research article effect of huanglian jiedu decoction on...

Research ArticleEffect of Huanglian Jiedu Decoction on Thoracic Aorta GeneExpression in Spontaneous Hypertensive Rats

Gui-Hua Yue1 Shao-Yuan Zhuo1 Meng Xia1 Zhuo Zhang2 Yi-Wen Gao2 and Yuan Luo2

1 Guangxi University of Chinese Medicine Nanning 530001 China2 Affiliated Ruikang Hospital Guangxi University of Chinese Medicine Nanning 530001 China

Correspondence should be addressed to Shao-Yuan Zhuo sdygh1969163com

Received 10 October 2013 Revised 13 January 2014 Accepted 9 February 2014 Published 13 March 2014

Academic Editor Samra Bashir

Copyright copy 2014 Gui-Hua Yue 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

Objective Hypertension is one of the most common cardiovascular disorders with high mortality Here we explored theantihypertension effects of Huanglian Jiedu Decoction (HJD) on thoracic aorta gene expression in spontaneous hypertensive ratsMethods A rat model of spontaneous hypertension was used The gene change profile of thoracic aorta after JHD treatment wasassessed by GeneChip(GC) analysis using the Agilent Whole Rat Genome Oligo Microarray Results Hypertension induced 441genes upregulated and 417 genes downregulated compared with the normal control group Treatment of HJD resulted in 76 genesdownregulated and 20 genes upregulated GC data analysis showed that the majority of change genes were involved in immunesystem process developmental process and cell death Conclusion Hypertension altered expression of many genes that regulatevarious biological functions HJD significantly reduced hypertension and altered the gene expression profiles of SHR rats Thesechanging genes were involved in many cellular functions such as regulating smooth muscle contraction Ca(2+) homeostasis andNO pathway This study provides the potential novel insights into hypertension and antihypertension effects of HJD

1 Introduction

Essential hypertension (primary hypertension or idiopathichypertension) is the most common type of hypertensionaffecting 95 of hypertensive patients The total number ofadults with hypertension in 2000 was 972 million and thenumber of adults with hypertension in 2025 was predictedto increase by about 60 to a total of 156 billion (292of the worldrsquos adult population) [1] The etiology of primaryhypertension is not fully understood Treatment of hyper-tension is facing the challenge because effective therapies ofhypertension are limited by availability cost and adverseeffects Chinese traditional medicine provides a potentialoption to overcome current challenge of antihypertensiontherapy Chinese medicine a system of ancient medical prac-tice that differs in substance methodology and philosophy tomodern medicine plays key role in health maintenance andhas become more frequently used

CM has been used to treat symptoms related to hyper-tension for more than 2500 years JHD is one of the effectiveformulas to combat hypertension in clinic HJD is an ancientChinese prescription first reported in the text Zhou H ıu Bei

J ı Fang (Emergency Standby Remedies) by Ge Hıng (281ndash341 CE Jın dynasty) This formula is commonly used for alltypes of Fire Toxin obstructing the three burners with highfever irritability and dry mouth and throat PharmacologicResearches of this formula show that it has antisepticiseddephlogisticate and enhances the immune system functionsOur previous studies show that HJD has antihypertensioneffects via regulating the inflammatory factors and alteringNO and SOD expression [2] Here this report is to studythe pathogenic mechanism of hypertension and its effects onthoracic aorta gene expression

2 Materials and Methods

21 Animals Models and Treatment The cohorts of 6-week-old male Spontaneous Hypertensive Rats (SHRs) were ran-domly placed into HJD group and SHR model group Thereare eight rats in each group All rats were from BeijingVital River Laboratory Animal Technology Co Ltd licenseNumber is SCXK2006-0009 SPF weight 160 plusmn 10 g Eight 6-week-old male normotensive Wistar-Kyoto(WKY) rats were

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 565784 9 pageshttpdxdoiorg1011552014565784

2 Evidence-Based Complementary and Alternative Medicine

used as the normal control group that were from Experi-mental Animal Center of GuangxiMedical University licenseNumber is SCXK2006-0003 SPF weight 160 plusmn 10 g

The animals were housed in a room with 20ndash24∘C 30ndash40 humidity Rats were ad lib to food and water Thisstudy was performed following the Guide for the Care andUse of Laboratory Animals of the P R China The protocolwas approved by the Committee on the Ethics of AnimalExperiments of the Guangxi University of Chinese Medicine

HJD was consisted of Rhizoma Coptidis Phellodendronamurense Rupr Radix Scutellariae andGardenia jasminoidesEllisin the ratio of 3 2 2 3 All the herbs were provided byRuikang Affiliated Hospital to Guangxi University of ChineseMedicine who bought from Yulin Herb Market in unify Thepurity of each componentwas determined to be above 98byhigh-performance liquid chromatography (HPLC) analysisHPLC-grade reagents methanol acetonitrile and water wereobtained from J T Baker (Phillipsburg NJ USA)These fourherbs were dried in 24 h and grinded into coarse powderThe herbs were cooked into concentrated juice (1mL juice =22 g raw herb) The rats were given HJD juice at 54 g(kgsdotd)by gavage The control group was given 09 saline withthe same volume All the treatments were administrated bygavage for 6 successive weeks

