Underlying anti-hypertensive mechanism of the Mizuhopecten yessoensis derived peptide NCW in spontaneously hypertensive rats via widely targeted kidney metabolomics

The angiotensin-converting enzyme(ACE)inhibitory peptide NCW derived from Mizuhopecten yessoensis has been demonstrated to have significant in vivo anti-hypertensive effects,however,its anti-hypertensive mechanism is still not fully clarified.This study established a UPLC-Q-TRAP-MS/MS-based widely targeted kidney metabolomics approach to explore the changes of kidney metabolic profiles and to clarify the antihypertensive mechanism of peptide NCW in spontaneously hypertensive rats(SHRs).Multivariate statistical analysis indicated that the kidney metabolic profiles were clearly separated between the SHR-NCW and SHRUntreated groups.A total of 85 metabolites were differentially regulated,and 16 metabolites were identified as potential kidney biomarkers,e.g.,3-hydroxybutyrate,malonic acid,deoxycytidine,and L-aspartic acid.The peptide NCW might regulate kidney metabolic disorder of SHRs to alleviate hypertension by suppressing inflammation and improving nitric oxide production under the regulation of linoleic acid metabolism,folate related pathways,synthesis and degradation of ketone bodies,pyrimidine metabolism,β-alanine metabolism,and retinal metabolism.

Epigallocatechin-3-gallate exerts antihypertensive effects and improves endothelial function in spontaneously hypertensive rats

Objective:To investigate the effect of epigallocatechin-3-gallate(EGCG)on endothelial dysfunction in spontaneously hypertensive rats(SHR).Methods:Wistar-Kyoto(WKY)rats and SHR were divided into four groups;WKY control,SHR control and SHR treated with EGCG(50 mg/kg/day)or losartan(10 mg/kg/day).The treatment was given daily for 4 weeks by oral gavage and the blood pressure was monitored by tail-cuff method every 3 days.Acetylcholineinduced endothelium-dependent relaxations were assessed in isolated phenylephrine-precontracted aortic rings at the end of treatment.The vascular levels of reactive oxygen species,nitric oxide,tetrahydrobiopterin,and cyclic guanosine monophosphate were also measured.Moreover,the expression of angiotensinⅡtype 1(AT_(1))receptor protein was determined.Results:The systolic blood pressure was significantly decreased in SHR treated with EGCG.The impaired endothelium-dependent relaxation was significantly improved in aortic ring isolated from the EGCG-treated SHR group.EGCG also significantly increased the levels of nitric oxide,tetrahydrobiopterin,and cyclic guanosine monophosphate,while decreasing the level of reactive oxygen species and the protein expression of AT_(1)receptor in SHR.Conclusions:EGCG attenuates endothelial dysfunction in SHR by decreasing oxidative stress and increasing vascular nitric oxide bioavailability,which may be modulated partly by inhibition of vascular AT_(1)receptors.An increase in endothelium-dependent relaxation may contribute to a decrease in blood pressure in hypertensive animals.

Effects of continuous intermedin infusion on blood pressure and hemodynamic function in spontaneously hypertensive rats

Objective To examine the effects of exogenously administered intermedin （IMD,adrenomedullin-2） on arterial blood pressure,cardiac function and the cardiovascular IMD receptor system in spontaneously hypertensive rats （SHRs） as well as to investigate the associated mechanisms.Methods Thirteen week-old male rats were divided in Wistar Kyoto （WKY） group （n =12）,SHR group （n =12）,IMD group （SHRs infused with IMD 1-47 500 ng/kg per hour,n =12）,and ADM group （SHRs infused with adrenomedullin 500 ng/kg per hour,n =12）.Results A two-week continuous administration of low dose IMD 1-47 via mini-osmotic pumps markedly reduced blood pressure,the maximal rates of increase and decrease of left-ventricle pressure development （LV ± dp/dtmax）,left ventricular systolic pressure and heart rate in SHRs.Furthermore,IMD also inhibited protein over-expression of cardiovascular IMD receptors,myocardial Receptor Activity-Modifying Proteins （RAMP1 and RAMP2）,aortic RAMP1,RAMP2,RAMP3,and calcitonin receptor-like receptor （CRLR）;suppressed up-regulation of aortic RAMP1,RAMP2,RAMP3 and CRLR gene expression; and markedly elevated the mRNA abundance of myocardial atrial natriuretic peptide （ANP） and myocardial brain natriuretic peptide （BNP）.Additionally,IMD 1-47 administration in SHRs increased aortic cAMP concentration and reduced myocardial cAMP concentration.Conclusion These findings support the speculation that IMD,as a cardiovascular active peptide,is involved in blood pressure reduction and cardiac function amelioration during hypertension.The mechanism underlying this effect may involve IMD binding of a receptor complex formed by RAMPs and CRLR,and consequential regulation of cAMP levels and other cardiovascular active factors,such as ANP and BNP.

