Anti-hypertensive and endothelia protective effects of Fufang Qima capsule (復(fù)方芪麻膠囊) on primary hypertension via adiponectin/adenosine monophosphate activated protein kinase pathway

ZHAO Zhiyue,SHI Zhenyu,ZHANG Zhenzhen,LI Yinghong,ZENG Xiaohui,CHEN Yuxing,YAO Nan,ZHOU Min,SU Hui,WANG Qinghai,JIN LiLi

ZHAO Zhiyue,SHI Zhenyu,ZHANG Zhenzhen,SU Hui,WANG Qinghai,JIN LiLi,Department of Cardiovascular,The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine (Guangdong Second Traditional Chinese Medicine Hospital),Guangzhou 510095,China

LI Yinghong,Department of the Electrocardiogram,The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine (Guangdong Second Traditional Chinese Medicine Hospital),Guangzhou 510095,China

ZENG Xiaohui,Department of Science and Education,The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine (Guangdong Second Traditional Chinese Medicine Hospital),Guangzhou 510095,China

CHEN Yuxing,YAO Nan,Pharmacological Laboratory,The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine (Guangdong Second Traditional Chinese Medicine Hospital),Guangzhou 510095,China

ZHOU Min,Department of Emergency,The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine(Guangdong Second Traditional Chinese Medicine Hospital),Guangzhou 510095,China

Abstract OBJECTIVE:To investigate the potential mechanism of the vascular remodeling effect and provide additional information about anti-hypertension activity of Fufang Qima capsule(復(fù)方芪麻膠囊，QM).METHODS:Spontaneous hypertensive rats (SHRs)were used to study the underlying mechanism of the anti-hypertension activity of QM.In this study,SHRs were randomly divided into 5 groups:model group,Telmisartan group (7.2 mg/kg,p.o.),and three QM groups (0.9298,1.8596,and 3.7192 g/kg,p.o.).Wistar Kyoto rats (WKY) were used as normal control group.Blood pressure(BP),aorta,perivascular adipose tissue (PVAT) histology were investigated to evaluate the effect of QM.Nitric oxide (NO)and endothelial nitric oxide synthase (eNOS) phosphorylation were measured.Adiponectin (APN) secretion,as well as APN signal pathway proteins including APN,adiponectin receptors (R1 and R2)and adenosine 5'-monophosphate-activated protein kinase(AMPK)were all analyzed.RESULTS:QM significantly reduced BP and ameliorated the vascular pathological change,i.e.intima media thicken and collagen fiber hyperplasia.Meanwhile,QM increased concentration of NO and the phosphorylation of eNOS in the aorta.The anti-hypertensive and endothelia-protective effect of QM could be attributed to activating APN/AMPK pathway by up-regulating the expression of APN in PVAT and APN Receptor 2,AMPKα and phosphorylated AMPKα in the aorta.CONCLUSION:The QM alleviation effect mechanism for primary hypertension was via modulating the APN/AMPK signal pathway.

Keywords:hypertension;adiponectin;AMP-activated protein kinases;nitric oxide synthase type III;Fufang Qima capsule

INTRODUCTION

Primary hypertension,95% patients suffering,is the most common type of hypertension.Nowadays,experts' research focusing on the primary hypertension has expanded from endothelial dysfunction to renin-angiotensin system (RAS),gene polymorphism,environment,and mentality aspects.The popularity of obesity has become the main factor of primary hypertension.Hence,researchers pay more attention to the attribution of adipose to hypertension.

Hypertension is a complex vascular disease,which mainly results from the decreased activity of NO.As one signal pathway factor,NO involved regulating the activity of the smooth muscle constriction,oxidative stress,and inflammation.1Perivascular adipose tissue(PVAT) is a kind of endocrinal adipose tissue,which has been recognized as an active contributor to vascular function involved in the control of BP.2PVAT interacts with other vascular fractions through the bidirectional information stream way including multiple active substances,i.e.adipocytokines,cytokines,reactive oxygen species (ROS).Adiponectin (APN) is one of the most influential PVAT-derived factor,which plays an important role in metabolic and cardiovascular homeostasis through insulin sensitizing action,anti-inflammatory and anti-atherogenic effects.3Thus,APN has been considered as a biomarker for hypertension and proposed as a new strategy target for checking BP.

