indicated that the totalRNA met the quality requirement of the miRNA microarrayanalysis (Figure 3 and Table 1)

34 Aberrant Expression of miRNAs in SHR Aortic TissueTo determine which miRNAs are potentially involved in theunderlyingmechanism of PQR treatment for essential hyper-tension we tested miRNA levels in all rats by microarrayanalysis We found that miRNA expression was remarkablyaberrant in the model group compared with that of thenormal group In the model group 32 of the 509 rat aortic


N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)

Normal group Model group PQR group00

05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)

Figure 2 (a) Masson staining of vascular tissue in each group (400x magnification) (b) HE staining of vascular tissue in each group (400xmagnification) (c) MT (d) MTLD N normal group M model group PQR PQR group MT medial thickness LD luminal diameter

miRNAs analyzed were differentially expressed (119875 lt 001)with 18 miRNAs upregulated and 14miRNAs downregulatedAfter 4 weeks of PQR treatment we found that 17 of the32 aortic miRNAs were differentially expressed seven wereupregulated and 10 were downregulated Significant time

course changes of miRNA expression were observed in theaortic tissue more than 468 miRNAs were dysregulated(down- or upregulated) after PQR treatment (Figure 2(a))All differential expression levels of miRNAs at three timepoints are listed in Figure 4 and Table 2 These data indicate


Table 2 Significantly upregulated and downregulated miRNAs in three groups

miRNA Expression level Modelnormal PQRmodelNormal group Model group PQR group

rno-miRNA-1 363 824 687 227 083rno-miRNA-10ab 85 212 118 249 056rno-miRNA-17-5p 121 933 289 771 031rno-miRNA-20a 327 6216 1215 1901 019rno-miRNA-96 432 7537 2113 1745 029rno-miRNA-126-5p 93 323 356 347 110rno-miRNA-139 197 428 334 217 078rno-miRNA-145 128 786 235 614 030rno-miRNA-153 68 1059 351 1557 033rno-miRNA-186a 355 2136 1788 652 084rno-miRNA-187 264 1366 334 517 024rno-miRNA-196ab 451 2097 612 465 029rno-miRNA-210 253 1988 386 786 019rno-miRNA-218 194 793 548 409 061rno-miRNA-221 225 895 298 398 033rno-miRNA-378 148 1253 387 847 031rno-miRNA-451 345 764 598 221 078rno-miRNA-486 71 235 228 331 097rno-miRNA-556 124 617 235 497 038rno-miRNA-15b 1643 238 289 014 121rno-miRNA-26ab 874 156 479 018 313rno-miRNA-30 795 323 948 041 293rno-miRNA-23ab 235 68 57 029 084rno-miRNA-29b 2562 459 1382 018 301rno-miRNA-98 1351 66 527 005 798rno-miRNA-122 1206 197 786 016 399rno-miRNA-125b 3786 1134 1782 029 157rno-miRNA-142-3p 996 487 469 049 096rno-miRNA-158 1328 298 342 022 115rno-miRNA-21 566 103 1427 018 1385rno-miRNA-330 3225 809 1186 025 147rno-let-7bc 786 174 152 022 087

N M PQR

28 S

18 S

Figure 3 Electrophoresis of total RNA N normal group M modelgroup PQR PQR group

that the development of essential hypertension involves awave of expression of sequential classes of miRNAs Thetemporal regulation of these miRNAs indicates that theymight play an important role in PQR treatment of essentialhypertension

35 Validation of miRNA Microarray Results Using qRT-PCRqRT-PCR is a quantitative and specific method that can beused to distinguish a single nucleotide difference betweenmiRNAs Thus involution was obtained by miChip analysisfor four selected miRNAs that showed either high (miR-145) or low (miR-30) signal intensities or high (miR-20a)or low (miRNA-98) differential expression values amongthe three groups The results of qRT-PCR analysis wereoften more reliable than those of the microarray analysisqRT-PCR showed that miR-145 and miR-20a expression wasdownregulated in the model group compared with theirexpression in the PQR group which was consistent with