Effects of Total Flavonoids ofHippophae RhamnoidesL.on Intracellular Free Calciumin Cultured Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats and Wistar-Kyoto Rats

To explore the effects of total flavonoids of Hippophae rhamnoides L. （TFH） quercetin （Que） and isorhamnetin （Isor） on the intracellular free calcium （[Ca^2＋]） in vascular smooth muscle cells （VSMC） of spontaneously hypertensive rats （SHR） and Wistar-Kyoto rats （WKY）. Metheds： Fluo 3-acetoxymethylester（Fluo-3/AM） was used to observe the effects of TFH （100mg/L） and its essential monomers, namely Que （10^-4mol/L） and Isor （10^-4mol/L） on changes of [Ca^2＋]1 in cultured SHR and WKY VSMC （abbr. to Ca-SHR ＆ Ca-WKY） following exposure to high K^＋, norepinephrine （NE） and angiotensin Ⅱ （AngⅡ）, and to compare with the effects of verapamil （Ver）. Results： （1） TFH, Que and Isor had inhibitory effects on resting Ca-SHR （P〈0.05）, but had no significant effects on Ca-WKY （P〉0.05）. （2） High K^＋ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）; TFH, Que and Isor could inhibit the elevation of [Ca^2＋]1 induced by high K^＋ -depolarization, with the effects similar to that of Ver, and the effect on Ca-SHR was more significant than that on Ca-WKY （P〈0.05）. （3） NE and Ang Ⅱ could increase Ca-SHR more significantly than Ca-WKY （P〈0.05）, TFH, Que and Isor had remarkably inhibitory effect on the elevation of Ca-SHR and Ca-WKY induced by NE or Ang Ⅱ. （4） In the absence of extracellular Ca^2＋ , TFH, Que and Isor also had certain inhibitory effect on Ca-SHR and Ca-WKY induced by NE, and the effect on the former was more significant than that on the latter（P〈0.05）. Ceaclusiea： TFH, Que and Isor might decrease the levels of [Ca^2＋], in VSMCs by blocking both voltage-dependent calcium channels （VDC） and receptoroperated calcium channels （ROC） in physiological or pathological state, which may be one of the important mechanisms of their hypotensive and protective effects on target organs in patients with hypertension.

The Expression of VIP and SP in the Cochlea of Spontaneously Hypertensive Rats and Its Implication

To investigate the expression of vasoactive intestinal peptide (VIP) and substance P (SP) in the cochlea of spontaneously hypertensive rat (SHR), and to assess the function of VIP and SP in the cochlea following the damage of hypertension, hearing thresholds of ABR were observed and the fixative (4% paraformaldehyde) was pumped through the circulatory system. Adult Wistar rats (3 months, n=20) served as the control group and SHRs (3 months, n=20) as the hypertension group. Bullas were taken out and cochleas were irrigated in vitro with the same fixative. The number of base turn's spiral ganglions in the sections was counted. The expression of VIP and SP were detected by SABC method and the images of the sections were analyzed. The number of base turn's spiral ganglsons in the hypertension group was significantly less than in the normal group (P<0.01). VIP and SP were expressed in the spiral ganglion cytoplasma and stria vascularis of the two groups. There were no significant difference in the expression of VIP and SP in spiral ganglion cytoplasma (P>0.05) between the two groups. However, in stria vascularis the expression of VIP in the hypertension group was higher than in the normal group (P<0.05), and no significant difference in SP was found between the two groups. It was suggested that VIP not only contributed to the regulation of the cochlea microcirculation, but also made the neurotransmitter in the pathway of the auditory system. However, SP made only the neurotransmitter in the pathway of the auditory system.