Based on Banxia Baizhu Tianma Tang(半夏白術(shù)天麻湯)and years of clinical practice,we developed the Fufang Qima capsule(復(fù)方芪麻膠囊，QM).It has been approved and applied as a hospital dosage form since 2002 (approval assign:Yue YZBA-2002-007).QM has been used for the treatment of hypertension which is characterized by deficiency ofQiand phlegm.In our clinical publishment,it has revealed that QM mainly cure isolated systolic hypertension (ISH) by lowering systolic pressure and adjusting the ambulatory BP to the normal level.4,5The treatment mechanism may be related to the activities of vessel endothelium protection and ventricular remodeling.6,7During ISH therapy,QM can attenuate the inflammation reaction by decreasing TNF-α and IL-6 level.8,9Moreover,the Spontaneous hypertensive rats (SHRs) model studies showed that the QM activity of controlling BP can be attributed to the effect of regulating NO,endothelin and angiotensin Ⅱfactors.10,11However,the molecular mechanisms of QM anti-hypertension have not been systematically studied.To address this research gap,we hypothesized that QM would play its anti-hypertensive effectviamodulating the adiponectin/adenosine 5'-monophosphate-activated protein kinase (APN/AMPK)signal pathway.

In TCM theory,it is a consensus that"Qi"disorder together with "Xue mai" injury is the main pathogenesis of primary hypertension.It closely relates to endothelium dysfunction,smooth muscle cell proliferation and metastasis abnormal,reconstitution of the artery outer membrane matrix and vascular elasticity decreased.What's more,it has been widely accepted that APN is one of the microscopic vehicles of "Qi".APN is a key regulator for vascular dysfunction in modern medicine.12The aim of this study was to investigate the potential molecule mechanism of the vascular remodeling effect and obtained more scientific knowledge for QM anti-hypertension effect.

MATERIALS AND METHODS

Drugs and reagents

Fufang Qima capsule (復(fù)方芪麻膠囊,1.72 g crude herbs per capsule,Lot.13063)was provided by Guangdong second traditional Chinese hospital (Guangzhou,China).Telmisartan was purchased from Boehringer Ingelheim Pharma GmbH&Co.,KG(Biberach,Germany).Anti-APN,anti-adiponectin receptor 1 (Adipo R1) and anti-adiponectin receptor 2 (Adipo R2) antibodies were purchased from Abcam (Cambridge,UK).Anti-APPL1 was purchased from Proteintech (Chicago,IL,USA).Anti-AMPKα,anti-phosphorylated-AMPKα (Thr 172) and anti-phosphorylated-endothelial nitric oxide synthase(eNOS)(Ser1177)antibodies were obtained from CST (Danvers,MA,USA).Other chemicals of analytical grade were purchased locally and used as received.

Animals

SHRs [certificate No.11400700060305,14 weeks,weighing (250 ± 20) g]and Wistar-Kyoto rats [certificate No.11400700060306,13 weeks,weighing(250±20) g]both of specific pathogen free grade were obtained from Vital River Laboratory Animal Co.,Ltd.(Beijing,China).All animals were adapted for 3 d before the experiment and raised for 8 weeks under specific pathogen free condition[(24±1)℃,40%-80%relative humidity and 12/12 h light/dark cycle].All the SHRs maintained the spontaneously hypertension until the 8th week,which were confirmed over 190 mm Hg by noninvasive rat tail BP device ((MRBP system,ⅡTC Life Science,Woodland Hills,CA,USA).All animal experimental protocols were approved by the Animal Ethics Committee of Guangdong Provincial Engineering Technology Institute of Traditional Chinese Medicine.