22 Measurement of Blood Pressure Noninvasive BP of ratswas measured using the Nexfin monitor (BMEYE BV Ams-terdam andTheNetherlands) with the latest implementationof the caudal arterymethod Ratsweremeasured for BP at day0 7 14 28 and 42 after HJD treatment each rat was measured3 times to obtain average BP value

23 RNA Isolation and Microarray Experiment After thelast HJD administration all rats were sacrificed and thenthe thoracic aortas were quickly dissected and stored in thefreezer tube in minus80∘C for RNA extraction Rat tissues werehomogenized in Trizol (Invitrogen Carlsbad CA) and thetotal RNA was extracted using chloroform and isopropylalcohol and purified using the RNeasy Mini Kit and RNase-free DNase Set (Qiagen Valencia CA) according to the man-ufacturersrsquo protocols The quality of the extracted total RNAwas measured using the Agilent 2100 Bioanalyzer (AgilentTechnologies Santa Clara CA USA) and the Nano-Chip-protocol before microarray experiments Gene Chip analysiswas performed using the Agilent Whole Rat Genome OligoMicroarray Labeling and hybridization were performed atShanghai Biotechnology Corporation

24 Data Analysis Microarray data were analyzed using theSBC Analysis system (httpsasebioservicecomindexjsp)

Gene expression profiles of SHR were compared to thosefrom WKY or HJD treatment group The changing wasselected by the criteria 119875 lt 005 and Fold Changes ge 2

3 Results

31 HJD Reduced Blood Pressure of SHR Rats The rat in SHRgroup had significantly higher blood pressures compared

020406080

100120140160180200

0 7 14 28 42

ControlModelHJD

Day

lowastlowast

lowast lowast

Figure 1 Effects of HJD on blood pressure in rats BP of rat wasmeasured with mmHg unit There were 8 rats in each group lowast119875 lt005 versus SHR group

with normal control group before HJD treatment (119875 lt 005)After one week administration the BP of HJD group beganto decrease than SHR group After 6 weeks of treatment therats in HJD group had almost similar blood pressure to therats in control group (Figure 1)

32Hypertension andHJDTreatmentCausedGene ExpressionChanges Volcano plot that was drawn by 119875 value and FoldChange checked by 119905-test showed the significant differenceof sample data between chipset groups (Figures 2 and3 described by Gene-Spring 110 119883-axis for Log

2(Fold

Change) 119884-axis formdashLog10

(119875 value) 119883-axis parallel line119875 = 005 119884-axis parallel line Fold Change = 20 Red Zone119875 lt 005 and Fold Change ge 20)

Gene microarray analysis showed that there were 858genes that alter in SHR group compared with normal controlgroups (Figure 2) There were 417 downregulated (Table 1)and there were 441 upregulated (Table 2)

JHD treatment altered 96 gene expressions pared withuntreated SHR group 119875 lt 005 and Fold Change ge 20 wereidentified between the HJD treatment group and the SHRmodel groups Of these 76 genes were downregulated and 20genes were upregulated (Table 3)

33 HJD Induced Gene Changes and Their Biological Con-sequences The genes that were altered after HJD treat-ment mainly belong to 12 classifications (Table 4) includinggenes that regulate immune system cellular functions andmetabolic and developmental functionsThere were 33 genesin the category of immune system development leukocyteactivation regulation of immune system process cell activa-tion and proliferation regulation of immune system processpositive regulation of biological process and so forth (119875 lt005) (Table 5)

Further functional pathway analysis showed that hyper-tension altered genes were involved in 22 KEGG pathways

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Downregulated genes in hypertension rats

Gene Abbr Gene Id Chro FC 119875

DRABUB01 rat DRG library Rattus norvegicuscDNA clone DRABUB01 51015840 mdash NA 9 0097 386119864 minus 03

Similar to RIKEN cDNA 1700001F09 (LOC289957) mdash NA 15 0092 947119864 minus 05

Mitochondrial ribosomal protein S2 Mrps2 362094 3 0081 649119864 minus 03

Q99LR9 MOUSE (Q99LR9) Riok3 protein(fragment) partial (13) mdash NA 18 0079 679119864 minus 04

AGENCOURT 26625193 NIH MGC 253 Rattusnorvegicus cDNA clone IMAGE7304073 51015840 mdash NA 5 0054 658119864 minus 03

Similar to 60S ribosomal protein L19 (LOC316856) mdash NA Un 0053 140119864 minus 04

Solute carrier family 4 (anion exchanger) member 1 Slc4a1 24779 10 0051 824119864 minus 03

Cell division cycle 25 homolog A (S pombe) Cdc25a 171102 8 0039 688119864 minus 04

Synuclein alpha (non-A4 component of amyloidprecursor) Snca 29219 4 0029 600119864 minus 03

Unknown mdash NA 7 0028 212119864 minus 04

Coproporphyrinogen oxidase Cpox 304024 11 0021 211119864 minus 06


Q8FIR6 ECOL6 (Q8FIR6) cytochrome b561homolog 2 partial (5) mdash NA 9 0009 205119864 minus 04