Effect of Salvia Miltiorrhiza Bge on Left Ventricular Hypertrophy and the Expression of Tumor Necrosis Factor-α in Spontaneously Hypertensive Rats

The effects of salvia miltiorrhiza Bge (SMB) on left ventricular hypertrophy (LVH) and the expression of tumor necrosis factor-α (TNF-α) in the left ventricle of spontaneously hypertensive rats and the action mechanism were investigated. Normal Wistar-kyoto (WKY) rats were used as negative control, and spontaneously hypertensive rats (SHR) were randomly assigned to receive pla- cebo or SMB. SMB (1 g/kg·d) was injected intraperitoneally for 12 weeks. Systolic blood pressure (SBP) and left ventricular mass index (LVMI) were measured. HE, VG and immunohistochemical staining combined with computed morphometry were employed to evaluate the cardiomyocyte size, diameter, the collagen volume fraction (CVF), perivascular circumferential area (PVCA), and tumor necrosis factor-α (TNF-α) expression in the left ventricular tissue. The results showed, as compared with WKY rats, the SBP, LVMI, cardiomyocyte size, diameter, CVF, PCVA, and TNF-α expression were increased markedly in the 20-week-old spontaneously hypertensive rats. SMB decreased LVMI (P<0.01), size of cardiomyocytes (P<0.01), collagen volume fraction (P<0.01), perivascular circum- ferential area (P<0.01), and TNF-α expression (P<0.01), but had no effect on SBP (P>0.05). It was suggested that chronic administration of SMB could inhibit and reverse the development of LVH in spontaneously hypertensive rats independent of BP. TNF-α may be involved in the reversal mecha- nism of LVH by SMB.

Effect of 2-Selenium Bridged β-Cyclodextrin,Glutathione Peroxidase Mimic on Stroke of Stroke-prone Spontaneously Hypertensive Rats

To investigate the treatment effect of 2-selenium bridged β -cyclodextrin(2-SeCD),a GPX mimic,on the stroke of stroke-prone spontaneously hypertensive rats(SHRSP),fifty-two SHRSP of 8-week old were randomly divided into four groups A,B,C and control group D. The rats of groups A,B,C and D were given 1.0%-1.5% NaCl mass fraction as drinking fluid. After onset of stroke,groups A,B and C were given \{orally\} 16.05,160.5 and 1605 mg·kg -1 ·day -1 of 2-SeCD,respectively,and group D was given water for \{2 weeks.\} The clinical score of stroke,systolic blood pressure(SBP),survival time of rats were recorded and the histopathologic examinations of their brain and carotid artery were made after decapitation. The clinical scores of stroke after treatment with 160.5 mg·kg -1 ·day -1 (Group B) and 1605 mg·kg -1 ·day -1 (Group C) of 2-SeCD are 2.55±0.98 and 1.98±0.79,respectively,those are obviously lower than that of group D(3.41±0.83,p<0.01). The survival days in group B(10.0±8.6) and group C(14.4±7.9) are longer than that for group D(4.7±2.9,p<0.01). The electron microscope study showed that the endothelium of carotid artery was near to normal in group B and group C,while it was seriously injured in control group D and mildly injured in group A. 2-SeCD may effectively be used to treat the stroke for SHRSP.