(a) (b)

Figure 4 Detection of miRNA by microarray analysis Total RNA extracted from three groups of rat aortic tissue were covalently labeledwith Cy3 (green) and Cy5 (red) and hybridized to the array The microarray slides contained two replicate subarrays (a) Normal group andmodel group (b) model group and PQR group

the microarray results Thus the miRNA expression profilesobtained by qRT-PCR fully support the results of miChipanalysis (Figure 5)

36 Results of miR-20a Target Gene Prediction We also per-formed a predicted target analysis formiRNA-20a which waschosen because it was highly expressed in the model groupand downregulated in the PQR group Potential target geneswere predicted using four software programs (miRandaTargetScan PicTar and DIANA-microT) To reduce falsepositive results genes predicted by at least three of these fourdatabases were selected as differentially expressed miRNAtargets for subsequent analysis Screening resulted in theselection of 38 target genes (Table 3)The target genes ofmiR-20a may be involved in the etiology of vascular remodelingthrough cell proliferation apoptosis migration and differen-tiation

4 Discussion

The observations reported here indicate that the underlyingmechanism of PQR treatment for essential hypertensiondoes not mediate vascular remodeling but strictly regulatesmiRNA expression Our previous studies have shown thatTCM (traditional Chinese medicine) treatment not onlyreduces high blood pressure in hypertension but also reversesboth cardiac and vascular smooth muscle cell hypertrophy[18] In the present study we demonstrated that PQR treat-ment fully prevented the development of hypertension aswell as cardiac hypertrophy and aorta remodeling It hasbeen argued that excessive use of PQR in hypertensionmightinterfere with some anatomical andor functional parametersthat are necessary to prevent blood pressure increase

A range of evidence has demonstrated that miRNAscould be used as clinical biomarkers in essential hypertension[19] The most robust multicenter study that provided suchevidence was conducted in Ghent Belgium and focusedon miRNA analysis of potential prognostic biomarkers in500 neuroblastoma patients [20] Although different tech-nological platforms have been used for miRNA profilingthere is significant overlap between prognostic signaturesdescribed in previous work and several miRNAs that werelater identified by more than three independent studies asbeing downregulated in essential hypertension or associatedwith vascular remodeling (eg miR-221 miR-26a miR-21miR-296-5p and miR-204) [21ndash24]

In the present study a microarray assay was appliedto obtain miRNA expression profiles for thoracic aorta inthree groups of SHR and qRT-PCR was used to verifythe microarray data A total of 32 miRNAs in SHR (18upregulated and 14 downregulated) and 17 miRNAs in thePQR treatment group (7 upregulated and 10 downregulated)were successfully identified Furthermore we also founddifferentially expressed miRNA-20a with 38 potential targetgenes in rats which demonstrated that miRNA expressionmight be significant in PQR treatment for rats with essentialhypertension In our studies the most frequently observedand the most promising miRNAs as potential treatmenttargets are miR-145 [11] and miR-208 [25] We found thatmiR-208 is upregulated in insulin-mediated proliferation ofvascular smooth muscle cells and may promote a switchfrom the G0G1 phase of the cell cycle to the S phase Thedirect target of miR-208 has been shown to be p21 [25]and p21 expression in vascular smooth muscle cells has beenshown to be crucial in limiting vascular proliferation invascular remodeling which is strongly associated with essen-tial hypertension [26] Interestingly some studies [27ndash29]


Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

upqRT-PCRmiChip assay

minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)