Modeling and grouping

Ten WKY rats (5 males,5 females) were used as control group.Fifty SHRs were randomly divided into five groups by random number table method (each group 10,5 males and 5 females):the model group,the Telmisartan group (7.2 mg/kg,p.o.) and three Fufang Qima capsule (QM) groups:0.9298,1.8596 and 3.7192 g/kg doses,respectively.

Drug administration,BP measurement and tissue sampling

QM (herbs suspended in pure water) and Telmisartan were administrated to SHRs for 8 weeks,WKY rats were given equal volume distill water.After administration,all rats were fasted for 24 h and measured BP.After the eighth week,rats were sacrificed and 5 mL blood was collected from the carotid.Serum was separated by centrifuge for 10 min at 3500 rpm and stored at－80 ℃.Aorta (including the ascending aorta,aortic arch,and thoracic aorta) was dissected and washed with pre-cold PBS.The aorta (2 cm containing PVAT) was fixed in neutral 10% formaldehyde.The rest tissue was stored at－80 ℃for RNA and protein extraction.

Hematein eosin(HE)staining

The paraffin embedded aorta segments were sectioned about 4 μm slides.The representative sections were stained with HE following published protocol.13The Software Image J software (National Institutes of Health,Bethesda,MD) was applied to observe the tissue structure changes.

Serum nitric oxide(NO)and APN measurement

The concentration of NO was determined using NO assay kit (Nanjing Jiancheng Bioengineering Institute,Nanjing,China)base on the protocols of the manufacturer.Serum APN was assayed by enzyme-linked immunosorbent assay kit (Shanghai Westang Bio-Tech Co.,Ltd.,Shanghai,China) according to the instruction of the manufactures.

Real-time polymerase chain reaction(RT-PCR)

The obtained aorta tissue was homogenized in Trizol(Invitrogen,Carlsbad,CA,USA).The total RNA was extracted according to the protocol of manufacturer.RNA quantity and purity were assessed by spectrophotometric analysis (Smart Spec plus,Bio-Rad,Hercules,CA,USA).Total RNA (1 μg/μL) reverse transcription was performed using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas;Thermo Fisher Scientific,Inc.,Pittsburgh,PA,USA) according to the producer instruction.A typical 25 μL reaction mixture consisted of 12.5 μL MaximaTMSYBR Green/Fluorescein qPCR Master Mix (2×),1 μL PCR forward primer (1 μM),1 μL PCR reverse primer (1 μM),1 μL cDNA,and 9.5 μL double-distilled water.A typical protocol was used following initial denaturation at 95 ℃for 5 min,followed by 45 cycles with denaturation at 95 ℃for 30 s,annealing at 55 ℃for 40 s,and elongation at 72 ℃for 50 s.Each sample for PCR was performed in triplicate.The sequences of primer were as follows:APN:F-5'-GGACAACAATGGACTCTA-3',R-5'-GCAATACAATCAACCTCTC-3';Adipo R1:F-5'-CATCTCTGCCATCATT-3',R-5'-TACAACACCACTCAAG-3';Adipo R2:F-5'-GAACGAATGGAAGAGT-3',R-5'-GCACAGGAAGAATACA-3';AMPKα1:F-5'-GTGTGCGAAGGAAGAA-3',R-5'-CGATAGTTGCTGATGGAT-3';AMPKα2:F-5'-TTGATGATGAGGCTGTGAA-3',R-5'-CTGGAGGCGAGGTAGA-3'.

Data normalization was obtained by subtracting CT value of the 18s from the target gene.The ΔΔCtwas calculated as the difference of the normalized Ctvalue(ΔCt) of the treatment and control group samples.ΔΔCt=ΔCttreatment-ΔCt control.The comparative expression level of target genes was equal to 2-ΔΔCt.