UI-R-C3-sf-g-04-0-UI mdash NA 1 0007 628119864 minus 03

Note Abbr gene abbreviation Chro chromosome Gene Id represents the identity (ID) of the gene in GenBank FC = SHR model group gene expressionlevelnormotensive WKY control group expression level ldquomdashNAUnrdquo indicates unknown

Table 2 Upregulated genes (model versus control)


Bone gamma-carboxyglutamate (gla) protein Bglap 25295 2 61402 196119864 minus 03

Uncharacterized protein mdash NA 2 25856 196119864 minus 05


Cellular retinoic acid binding protein 2 Crabp2 29563 2 20657 473119864 minus 03

Major histocompatibility complex assembledfrom 40 BACs strain Brown Norway(BNssNHsd) RT1n haplotype segment 211

mdash NA 20 19648 152119864 minus 03

V-set domain containing T cell activationinhibitor 1 Vtcn1 295322 2 18951 115119864 minus 03

Interferon-induced protein with tetratricopeptiderepeats 1 Ifit1 56824 1 17991 151119864 minus 04



Similar to SMAD-interacting zinc finger protein 2 LOC679126 679126 13572 821119864 minus 05

Transient receptor potential cation channelsubfamily C member 4 Trpc4 84494 2 13374 820119864 minus 04

Q3X0T1 9ACTN (Q3X0T1) 60 kDainnermembrane protein partial (6) mdash NA 19 12417 435119864 minus 03

CJ032 MOUSE (Q9CRC6) protein C10orf32homolog partial (15) mdash NA 1 11726 863119864 minus 04

TL0ACA45YL24 mdash NA X 11407 180119864 minus 03



Table 3 The upregulated and downregulated genes after the treatment of HLJDD


Solute carrier family 4 (anion exchanger)member 1 Slc4a1 24779 10 3871 674119864 minus 03

Protein phosphatase 1 regulatory subunit 3D Ppp1r3d 689995 3 2566 278119864 minus 03

Ribonucleotide reductase M2 Rrm2 362720 6 2551 630119864 minus 03

pim-1 oncogene Pim1 24649 20 2529 512119864 minus 03

Spectrin beta erythrocytic Sptb 314251 6 2470 466119864 minus 03

Transmembrane and coiled-coil domain family 2 Tmcc2 305095 13 2444 338119864 minus 03


UI-R-C4-alc-g-09-0-UI mdash NA Un 0235 360119864 minus 03

UI-R-BJ2-bql-b-07-0-UI mdash NA 13 0385 314119864 minus 03

Potassium voltage-gated channel subfamily H(eag-related) member 1 Kcnh1 65198 13 0393 998119864 minus 03

Nuclear receptor subfamily 4 group A member 3 Nr4a3 58853 5 0456 987119864 minus 03

Note Abbr gene abbreviation Chro chromosome Gene Id represents the identity (ID) of the gene in GenBank FC = HLJDT treatment groupSHR modelgroup gene expression level ldquomdashNAUnrdquo indicates unknown

Table 4 Functional catalogers of hypertension-induced gene change profiles

GO Id Term Hit genes 119875

GO0032501 Multicellular organismal processGO0051239 Regulation of multicellular organismal process 56 00033GO0048771 Tissue remodeling 14 00068GO0001816 Cytokine production 15 00243GO0003008 System process 77 00485GO0032502 Developmental processGO0001503 Ossification 18 00005GO0050793 Regulation of developmental process 66 00045GO0051704 Multiorganism processGO0051707 Response to other organism 23 00026GO0044419 Interspecies interaction between organisms 7 00059GO0050896 Response to stimulusGO0009607 Response to biotic stimulus 24 00074GO0042221 Response to chemical stimulus 97 00214GO0010926 Anatomical structure formationGO0022607 Cellular component assembly 41 0042GO0050789 Regulation of biological processGO0051239 Regulation of multicellular organismal process 56 00033GO0050793 Regulation of developmental process 66 00045GO0048518 Positive regulation of biological process 86 00361

(Table 6) while HJD altered genes were mainly related to 10KEGG pathways (119875 lt 005) (Table 7)

4 Discussion and Conclusions

prehypertension criterion is systolic pressure 120sim139mmHg(1mmHg = 0133 kPa) andor diastolic pressure 80sim89mmHg As the important pathogenic risk factor of manycardiovascular and cerebrovascular diseases prehyperten-sion can affect the structure and function of heart brainand liver compared to the ideal blood pressure Duringprehypertension the elasticity of large artery trunks was

already obviously damaged and the relaxation function ofleft ventricle was slightly damaged which are the damagesymbol of target organs in hypertension [3] Our previousdata showed that SHR arterial elasticity and endothelialfunctions in SHR rats were impaired in early stage [3] Andthe gene array studies coincidences showed that multiplegenes in aorta thoracic in young SHR were changed intranscription level compared with normotensive WKY Inparticular expression genes related to smoothmuscles shrinkor cytoskeletons were significantly increased or decreasedThese genes were Chrm3 MRPS2 TRPC4 P2rx4 Slc8a3Htr5a Chp and so forthThemuscarinic cholinergic receptor