Figure 5 Validation of miRNA microarray data by qRT-PCR (a) miR-20a (b) miR-145 (c) miRNA-98 (d) miR-30The relative expressionof four miRNAs was normalized to the expression of the internal control gene (U6)

have shown that miR-143 andmiR-145 play an important rolein switching the phenotypes of smooth muscle cells duringvascular remodeling The function of these miRNAs is likelymediated by the degradation of many transcription factorsincluding KLF4 KLF5 Elk-1 and other transcription factorsinvolved in Jagged-1Notch signaling [30] which have beenlinked to the inhibition of differentiation of smooth musclecells MiR-20a a member of the miR-17ndash92 cluster is a highlyconserved miRNA within a noncoding RNA encoded by thec13 or f25 host gene localized on chromosome 13 [31] Thefunctions of each cluster member in essential hypertensionhave not been clearly established Recently Pin et al foundthat miR-20a can inhibit the expression of MKK3 anddownregulate p38 pathway-mediated and VEGF-induced

endothelial cell migration and angiogenesis [32] miR-20a has also been shown to play an important role invascular remodeling [33] In contrast several function-ally well-characterized miRNAs that had previously beenobserved in other diseases were later identified in SHR forthe first time with a high level of statistical significance indi-cating their potential involvement in essential hypertensionpathogenesis These included miR-20a miR-18b miR-375and miR-215 [34]

In conclusion our study demonstrates that PQR hasbeneficial effects in reducing blood pressure and vascu-lar remodeling in SHR The underlying mechanism mightbe related to the modulation of 18 upregulated and 14downregulated miRNAs in particular miR-20a miR-145


Table 3 Predicted target genes of miRNA-20a

Target gene Accession no Target gene nameZNFX1 NM 021035 Zinc finger NFX1-type containing 1IL25 NM 022789 Interleukin 25MAP3K2 NM 006609 Mitogen-activated protein kinase kinase kinase 2AMPD3 NM 001025390 Adenosine monophosphate deaminase 3GPR137C NM 001099652 G protein-coupled receptor 137CACTBL2 NM 001017992 Actin beta-like 2MFAP3L NM 001009554 Microfibrillar-associated protein 3-likeTRIP11 NM 004239 Thyroid hormone receptor interactor 11DGUOK NM 080918 Deoxyguanosine kinaseMFN2 NM 001127660 Mitofusin 2VPS36 NM 004755 Vacuolar protein sorting 36 homologPLS1 NM 001145319 Plastin 1ARHGAP12 NM 018287 Rho GTPase activating protein 12FZD3 NM 017412 Fizzled family receptor3PDK4 NM 002612 Pyruvate dehydrogenase kinase isozyme 4KIF23 NM 004856 Kinesin family member 23VLDLR NM 003383 Very low density lipoprotein receptorFBXO4B NM 001024680 F-box protein 4BZNF652 NM 014897 Zinc finger protein 652RASD1 NM 016048 RAS dexamethasone-induced 1RS1 NM 000330 Retinoschisin 1TNFRSF21 NM 014452 Tumor necrosis factor receptor superfamily member 21FGL1 NM 004467 Fibrinogen-like 1CCND2 NM 001759 Cyclin D2TMEM133 NM 032021 Transmembrane protein 133LPGAT1 NM 014873 Lysophosphatidylglycerol acyltransferase 1IPO7 NM 006391 Importin 7GUCY1A3 NM 000856 Guanylate cycle 1 souble alpha 3TSPAN9 NM 001168320 Tetraspanin 9KLF12 NM 007249 Kruppel-like factor 12SMOC2 NM 001166412 SPARC related modular calcium binding 2MAP3K3 NM 002401 Mitogen-activated protein kinase kinase kinase 3NRP2 NM 018534 Neuropilin 2SOCS6 NM 004232 Suppressor of cytokine signaling 6SLC16A6 NM 001174166 Solute carrier family 16 member 6 (monocarboxylic acid transporter 7)PRR14L NM 173566 Proline rich 14-likeANO6 NM 001025356 Anoctamin 6ZBTB43 NM 001135776 Zinc finger and BTB domain containing 43

miR-30 andmiR-98We suggest that the target genes of miR-20a may be involved in the etiology of vascular remodel-ing through cell proliferation apoptosis migration anddifferentiation However the underlying mechanisms shouldbe further investigated through basic research and well-controlled clinical trials