Western blotting

APN in PVAT,Adipo R1,Adipo R2,APPL1,AMPKα,phosphorylated-AMPKα (p-AMPKα),phosphorylated-eNOS (p-eNOS) in aorta protein expression were analyzed by western blot.Tissue samples(40 mg)were homogenized in ice cold lysis buffer with phosphatase inhibitor cocktail (Thermo Scientific,Madison,WI,USA).Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis,then transferred on nitrocellulose membrane filters.After blocking 1 h with 5% bovine serum albumin (BSA) in TBST (tris-buffered saline (pH 7.6) containing 0.1%Tween 20),membranes were probed overnight at 4 ℃with primary antibody (anti-APN (1∶1000),anti-Adipo R1(1∶2000),anti-Adipo R2(1∶2000),anti-APPL1(1∶2500),anti-AMPKα (1∶2500),anti-phosphorylated AMPKα(Thr172,1∶2000),and anti-phosphorylated eNOS (Ser1177,1∶2000).The membranes were washed with TBST and incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1∶2500 dilution,Santa Cruz).The antibody-antigen complexes were visualized by enhanced chemiluminescence ChemiDoc MP (BIO-RAD Hercules,CA,USA).The density of each band was quantified by densitometry utilizing Image J software (National Institutes of Health,Bethesda,MD,USA).

Statistical analysis

The results are expressed as mean ± standard deviation(±s).Statistical analysis was performed by one-way analysis of variance using SPSS 17.0 (International Business Machines Corp.,Armonk,NY,USA).A post-hoc least significant difference test was applied to analyze the difference among the groups under homogeneity of variance.If not met,a Dunnett's test would be applied(aP<0.05 as compared to WKY group;bP<0.05 as compared to model group.A value ofP<0.05 was set as statistically significant.).

RESULTS

QM lowered the BP without affecting the bodyweight of SHRs

As shown in Supplementary Figure 1A,systolic blood pressure (SBP) of WKY rats maintained about 120 mm Hg before administration (0 week),while the SBP of SHRs kept rising over 190 mm Hg.The SBP in the Telmisartan group was significantly reduced after 1 week of administration compared with the SHRs model group(P<0.01).Three QM dose groups showed no significant difference (P >0.05) compared with the SHRs model group.After 4 weeks of administration,QM low-dose group decreased SBP obviously compared with the SHRs model group (P <0.05).After 6 weeks of administration,QM group (1.8596 g/kg dose) showed significantly lower SBP than SHRs model group (P <0.01).After 8 weeks of administration,there was no significant difference of three QM groups SBP compared with SHRs model group (P >0.05).What's more,three dose QM groups showed significantly lower SBP than before administration and QM antihypertensive effect dose-dependent is not obvious.Besides,the bodyweight (Supplementary Figure 1B) of the 6 groups showed no obvious obesity or weight loss during the experiment,which meant that QM has no impact on the animal weight.

QM ameliorated aorta pathological change

HE staining (Supplementary Figure 2A) revealed aorta histology difference between WKY and SHRs groups.As shown in Supplementary Figure 2B,the aortas of SHRs were characterized by the increasing intima media thicken (IMT) along with the hyperplastic and disorganized collagen fibers compared with WKY group.Furthermore,some smooth muscle cells (SMCs) in SHRs aorta were found to be acidophily pathological change.By contrast,Telmisartan improved the pathological features including the intima media thickening,collagen fiber hyperplasia and SMCs acidophily.QM groups (0.9298 and 1.8596 g/kg does) were beneficial on SHRs,which decreased the IMT and the collagen fibrosis.The inner diameter of aorta in SHRs group was different from WKY rats,no atherosclerosis and calcification were observed in the results(Supplementary Figure 2C).In addition,PVAT hasn't obviously changed(Supplementary Figure 3A) in each group and the size of BAT and WAT also didn't show significantly different(Supplementary Figure 3B,3C).