Table 5 Functional category of HJD-induced gene change profiles


GO0002376 Immune system processGO0002520 Immune system development 10 00001GO0045321 Leukocyte activation 12 00001GO0002682 Regulation of immune system process 12 00001GO0006955 Immune response 10 00003GO0002684 Positive regulation of immune system process 7 00006GO0045058 T cell selection 2 00046GO0002253 Activation of immune response 3 0026GO0009987 Cellular processGO0001775 Cell activation 13 00001GO0008283 Cell proliferation 15 00001GO0048522 Positive regulation of cellular process 22 00002GO0008219 Cell death 13 00016GO0048523 Negative regulation of cellular process 16 00086GO0016043 Cellular component organization 19 00181GO0007049 Cell cycle 7 00453GO0008152 Metabolic processGO0009058 Biosynthetic process 28 00005GO0009893 Positive regulation of metabolic process 11 00361GO0006807 Nitrogen compound metabolic process 5 00413GO0019748 Secondary metabolic process 2 00423GO0032502 Developmental processGO0050793 Regulation of developmental process 17 00008GO0051093 Negative regulation of developmental process 10 00026GO0048856 Anatomical structure development 21 00086GO0009790 Embryonic development 8 00154GO0007275 Multicellular organismal development 20 00249GO0001503 Ossification 4 00251GO0009653 Anatomical structure morphogenesis 12 00409GO0016265 DeathGO0008219 Cell death 13 00016GO0032501 Multicellular organismal processGO0001816 Cytokine production 7 00003GO0051240 Positive regulation of multicellular organismal process 7 00017GO0051239 Regulation of multicellular organismal process 14 00021GO0007275 Multicellular organismal development 20 00249GO0050896 Response to stimulusGO0006955 Immune response 10 00003GO0009605 Response to external stimulus 11 00084GO0009607 Response to biotic stimulus 6 00201GO0048584 Positive regulation of response to stimulus 5 00209GO0048585 Negative regulation of response to stimulus 3 00445GO0050789 Regulation of biological processGO0002682 Regulation of immune system process 12 00001GO0048518 Positive regulation of biological process 24 00001GO0050793 Regulation of developmental process 17 00008GO0051239 Regulation of multicellular organismal process 14 00021GO0048519 Negative regulation of biological process 18 00045GO0032844 Regulation of homeostatic process 3 0045GO0048518 Positive regulation of biological process 24 00001GO0048519 Negative regulation of biological process 18 00045GO0010926 Anatomical structure formation 12 00254


Table 6 Hypertension altered genes in 22 KEGG pathways

KEGG pathway Hit genes 119875

Olfactory transduction 29 0Neuroactive ligand-receptorinteraction 13 00002

Cytokine-cytokine receptor interaction 10 00009Focal adhesion 9 00009Tight junction 6 00061RIG-I-like receptor signaling pathway 4 00081Ether lipid metabolism 3 00083Calcium signaling pathway 7 00086Leishmania infection 4 00089Amyotrophic lateral sclerosis (ALS) 4 00113Arrhythmogenic right ventricularcardiomyopathy (ARVC) 4 0014

ABC transporters 3 00171Aldosterone-regulated sodiumreabsorption 3 00213

Hypertrophic cardiomyopathy (HCM) 4 00231Dilated cardiomyopathy 4 00247Glycosphingolipidbiosynthesismdashganglio series 2 00265

Glutathione metabolism 3 00284Apoptosis 4 0032Wnt signaling pathway 5 00324Natural killer cell mediatedcytotoxicity 5 00339

Maturity onset diabetes of the young 2 00415MAPK signaling pathway 7 0047

Table 7 HJD altered genes in 10 KEGG pathways


Hematopoietic cell lineage 3 00004Metabolic pathways 8 00016Natural killer cell mediatedcytotoxicity 3 00027

NOD-like receptor signaling pathway 2 00066Cytokine-cytokine receptor interaction 3 00105Pyrimidine metabolism 2 00148T cell receptor signaling pathway 2 00192Axon guidance 2 00254Jak-STAT signaling pathway 2 00308Purine metabolism 2 00379

3 also known as Chrm3 (FC = 4343) controls smoothmuscle contraction and its stimulation causes secretion ofglandular tissue [4] MRPS2 (FC = 0081) encodes mito-chondrial ribosomal protein S2 [5 6] Genes related toCalcium signaling pathway like TRPC4(FC = 13374) P2rx2(FC = 4075) and P2rx4 (FC = 3329) expression increasedwhereas Slc8a3 (FC = 0313) Htr5a (FC = 0332) and Chp

7

6

5

4

3

2

1

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9

10

11

12

13

log2(fold change)

log10(P

val

ues)

Figure 2 Hypertension induced gene changes compared withnormal control rats Note model = spontaneous hypertension rats(SHR) model group and control = normotensive Wistar-Kyoto ratscontrol group

6

5

4

3

2

1

minus13

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9101112131415

log2(fold change)

log10(P

val

ues)

Figure 3 HJD treatment caused gene changes compared withuntreated SHR rats versus SHR Note model = spontaneous hyper-tension rats (SHR) model group medication = HJD treatmentgroup