5 Conclusion

Taken together our findings indicated that PQR could exertits antihypertensive effect through deterioration of the vascu-lar remodeling process The mechanism might be associated

with regulating differentially expressed miRNAs in aortatissue

Conflict of Interests

The authors claim no conflict of interests involved in thestudy

Acknowledgments

This work was supported by research grants from theNational Natural Science Foundation of China (30506644


and 30407125) andChineseMedicine and Pharmacy PlannedProject ofHunanProvince P R China (2009047 and 201245)The authors thank Dr Joen-Rong Sheu for critical reading ofthis paper

References

[1] D Lloyd-Jones R Adams M Carnethon et al ldquoHeart diseaseand stroke statisticsmdash2009 update A report from the Ameri-can heart association statistics committee and stroke statisticssubcommitteerdquo Circulation vol 119 no 3 pp 480ndash486 2009

[2] FHMesserli BWilliams andE Ritz ldquoEssential hypertensionrdquoThe Lancet vol 370 no 9587 pp 591ndash603 2007

[3] Y-J Lv G-L Liu X-M Ji et al ldquoQindan capsule changesadventitial collagen synthesis in spontaneously hypertensiveratsrdquo Chinese Journal of Integrative Medicine vol 19 no 9 pp689ndash695 2013

[4] G-W Zhong M-J Chen Y-H Luo et al ldquoEffect of Chineseherbal medicine for calming Gan and suppressing hyperactiveyang on arterial elasticity function and circadian rhythm ofblood pressure in patients with essential hypertensionrdquo ChineseJournal of Integrative Medicine vol 17 no 6 pp 414ndash420 2011

[5] G W Zhong Y H Luo L L Xiang et al ldquoClinical efficacystudy on calming liver and restraining Yang formula in treatingpatients with mild or moderate degree of essential hyperten-sionrdquo China Journal of Chinese Materia Medica vol 16 no 9pp 776ndash778 2010

[6] G W Zhong W Li M J Chen et al ldquoEffeets on the vascularremodeling and adiponectin expression in aorta in the spon-taneously hypertensive rats by Chinese herb mixture methodrdquoChinese Journal of Hypertension (China) vol 16 no 9 pp 812ndash816 2008

[7] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[8] H-W Hwang and J T Mendell ldquoMicroRNAs in cell prolifera-tion cell death and tumorigenesisrdquo British Journal of Cancervol 94 no 6 pp 776ndash780 2006

[9] T Kunej I Godnic S Horvat M Zorc and G A Calin ldquoCrosstalk between MicroRNA and coding cancer genesrdquo CancerJournal vol 18 no 3 pp 223ndash231 2012

[10] D Catalucci P Gallo and G Condorelli ldquoAdvances in molecu-lar genetics genomics proteomics metabolomics and systemsbiology microRNAs in cardiovascular biology and heart dis-easerdquoCirculation CardiovascularGenetics vol 2 no 4 pp 402ndash408 2009

[11] K R Cordes N T SheehyM PWhite et al ldquoMiR-145 andmiR-143 regulate smooth muscle cell fate and plasticityrdquo Nature vol460 no 7256 pp 705ndash710 2009

[12] S K Gupta C Bang and TThum ldquoCirculating MicroRNAs asbiomarkers and potential paracrinemediators of cardiovasculardiseaserdquo Circulation Cardiovascular Genetics vol 3 no 5 pp484ndash488 2010

[13] S Li J Zhu W Zhang et al ldquoSignature microRNA expressionprofile of essential hypertension and its novel link to humancytomegalovirus infectionrdquo Circulation vol 124 no 2 pp 175ndash184 2011

[14] D Torella C Iaconetti D Catalucci et al ldquoMicroRNA-133controls vascular smoothmuscle cell phenotypic switch in vitroand vascular remodeling in vivordquo Circulation Research vol 109no 8 pp 880ndash893 2011

[15] X-P Li Y-H Luo G-W Zhong L-L Xiang and Y-H LildquoPharmacodynamic studies on formula for calming the liverand suppressing yang in treating spontaneous hypertensionratsrdquo China Journal of Traditional Chinese Medicine and Phar-macy vol 26 no 4 pp 710ndash715 2011