Telmisartan Attenuates the Growth of Epithelium-like Cells and Glomerular Injury in Spontaneously Hypertensive Rats

The abnormal growth of epithelium-like cells has been noticed in spontaneously hypertensive rats(SHRs)with hypertensive nephropathy.However,the characteristics of abnormal epithelium-like cells and their pathogenesis in hypertensive nephropathy are not fully understood.In the present study,we investigated the correlation of epithelium-like cells with glomerular injury,and the effects of early drug intervention with telmisartan,an anti-hypertensive drug,on the growth of epithelium-like cells.The results showed that the epithelium-like cells were obviously observed lining along the luminal surface of Bowman’s capsule in glomeruli,significantly resulting in the atrophy of the glomerular tuft.Some of the epithelium-like cells strongly expressed proliferating cell nuclear antigen(PCNA)and vimentin,indicating active cellular proliferation.The incidence of epithelium-like cells varied from 13.6%to 54.4%of glomeruli in 48-week-old SHRs,and from 5.1%to 18.0%of glomeruli in age-matched Wistar-Kyoto(WKY)rats(P<0.01).The linear regression analysis further confirmed an obvious correlation between the incidence of epithelium-like cells and the glomerular injury.Moreover,early intervention with telmisartan could dramatically attenuate the progression of epithelium-like cells growth.However,no significant effect of telmisartan on the established epithelium-like cells was observed.Taken together,we demonstrated the involvement of abnormal epithelium-like cells growth in glomerular injury during hypertensive nephropathy in SHRs,and firstly showed the positive effects of the anti-hypertensive drug on the progression of epithelium-like cells growth.

Effect of Salvia Miltiorrhiza on Left Ventricular Hypertrophy and Cardiac Aldosterone in Spontaneously Hypertensive Rats

Chronic treatment with Salvia Miltiorrhiza preventing left ventricular hypertrophy (L VH) and its possible mechanism- inhibiting the action of cardiac aldosterone in spontaneously hypertensive rats (SHR) were investigated.Normotensive Wistar- kyoto (WKY ) rats and SHRs were used.Part of SHRs was treated with Salvia Miltiorrhiza for 12 weeks.Systolic blood pres- sure (SBP) and left ventricular mass index were measured.Sections of heart tissue were stained with HE method and Van Gieson method.Collagen volume fraction was determined in the leftven- tricle by automatically quantitative m orphometry.Cardiac aldosterone concentration was measured by radioimm unoassay.The results indicated thatcom pared with WKY rats,SHRs exhibited high- er SBP,left ventricular collagen volume fraction,and aldosterone concentration (all P<0 .0 5 ) . After the treatm ent with Salvia Miltiorrhiza,SBP,left ventricular collagen volum e fraction,and aldosterone concentration in SHR were decreased as compared with control group (P<0 .0 5 ) ex- cept SBP.It was concluded thatchronic treatment with Salvia Miltiorrhiza could preventleftven- tricular hypertrophy in SHR,significantly inhibit collagen compositions in left ventricle.The m echanism was probably related with the inhibition of the cardiac aldosterone action.

POTENT HYPOTENSIVE EFFECTS OF ORPHANIN FQ IN CONSCIOUS STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

Orphanin FQ（OFQ） or nociceptin is a novel neuropeptide consisting of 17 amino acids. This peptide has a primary structure reminiscent of that of opioid peptide but exhibits an opposite effect to make animals hyperreactive. The effect of this new peptide on cardiovascular function are not completely known. The present study was conducted to investigate the effect of intravenous bolus injection of orphanin FQ on mean arterial blood presure （MABP） in conscious stroke-prone spontaneously hypertensive rats (SHRsp). Adult male SHRsp and Wistar normotensive rats （250～300 g body weight, 2. 5～3 months old） were used in this study. The MABP was measured in the conscious state by a tail-cuff method. In SHRsp model, intravenous bolus injection of orphanin FQ or Tyr1-orphanin FQ （0. 5 mg/kg） induced a prolonged and marked reduc- tion in MABP. The maximum changes in MABP were -30. 2±4. 2 mmHg by orphanin FQ and -28. 2± 4. 7 mmHg by Tyr1-orphanin FQ at 10 min after administration,and this effect lasted over 30 min. The Phe1→Tyr substitution in orphanin FQ was found to retain almost fully hypotensive activity. Pretreatment of SHRsp with naloxone-HCI（60 μg/kg）, 5 min before the injection of orphanin FQ, did not block the hy- potensive effect of orphanin FQ. Therefore, opioid receptors could not account for the hypotensive effect of orphanin FQ in SHRsp. In Wistar rats, intravenous bolus injection of the same dose of orphanin FQ did not cause a change in MABP. These observations suggest that orphanin FQ is a novel hypotensive peptide and may have some role in the regulation of blood pressure in SHRsp, rather than in normotensive rats. The ex-act underlying mechanisms are waiting to be clarified.