(FC = 0398) expression decreased which will cause theimbalance of Ca(2+) in endothelial cells TRPC4 is wellrecognized as a prominent Cation channel in the vascularendothelium which suggested to serve stimulated Ca(2+)entry in a specific endothelial state during the transition froma proliferating to a quiescent phenotype Thus TRPC4 mayadopt divergent as yet unappreciated functions in endothelialCa(2+) homeostasis and emerges as a potential key playerin endothelial phenotype switching and tuning of cellulargrowth factor signaling [7] P2X receptors are ATP activatedchannels that allow the passage of ions across cell membranesand participate in regulating renal microvascular functionautoregulation and hypertension-associated renal vascularinjury [8]

The ATP-gated P2X4 ion channel expressed on endothe-lial cells and encoded by P2rx4 gene is crucial to flow-sensitive mechanisms that regulate blood pressure and vas-cular remodeling P2rx4 (minusminus) mice do not have normalendothelial cell responses to flow such as influx of Ca(2+)and subsequent production of the potent vasodilator nitricoxide (NO) Additionally vessel dilation induced by acuteincreases in blood flow ismarkedly suppressed in P2rx4 (minusminus)mice Furthermore P2rx4 (minusminus) mice have higher blood


pressure and excrete smaller amounts of NO products intheir urine than wild-type mice do Moreover no adaptivevascular remodeling that is a decrease in vessel size inresponse to a chronic decrease in blood flow was observedin P2rx4 (minusminus) mice [9] SLC8A3 solute carrier family 8(sodiumcalcium exchanger) members 3 encodes a memberof the sodiumcalcium exchanger integral membrane proteinfamily Three mammalian isoforms in family 8 (SLC8A1SLC8A2 and SLC8A3) and their splice variants are expressedin a tissue-specific manner to mediate Ca(2+) fluxes acrossthe cell membrane and thus significantly contribute tomaintain Ca(2+) homeostasis in a wide variety of cell types[10] The gene described in this record is a member of 5-hydroxytryptamine receptor family and encodes a multi-pass membrane protein that functions as a receptor for 5-hydroxytryptamine and couples to G proteins negativelyinfluencing cAMP levels via GI and Go [11] This proteinhas been shown to function in part through the regulationof intracellular Ca(2+) mobilization [12] Calcium bindingprotein p22 (Chp) which belongs to the EF-hand superfamilyof Ca(2+)-binding proteins may act by transducing cellularCa(2+) signals to downstream effectors in many cell types[13] It is also known to be an endogenous inhibitor ofcalcineurin activity [14] and thus inactivates the T cells of theimmune system Prehypertension showed the symptoms ofdizziness headache anxiety and irritability [15] 300 casesof prehypertension were investigated [16] on TCM syndromeon the criterion of Subhealth of TCM clinical guidelineswhichwas announced in 2006 The results showed that most of thecases were belonging to patterns of depression of the livergenerating pathogenic fire stagnation of liver qi and spleendeficiency and interior disturbance of phlegm heat

HJD were first reported in Gehongrsquo book of Handbookof Prescription for Emergency but it did not give the nameof this prescription however the first name SLC8A3 solutecarrier family 8 (sodiumcalcium exchanger) members 3encodes a member of the sodiumcalcium exchanger integralmembrane protein family Three mammalian isoforms infamily 8 (SLC8A1 SLC8A2 and SLC8A3) and their splicevariants are expressed in a tissue-specific manner to mediateCa(2+) fluxes across the cell membrane and thus signifi-cantly contribute to maintain Ca(2+) homeostasis in a widevariety of cell types [10] The gene described in this recordis a member of 5-hydroxytryptamine receptor family andencodes a multipass membrane protein that functions as areceptor for 5-hydroxytryptamine and couples to G proteinsnegatively influencing cAMP levels via GI and Go [11] Thisprotein has been shown to function in part through theregulation of intracellular Ca(2+) mobilization [12] Calciumbinding protein p22 (Chp) which belongs to the EF-handsuperfamily of Ca(2+) binding proteins may act by transduc-ing cellular Ca(2+) signals to downstream effectors in manycell types [13] It is also known to be an endogenous inhibitorof calcineurin activity [14] and thus inactivates the T cells ofthe immune system Prehypertension showed the symptomsof dizziness headache anxiety and irritability [15] 300 casesof prehypertension were investigated [16] on TCM syndromeon the criterion of Subhealth of TCM clinical guidelineswhichwas announced in 2006 The results showed that most of the

cases were belonging to patterns of depression of the livergenerating pathogenic fire stagnation of liver qi and spleendeficiency and interior disturbance of phlegm heat

HJD were first reported in Gehongrsquo book of Handbook ofPrescription for Emergency but it did not give the name ofthis prescription however the first name occurred in Wang-taorsquo book of Waitaimiyao This prescription was made upof Rhizoma Coptidis Phellodendron amurense Rupr RadixScutellariae and Gardenia jasminoides Ellis in proportionRhizoma Coptidis exerts as the sovereign to purge heart fireand middle energizers fire Phellodendron amurense Ruprworks as the minister to clear away lung heat and theupper energizers fire Radix Scutellariae clears away the lowerenergizers fire and Gardenia jasminoides Ellis clears awaythe triple energizers fire They work together to removethe toxin HJD have played more and more important rolein cardiovascular diseases that have already used HJD intreatment of hypertension in clinic for many years