QM promoted the APN protein and mRNA expression

Serum APN secretion was significantly changed under primary hypertension (Supplementary Figure 4A).Compared with WKY group,APN secretion was reduced significantly to (16 ± 3) μg/mL in the model group.After treatment,serum APN in Telmisartan group was increased to (22 ± 4) μg/mL.QM groups were also increased like the WKY group,without noticeable dose dependency.In addition,mRNA and protein expression of APN in PVAT were analyzed to investigate the potential mechanism of QM increasing APN.As Supplementary Figure 4C and 4D demonstrated,the model group APN protein expression in PVAT was lower than WKY group.In comparison,both Telmisartan and QM significantly enhanced the APN protein expression in PVAT,which was consistent with the mRNA results (Supplementary Figure 4B).Protein and mRNA expression of Adipo R1,as well as Adipo R2 were down-regulated in SHRs group(Figure 1A,1D).Telmisartan could reverse the mRNA reduction of both receptors,especially Adipo R1 (Figure 1A-1C);But telmisartan has not significantly affected Adipo R1 and Adipo R2 protein expression (Figure1B,1C,1E,1F).Similarly,QM increased mRNA expression of both receptors,particularly the 0.9298 and 1.8596 g/kg dose groups(Figure 1A,1D).Overall,QM groups (0.9298 and 1.8596 g/kg dose) both increased the Adipo R1 and Adipo R2 protein expression.

QM increased the NO secretion and upregulated protein expression of p-eNOS

As revealed in Supplementary Figure 5A,concentration of NO in the model group was significantly reduced when compared with WKY group.In contrast,Telmisartan group significantly increased the serum NO content,accompanied with the p-eNOS up-regulation in the aorta (Supplementary Figure 5B,5C).QM groups,especially the 1.8596 and 0.9298 g/kg dose,could promote the NO concentration in serum and up-regulate the p-eNOS protein expression (P <0.01).As a result,QM anti-hypertension effect could be attributed to promoting NO secretion.

QM increased the AMPKα and p-AMPKα protein expression

To further investigate the anti-hypertension effect of QM,we examined the APN key downstream AMPK,which amplified its effect on vasodilatation.AMPKα and p-AMPKα in the model group were significantly reduced compared with WKY group(P <0.01).Telmisartan increased the protein AMPKα expression (P <0.01) but not significantly increased p-AMPKα protein expression (Figure 2A,2B,2D,2E).The AMPKα and p-AMPKα mRNA expression in Telmisartan group significantly increased (Figure 2C,2F).By contrast,all QM groups clearly showed the increased effect of AMPKα and p-AMPKα (Figure 2B,2D),particularly 1.8596 and 0.9298 g/kg dose obviously observed (P <0.01).Moreover,mRNA expression of AMPKα1 and AMPKα2 exhibited a significant increasing trend(P <0.05) after different treatment groups (Figure 2C,2F).Compared with the WKY group,the APPL1 expression (Supplementary Figure 6A,6B) in the model group was significantly reduced (P <0.01).QM showed a slight up-regulation of APPL1 expression.

Figure 1 Effect of QM on protein and mRNA expression of adiponecin receptors

Figure 2 Influence of QM on expression AMPKα and phosphorylated AMPKα

DISCUSSION

The theory about adipose tissue has becoming a hotspot.Base on this view,we proposed the potential mechanism of QM anti-hypertensive effect.PVAT tightly adheres to the outer membrane of the vascular.More and more evidence confirms that PVAT plays a key role in the regulation of vascular function.14Because it can peri-secrete the PVAT-derived relaxing factors (PVRF) into the vessel to control the vascular.15,16As early as 1991,Soltiset al17have found that PVAT could antagonist the vasoconstriction by adrenaline and noradrenaline.Later,Lohnet al18proved that one relaxation factor derived from PVAT could dilate the vesselviaactivating the tyrosine kinase dependent K+channel in the SMCs.On one hand,the morphology and function of PVAT would be obviously changed under hypertension.The PVAT anti-constriction effect on vessel would reduce when the people were suffering from hypertension.19It could be attributed to these reasons:decreasing the size of PVAT,macrophage infiltration,reducing K+channel expression in the vessel smooth muscle,etc.20As our study showed,QM could decrease the BP in the SHRs after 8 weeks of administration,reduce the intima media thicken and improve collagen fibrosis.However,the size of BAT and WAT in all the SHRs groups were not significantly different from the WKY group,so we inferred that the size of BAT and WAT was not obviously related to the PVAT anti-hypertension function.