[16] E L Schiffrin ldquoRemodeling of resistance arteries in essentialhypertension and effects of antihypertensive treatmentrdquo Amer-ican Journal of Hypertension vol 17 no 12 pp 1192ndash1200 2004

[17] A Krek D Grun M N Poy et al ldquoCombinatorial microRNAtarget predictionsrdquo Nature Genetics vol 37 no 5 pp 495ndash5002005

[18] G-W Zhong W Li Y-H Luo et al ldquoEffects of the calmingliver and suppressing yang method on proliferation and theexpression of heat shock protein 27 in vascular smooth musclecells of spontaneously hypertensive ratsrdquo Chinese Journal ofGerontology vol 29 no 2 pp 385ndash388 2009

[19] Y DrsquoAlessandra P Devanna F Limana et al ldquoCirculatingmicroRNAs are new and sensitive biomarkers of myocardialinfarctionrdquo European Heart Journal vol 31 no 22 pp 2765ndash2773 2010

[20] J Bienertova-Vasku P Mazanek R Hezova et al ldquoExtensionof microRNA expression pattern associated with high-riskneuroblastomardquo Tumor Biology vol 34 no 4 pp 2315ndash23192013

[21] N J Leeper A Raiesdana Y Kojima et al ldquoMicroRNA-26ais a novel regulator of vascular smooth muscle cell functionrdquoJournal of Cellular Physiology vol 226 no 4 pp 1035ndash10432011

[22] H Kang B N Davis-Dusenbery P H Nguyen et al ldquoBonemorphogenetic protein 4 promotes vascular smooth musclecontractility by activatingmicroRNA-21 (miR-21) which down-regulates expression of family of dedicator of cytokinesis(DOCK) proteinsrdquoThe Journal of Biological Chemistry vol 287no 6 pp 3976ndash3986 2012

[23] X Liu Y Cheng J Yang L Xu and C Zhang ldquoCell-specificeffects of miR-221222 in vessels molecular mechanism andtherapeutic applicationrdquo Journal of Molecular and CellularCardiology vol 52 no 1 pp 245ndash255 2012

[24] R-R Cui S-J Li L-J Liu et al ldquoMicroRNA-204 regulatesvascular smooth muscle cell calcification in vitro and in vivordquoCardiovascular Research vol 96 no 2 pp 320ndash329 2012

[25] Y Zhang Y Wang X Wang et al ldquoInsulin promotes vascularsmooth muscle cell proliferation via microRNA-208-mediateddownregulation of p21rdquo Journal of Hypertension vol 29 no 8pp 1560ndash1568 2011

[26] E M Jeon H C Choi K Y Lee K C Chang and Y J KangldquoHemin inhibits hypertensive rat vascular smooth muscle cellproliferation through regulation of cyclin D and p21rdquo Archivesof Pharmacal Research vol 32 no 3 pp 375ndash382 2009

[27] B N Davis-Dusenbery M C Chan K E Reno et al ldquoDown-regulation of Kruppel-like Factor-4 (KLF4) by microRNA-143145 is critical for modulation of vascular smooth musclecell phenotype by transforming growth factor-120573 and bonemorphogenetic protein 4rdquo The Journal of Biological Chemistryvol 286 no 32 pp 28097ndash28110 2011

[28] M Xin E M Small L B Sutherland et al ldquoMicroRNAsmiR-143 and miR-145 modulate cytoskeletal dynamics andresponsiveness of smooth muscle cells to injuryrdquo Genes ampDevelopment vol 23 no 18 pp 2166ndash2178 2009

[29] Y Cheng X Liu J Yang et al ldquoMicroRNA-145 a novelsmoothmuscle cell phenotypic marker andmodulator controls


vascular neointimal lesion formationrdquoCirculation Research vol105 no 2 pp 158ndash166 2009

[30] JM Boucher SM Peterson SUrs C Zhang andL Liaw ldquoThemiR-143145 cluster is a novel transcriptional target of Jagged-1Notch signaling in vascular smooth muscle cellsrdquo Journal ofBiological Chemistry vol 286 no 32 pp 28312ndash28321 2011