Hypertension induces endothelial dysfunction resultingin impairment of endothelium-dependent vasodilatationand widespread abnormalities in endothelial integrity andhomeostasis [17] Endothelial dysfunction exhibits severalpathological conditions including altered anticoagulant andanti-inflammatory properties of the endothelium impairedmodulation of vascular growth and dysregulation of vascularremodelingThemajor cause of impairment of endothelium-dependent vasorelaxation is loss of nitric oxide (NO) bioac-tivity in the vessel wall [18]

Our previous studies showed thatrsquo spontaneous hyperten-sion ratsrsquo blood pressure increases at six-week-old age andthe tail-arterial-systolic-pressure increase at 12-weeksof ageHypertension decreases plasma NO and SOD and increaseof MDA and Von Wille brand factor (vWF) at the 12 weeksof age HJD treatment significantly reversed hypertension-induced NO and SOD [2]

HJD treatment alteredmany genes related to cell responseprocesses hyperplasia and immune response such asIL1b(FC = 2519) Klrk1(FC = 2001) Snca(FC = 2665) andGch1(FC = 2164) It has been reported that interleukin(IL)-1 beta induces the synthesis of both nitric oxide (NO)and prostaglandin (PG)E2 in cultures of dispersed ovariancells and exerts cytotoxic effects on these cells [19 20]Furthermore IL-1 beta can interact with NO and PGE2during steroid genesis [21] Klrk1 is involved in positiveregulation of nitrogen compound metabolic process (GeneOntology 0051173) Alpha-synuclein (ASN) encoded by Sncagene is a small soluble acidic protein and plays an importantrole in the regulation of synaptic nerve terminal function Ithas been shown that ASN stimulated NOS activity and NOrelease in the rat brain [22] GTP cyclohydrolase 1 (GCH1)is the first and rate-limiting enzyme in the synthesis oftetrahydrobiopterin (BH4) an essential cofactor for tyrosinehydroxylase (TH) and NOS in the catecholamine and NOproduction pathways respectively thus playing a crucial rolein the sympathoadrenal and endogenous nitro vasodilatorsystems [23]

According to the GO analyses comparedwith normoten-sive WKY the biological procedure which SHR differen-tial expression genes attended mainly focused on 6 items


of secondary classification such as multicellular organis-mal process developmental process multiorganism processresponse to stimulus anatomical structure formation andregulation of biological process mainly involving 12 itemsof third classification for example ossification responseto other organism regulation of multicellular organismalprocess regulation of developmental process interspeciesinteraction between organisms tissue remodeling responseto biotic stimulus and so froth (119875 lt 005) (Table 5)

HJD can upregulate NO expression in the blood plasma[4] Here HJD also altered the NO pathway related genesincluding IL1b Klrk1 Snca and Gch1 Taking togetherthese findings suggest that HJD may reduce hypertension byregulating NO pathways

In conclusion HJD can significantly reduce hypertensionand alter the gene profiles in aorta thoracic in rats Theseresults provide the potential gene changes and pathwaysunderlying hypertension and antihypertension effects of HJDin treatment of hypertension

Conflict of Interests

Theauthors declare no conflict of interests regarding the pub-lication of this paper

Acknowledgments

The authors are supported by Chinese Nature Science Foun-dationGrant (no 81060294) Talents inGuangxi Colleges andUniversities supported plans (J11064) andThe key project ofGuangxi University of Chinese Medicine Affiliated RuikangHospital (RKZ201015)

References

[1] P M Kearney M Whelton K Reynolds P Muntner P KWhelton and J He ldquoGlobal burden of hypertension analysisof worldwide datardquo The Lancet vol 365 no 9455 pp 217ndash2232005

[2] G Yue h Z Zhang w and Z Meng y ldquoEffect of Huanglian JieduDecoction on endothelial function in younger spontaneouslyhypertensive ratsrdquo Chinese Journal of Basic Medicine in Tradi-tional Chinese Medicine no 4 pp 303ndash305 2010

[3] D Erdogan I Yildirim O Ciftci et al ldquoEffects of normal bloodpressure prehypertension and hypertension on coronary mic-rovascular functionrdquo Circulation vol 115 no 5 pp 593ndash5992007

[4] S Onodera T Chiba T Sugai andW Habano ldquoA genetic asso-ciation between 1205733-aderenoceptor and cholinergic receptormuscarinic 3 polymorphisms in irritable bowel syndromerdquoHepato-Gastroenterology vol 58 no 110-111 pp 1474ndash1478 2011

[5] M A AlimM S Hossain K Arima et al ldquoTubulin seeds 120572-sy-nuclein fibril formationrdquo Journal of Biological Chemistry vol277 no 3 pp 2112ndash2117 2002

[6] M A Alim Q-LMa K Takeda et al ldquoDemonstration of a rolefor 120572-synuclein as a functionalmicrotubule-associated proteinrdquoJournal of Alzheimerrsquos Disease vol 6 no 4 pp 435ndash442 2004

[7] A Graziani M Poteser W-M Heupel et al ldquoCell-cell contactformation governs Ca2+ signaling by TRPC4 in the vascular

endothelium evidence for a regulatory TRPC4-120573-catenin inter-actionrdquo Journal of Biological Chemistry vol 285 no 6 pp 4213ndash4223 2010