APN is considered as a key PVAT factor that is closely associated with various risk factors for hypertension.APN is encoded by APM1 locating on chromosome 3q27,which is closely related to the sensitivity of type 2 diabetes and cardiovascular diseases.21APN would increase if the renin-angiotensin system was blocked in essential hypertension patients.22Meanwhile,APN could suppress the sympathetic nervous system to reverse the vasoconstriction via activating the central nervous system.It has been found that APN could effectively promote the NO secretion and the subsequent anti-inflammation activity to maintain endothelium function in an APN deficient mice model.23,24Moreover,APN not only facilitated the eNOS activity and resulting NO secretionviaimproving the stability of eNOS mRNA and promoting the phosphorylation of eNOS at Ser1177 site,but also enhanced the interaction between eNOS and heat-shock protein 90(hsp90).25-27Hence,the APN decreasing was considered as an unfavorable indicator for primary hypertension.28According to our current work,QM increased APN secretion,promoted APN protein and mRNA expression.Meanwhile,it was observed that QM treatment groups could significantly increase the NO content,as well as elevated the phosphorylation of eNOS protein expression.The QM anti-hypertension effect on primary hypertension could be attributed to regulation of the APN and NO secretion rather than the structure and composition remodeling of PVAT.Actually,the APN exists in the blood as the full-length APN form(fAPN)and spherical APN form(gAPN).29,30

APN exerts its amelioration on vascular dysfunction in various vasculatures and anti-diabetic effects on insulin-sensitive organs when it binds to the two distinct APN receptors and acts the downstream protein APPL1 (adaptor protein containing pleckstrin homology domain,phosphotyrosine-binding domain and leucine zipper motif 1)viaLBK1 and AMPK signal pathway.31,32So far,three specific APN receptors (the AdipoR1,the AdipoR2 and the T-cadherin) have been found.However,researchers paid more attention to AdipoR1 and AdipoR2 than T-cadherin because T-cadherin was more than one binding ligand.In vitroandin vivoexperiments,the results demonstrated AdipoR1 and AdipoR2,as critical mediators for adiponectin signal,in which they increased AMPK activity,the fatty-acid oxidation and glucose uptake.33,34However,AdipoR1 and AdipoR2 are quite different in terms of distribution and affinity to APN.For instance,AdipoR1 is more ubiquitously expressed and enriched in skeletal muscle,testis,and adipose,and has a stronger affinity to gAPN.Whereas AdipoR2 is mainly expressed in the liver and tends to bind to fAPN.35,36In this study,QM reversed APN serum secretion decreasing via AdipoR2 protein and mRNA expression.So QM anti-hypertension effect could attribute to promoting the APN and its receptor binding.

APPL1 is an adaptor molecule with a pleckstrin homology domain and a phosphotyrosine binding domain.Due to its ubiquitous expression,APPL1 promotes the widespread adiponectin stimulated AMPK activation,glucose uptake and β-oxidation.APPL1 also be involved in the crosstalk between adiponectin and insulin signal transduction cascade.37,38One key molecular involved in APN signaling pathway is AMPK.39,40Besides the role as a metabolic master switch that regulates several intracellular systems,AMPK also is a critical regulator of vascular homeostasis and media the phosphorylation downstream of APN signal.41,42QM had a slight up-regulation of APPL1 expression effect,which could be verified by further study in the future.APN in QM groups increased and accompanied with a remarkable increasing of AMPKα and p-AMPKα.These results may provide evidence and research ideas for the QM treatment mechanism of primary hypertensionviaenhancing the APN and its receptor binding and activating the AMPK involved APN signal pathway.

In conclusion,taken together,it was rational to propose that the QM achieved the treatment for primary hypertensionviamodulating the APN/AMPK signal pathway.QM improved the secretion of APN in PVAT and promoted phosphorylation of AMPKα,thereby stimulated the phosphorylation of eNOS as well as the NO production.Due to the NO vasodilation,QM decreased BP and improved the vascular pathological including intima media thicken and collagen fibrosis.As a result,QM brought benefits to the patients suffering from primary hypertension.Moreover,this study would help us to understand the risk factors,such as RAS activity and pre-inflammatory cytokines in clinical patients.