[31] C Doebele A Bonauer A Fischer et al ldquoMembers of themicroRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenicfunction in endothelial cellsrdquo Blood vol 115 no 23 pp 4944ndash4950 2010

[32] A-L Pin F Houle M Guillonneau E R Paquet M J Simardand J Huot ldquomiR-20a represses endothelial cell migration bytargeting MKK3 and inhibiting p38 MAP kinase activation inresponse to VEGFrdquo Angiogenesis vol 15 no 4 pp 593ndash6082012

[33] D Frank J Gantenberg I Boomgaarden et al ldquoMicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involvingits novel target Egln3PHD3rdquo Journal of Molecular and CellularCardiology vol 52 no 3 pp 711ndash717 2012

[34] J Song D Kim C-H Chun and E-J Jin ldquoMicroRNA-375a new regulator of cadherin-7 suppresses the migration ofchondrogenic progenitorsrdquoCellular Signalling vol 25 no 3 pp698ndash706 2013

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


a significant role in the renin-angiotensin system (RAAS)mdashan important modulator of systemic blood pressure [11]Some miRNAs including miR-1 miR-145 miR-122 miR-221 and miR-222 have been linked to vascular endothelialdysfunction [12] Others have been linked to the regulationof vascular smooth muscle cells these include miR-145 let-7d miR-24 miR-26a and miR-146 [13] The miRNAs miR-1miR-155 and miR-208 have significant effects on the RAAS[14] Therefore a new strategy for hypertension treatmentmight involve maintenance and restoration of stability bytargeting corresponding miRNA expression in the organ ofinterest

To elucidate the association between miRNA expressionand PQR treatment for essential hypertension we carried outanalysis of miRNA gene expression in aortic tissue from SHRthat had received PQR interventionWe tested the hypothesisthat PQRplays an antihypertensive role by regulatingmiRNAexpression in rat aortic tissueThis research may also providenew insights into potential therapeutic targets to prevent andtreat hypertension

2 Materials and Methods

21 Animals and Drugs Forty 16-week-old male sponta-neously hypertensive rats (SHR) and 20 male Wistar (WKY)rats (Vital River Laboratory Animal Technology Co LtdBeijing China) of the same age were housed in a sterileenvironment at a temperature of 21 plusmn 1∘C and a relativehumidity of 50 plusmn 10 in a 12-hour day-night cycle Bothgroups of rats had been fed standard rat chow and wateruntil they were 16 weeks old All animal study protocolswere approved by the Animal Care and Use Committee ofCentral SouthUniversity (201303117) and followed the animalmanagement rules set out by the Ministry of Health Chinaand the US National Institutes of Health Guide for the Careand Use of Laboratory Animals The PQR medication recipewas composed of Rhizoma Gastrodiae Ramulus Uncariaecum Uncis Concha Haliotidia Concha Ostreae and RadixAchyranthis Bidentatae all componentswere purchased fromthe Department of Pharmacy Xiangya Hospital CentralSouth University One gram of extract was equal to 425 g ofcrude material

22 Animal Groupings and Treatments The WKY rats andSHR were arbitrarily separated into three groups the normalgroup (119899 = 20) the model group (119899 = 20) and the PQRgroup (119899 = 20) Rats in the PQR group were administeredPQR at a dose of 50mgsdotkgminus1sdotdminus1 by gastrogavageThe otherswere given an equal volume of distilled water For all groupsthe administration course lasted 4 weeks All animals wereused for the miRNA analysis and verification study FortySHR were randomly divided into two groups and were given50mgkg of PQR by gastrogavage once daily for 4 weeksnormal saline was given as the negative control

23 Blood Pressure Detection Systolic blood pressure (SBP)was measured in all rats as previously described [15] Tail-cuff plethysmography (TCP) with a rat tail blood pressuremonitor was used The SBP of each rat was measured five

timesmdashonce before treatment and 1 2 3 and 4 weeks aftertreatment At every time point the mean of the lowest threevalues within 5mmHg was regarded as the SBP value