[8] Z Guan and EW Inscho ldquoRole of adenosine 51015840-triphosphate inregulating renal microvascular function and in hypertensionrdquoHypertension vol 58 no 3 pp 333ndash340 2011

[9] K Yamamoto T Sokabe T Matsumoto et al ldquoImpaired flow-dependent control of vascular tone and remodeling in P2X4-deficient micerdquoNatureMedicine vol 12 no 1 pp 133ndash137 2006

[10] D Khananshvili ldquoThe SLC8 gene family of sodium-calciumexchangers (NCX) - structure function and regulation inhealth and diseaserdquo Molecular Aspects of Medicine vol 34 no2-3 pp 220ndash235 2013

[11] B J B FranckenM Jurzak J F M VanhauweW HM L Luy-ten and J E Leysen ldquoThe human 5-ht(5A) receptor couples toG(i)G(o) proteins and inhibits adenylate cyclase in HEK 293cellsrdquo European Journal of Pharmacology vol 361 no 2-3 pp299ndash309 1998

[12] N C Schanen S W Scherer L-C Tsui and U Francke ldquoAssi-gnment of the 5-hydroxytryptamine (serotonin) receptor 5Agene (HTR5A) to human chromosome band 7q361rdquoCytogenet-ics and Cell Genetics vol 72 no 2-3 pp 187ndash188 1996

[13] M R Barroso K K Bernd N D DeWitt A Chang K Millsand E S Sztul ldquoA novel Ca2+-binding protein p22 is requiredfor constitutive membrane trafficrdquo Journal of Biological Chem-istry vol 271 no 17 pp 10183ndash10187 1996

[14] M Jimenez-Vidal J Srivastava L K Putney and D L BarberldquoNuclear-localized calcineurin homologous protein CHP1interacts with upstream binding factor and inhibits ribosomalRNA synthesisrdquo Journal of Biological Chemistry vol 285 no47 pp 36260ndash36266 2010

[15] W J Xiong ldquoThe application of the classical formula in the trea-tment of hypertensionrdquoThe Journal of Traditional ChineseMed-icine vol 11 no 20 pp 1836ndash1839 2013

[16] H Gu J Chen and J Wang ldquoThe invertigate of the risk fac-tors for the patients with prehypertension and cardiovasculardiseaserdquo Chinese Orthodox Medicine Journal vol 1 pp 33ndash352012

[17] H Brunner J R Cockcroft J Deanfield et al ldquoEndothelial fun-ction and dysfunction Part II association with cardiovascularrisk factors and diseases A statement by theWorking Group onEndothelins and Endothelial Factors of the European Society ofHypertensionrdquo Journal of Hypertension vol 23 no 2 pp 233ndash246 2005

[18] H Cai and D G Harrison ldquoEndothelial dysfunction in cardio-vascular diseases the role of oxidant stressrdquo Circulation Rese-arch vol 87 no 10 pp 840ndash844 2000

[19] C Ellman J A Corbett T P Misko M McDaniel and K PBeckerman ldquoNitric oxidemediates interleukin-1-induced cellu-lar cytotoxicity in the rat ovary a potential role for nitric oxidein the ovulatory processrdquo Journal of Clinical Investigation vol92 no 6 pp 3053ndash3056 1993

[20] I Ben-Shlomo E Y Adashi and D W Payne ldquoThe morphoge-niccytotoxic and prostaglandin-stimulating activities of inte-rleukin-1120573 in the rat ovary are nitric oxide independentrdquo Journalof Clinical Investigation vol 94 no 4 pp 1463ndash1469 1994

[21] S AhsanM Lacey and S AWhitehead ldquoInteractions betweeninterleukin-1(120573) nitric oxide and prostaglandin E2 in therat ovary effects on steroidogenesisrdquo European Journal ofEndocrinology vol 137 no 3 pp 293ndash300 1997

[22] A Adamczyk A Kazmierczak and J B Strosznajder ldquo120572-Synu-clein and its neurotoxic fragment inhibit dopamine uptake into


rat striatal synaptosomes relationship to nitric oxiderdquo Neuro-chemistry International vol 49 no 4 pp 407ndash412 2006

[23] L Zhang F Rao K Zhang et al ldquoDiscovery of common humangenetic variants of GTP cyclohydrolase 1 (GCH1) governingnitric oxide autonomic activity and cardiovascular riskrdquo Jour-nal of Clinical Investigation vol 117 no 9 pp 2658ndash2671 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


used as the normal control group that were from Experi-mental Animal Center of GuangxiMedical University licenseNumber is SCXK2006-0003 SPF weight 160 plusmn 10 g

The animals were housed in a room with 20ndash24∘C 30ndash40 humidity Rats were ad lib to food and water Thisstudy was performed following the Guide for the Care andUse of Laboratory Animals of the P R China The protocolwas approved by the Committee on the Ethics of AnimalExperiments of the Guangxi University of Chinese Medicine

HJD was consisted of Rhizoma Coptidis Phellodendronamurense Rupr Radix Scutellariae andGardenia jasminoidesEllisin the ratio of 3 2 2 3 All the herbs were provided byRuikang Affiliated Hospital to Guangxi University of ChineseMedicine who bought from Yulin Herb Market in unify Thepurity of each componentwas determined to be above 98byhigh-performance liquid chromatography (HPLC) analysisHPLC-grade reagents methanol acetonitrile and water wereobtained from J T Baker (Phillipsburg NJ USA)These fourherbs were dried in 24 h and grinded into coarse powderThe herbs were cooked into concentrated juice (1mL juice =22 g raw herb) The rats were given HJD juice at 54 g(kgsdotd)by gavage The control group was given 09 saline withthe same volume All the treatments were administrated bygavage for 6 successive weeks

22 Measurement of Blood Pressure Noninvasive BP of ratswas measured using the Nexfin monitor (BMEYE BV Ams-terdam andTheNetherlands) with the latest implementationof the caudal arterymethod Ratsweremeasured for BP at day0 7 14 28 and 42 after HJD treatment each rat was measured3 times to obtain average BP value

23 RNA Isolation and Microarray Experiment After thelast HJD administration all rats were sacrificed and thenthe thoracic aortas were quickly dissected and stored in thefreezer tube in minus80∘C for RNA extraction Rat tissues werehomogenized in Trizol (Invitrogen Carlsbad CA) and thetotal RNA was extracted using chloroform and isopropylalcohol and purified using the RNeasy Mini Kit and RNase-free DNase Set (Qiagen Valencia CA) according to the man-ufacturersrsquo protocols The quality of the extracted total RNAwas measured using the Agilent 2100 Bioanalyzer (AgilentTechnologies Santa Clara CA USA) and the Nano-Chip-protocol before microarray experiments Gene Chip analysiswas performed using the Agilent Whole Rat Genome OligoMicroarray Labeling and hybridization were performed atShanghai Biotechnology Corporation

24 Data Analysis Microarray data were analyzed using theSBC Analysis system (httpsasebioservicecomindexjsp)

Gene expression profiles of SHR were compared to thosefrom WKY or HJD treatment group The changing wasselected by the criteria 119875 lt 005 and Fold Changes ge 2

3 Results

31 HJD Reduced Blood Pressure of SHR Rats The rat in SHRgroup had significantly higher blood pressures compared

020406080

100120140160180200

0 7 14 28 42

ControlModelHJD

Day

lowastlowast

lowast lowast

Figure 1 Effects of HJD on blood pressure in rats BP of rat wasmeasured with mmHg unit There were 8 rats in each group lowast119875 lt005 versus SHR group

with normal control group before HJD treatment (119875 lt 005)After one week administration the BP of HJD group beganto decrease than SHR group After 6 weeks of treatment therats in HJD group had almost similar blood pressure to therats in control group (Figure 1)

32Hypertension andHJDTreatmentCausedGene ExpressionChanges Volcano plot that was drawn by 119875 value and FoldChange checked by 119905-test showed the significant differenceof sample data between chipset groups (Figures 2 and3 described by Gene-Spring 110 119883-axis for Log

2(Fold

Change) 119884-axis formdashLog10

(119875 value) 119883-axis parallel line119875 = 005 119884-axis parallel line Fold Change = 20 Red Zone119875 lt 005 and Fold Change ge 20)

Gene microarray analysis showed that there were 858genes that alter in SHR group compared with normal controlgroups (Figure 2) There were 417 downregulated (Table 1)and there were 441 upregulated (Table 2)

JHD treatment altered 96 gene expressions pared withuntreated SHR group 119875 lt 005 and Fold Change ge 20 wereidentified between the HJD treatment group and the SHRmodel groups Of these 76 genes were downregulated and 20genes were upregulated (Table 3)

33 HJD Induced Gene Changes and Their Biological Con-sequences The genes that were altered after HJD treat-ment mainly belong to 12 classifications (Table 4) includinggenes that regulate immune system cellular functions andmetabolic and developmental functionsThere were 33 genesin the category of immune system development leukocyteactivation regulation of immune system process cell activa-tion and proliferation regulation of immune system processpositive regulation of biological process and so forth (119875 lt005) (Table 5)

Further functional pathway analysis showed that hyper-tension altered genes were involved in 22 KEGG pathways


























mdash NA 20 19648 152119864 minus 03



















































7

6

5

4

3

2

1

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9

10

11

12

13

log2(fold change)

log10(P

val

ues)


6

5

4

3

2

1

minus13

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9101112131415

log2(fold change)

log10(P

val

ues)



















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































mdash NA 20 19648 152119864 minus 03



















































7

6

5

4

3

2

1

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9

10

11

12

13

log2(fold change)

log10(P

val

ues)


6

5

4

3

2

1

minus13

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9101112131415

log2(fold change)

log10(P

val

ues)



















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































7

6

5

4

3

2

1

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9

10

11

12

13

log2(fold change)

log10(P

val

ues)


6

5

4

3

2

1

minus13

minus12

minus11

minus10

minus9

minus8

minus7

minus6

minus5

minus4

minus3

minus2

minus1 0 1 2 3 4 5 6 7 8 9101112131415

log2(fold change)

log10(P

val

ues)



















Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































Acknowledgments


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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