24 Histological and Morphological Assay Rats were anes-thetizedwith 10 chloral hydrate (400mgkg intraperitonealinjection) at the end of each week of whole-day drug admin-istration The thoracic aorta below the aortic arch of each ratwas stripped and clipped A portion was fixed in 8 neutralformaldehyde embedded in paraffin sectioned at 5 120583mand stained with the hematoxylin-eosin (HE) and Massonmethods [16] Light microscopy was used to image eachcross-sectional slice of which there were five per rat Eachvascular ring in the perpendicular position and the vesselmedia wall were observed The images were observed undera Leica imaging system (LeicaMicrosystems GmbHWetzlarGermany) The media thickness (MT) and inner diameter(LD)weremeasured and the ratio ofmedia thickness to innerdiameter (MTLD)was calculatedOther parts of the thoracicaorta were removed from the adventitia and were promptlyrefrigerated at minus80∘C for miRNA assay

25 RNA Microarray and Hybridization

RNA Extraction Total RNA was extracted by a one-stepmethod using TRIzol (Invitrogen USA) following the manu-facturer protocol concentrated using isopropanol precipita-tion and quantified using a spectrophotometer and agarosegel electrophoresis The polyethylene glycol (PEG) methodwas used to isolate and purify 50 120583g of total RNA

Fluorescently Labeled miRNA miRCURY LNA array labelingkit (Exiqon Denmark) was used Total RNA (10 120583g) wasadded to 2 120583L of Hy

3fluorescent label solution and 2 120583L of

labeling enzyme mixed by pipetting and then incubated at65∘C for 15min to terminate the labeling process

miRNA Microarray Hybridization A miRCURY LNA arraylabeling kit using Macro Kit (ID 208000V71) and hybridbox II (ID 40080) was purchased from Exiqon Biochipslides and cover slips were purchased from Ambion Inc(USA) miRNA microarray hybridization was performedaccording to the miRCURY LNA array kit instructions10 120583L of total RNA was added to 10 120583L of 2x hybridizationbuffer and incubated for 3ndash5min at 95∘C Then 20120583L of thehybridization solution was placed on a microarray slide andcompletely covered with a Bioarray Lifter Slip coverslip Themicroarray slide was placed into the Hybridization ChamberII in a horizontal orientation and bathed at 60∘C for 16 hFollowing incubation hybridization samples were removedfrom the microarray slides with a wash solution Each of 509miRNAs was detected by three replicate probe spots on eachmicroarray slide for a total of six measurements per miRNAper sample after repeated fluorescence exchange

Image Acquisition andQuantification Eachmicroarray (chip)was rinsed and immediately dried then illuminated by a sin-gle 635 nm beam and scanned by a GenePix 4000B dual laserscanner (Molecular Devices LLC USA) Image files were






3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results



0 1 2 3 4

0

100

200

300


SBP

(mm

Hg)

998771

Week










N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N M PQR

(a)

N M PQR

(b)

PQR group0

50

100

150

P = 002 P = 004

MT

(120583m

)

Model group

m)

Normal group

(c)


05

10

15

20

25

P = 0009 P = 003

MT

LD

(d)








N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















N M PQR

28 S

18 S





(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)




4 Discussion





Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

minus10

0

10

20

qRT-PCRmiChip assay

miRNA-20aFo

ld ch

ange

s

(a)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus5

0

5

10

miRNA-145

Fold

chan

ges

(b)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro

up

qRT-PCRmiChip assay

minus30

minus20

minus10

0

10miRNA-98

Fold

chan

ges

(c)

Mod

el g

roup

nor

mal

gro

up

PQR

grou

pm

odel

gro


minus4

minus2

0

2

4 miRNA-30Fo

ld ch

ange

s

(d)









5 Conclusion





Acknowledgments




References










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















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
















research article effects of the pinggan qianyang recipe on...

Documents