Expression of p-PPARγ in the aging thoracic aorta of spontaneously hypertensive rat and inhibitory effect of rosiglitazone

Objective:To investigate the expression of phosphorylated peroxisome proliferators-activated receptor y(p-PPARY) in the aging thoracic aorta of spontaneously hypertensive rat(SHR) and the inhibitory effect of rosiglitazone on the phosphorylation of PPART.Methods:16,32 and 64 week-old Wistar-Kyoto rats(WKY) and SHR were randomly and respectively divided into WKY,SHR and SHR+rosiglitazone group(9 in each group).The rats in SHR+rosiglitazone group were treated with rosiglitazone(5 mg/kg,intragastrically) for 56 d,whereas normal saline was applied in WKY and SHR groups.Systolic blood pressure(SBP)of rats was measured by tail cuff method.Histopathological damage of thoracic aorta was analyzed using Hematoxylin-Eosin(HE) staining.Immunohistochemical staining and western blot were performed to test the level of p-PPARY protein in the thoracic aorta arising from each group.Results:The SBP in 16,32 and 64 week-old SHR were significantly higher as compared with those in matched WKY rats(P【0.05,respectively).HE staining showed increased content of smooth muscle cell,wrinkled lining endothelium and increased thickness of internal elastic lamina in the thoracic aorta of SHR.Immunohistochemical staining and western blot indicated that the levels of p-PPARY in the thoracic aorta arising from SHR were obviously higher than those in the thoracic aorta arising from WKY rats(P【0.05,respectively).Importantly,the high SBP,histopathological abnormalities of the thoracic aorta and elevated p-PPARY expression were prominently abrogated by rosiglitazone treatment in SHR(P【0.05,respectively).Furthermore,the SBP,histopathological abnormalities of the thoracic aorta and p-PPARY expression were positively correlated with age in SHR(P【0.05,respectively).Conclusions:The PPARY phosphorylation was observed in the thoracic aorta of SHR and its expression was increased by the increase of age.Furthermore,rosiglitazone inhibited the PPARY phosphorylation and suppressed vascular aging in SHR.

Effect of Allocryptopine on Late sodium current of atrial myocytes in spontaneously hypertensive rats

Objective To explore the effect of allocryptopine （All） on the Late sodium current （INa,Late） of atrial myocytes in spontaneously hyper- tensive rats （SHR）. Method The enzyme digestion method was used to separate single atrial myocytes from SHR and Wistar-Kyoto rat （WKY） rats. INa,Late was record by patch-clamp technique and the effect of All on the current was evaluated. Results Comparing with WKY cells, markedly increasing of INa,Late current in SHR myocytes was found from 0.24 ± 0.02 pA/pF of WKY cells to 1.73± 0.04 pA/pF of SHR cells （P 〈 0.01, n = 15）. After treament with 30 μmol/L All; the current densities was reduced to 0.92 ± 0.03 pA/pF. The ratio of INa,Late/INa,peak of WKY and SHR were 0.09% ± 0.01% and 0.71% ± 0.02%, INa, Late/INa,peak of SHR was reduced to 0.37% ± 0.02% by 30 μmol/L All （P 〈 0.01, n = 15）. We also determined the effect of All on the gating mechanism of the INa,Late in the SHR cells. It was found that All decreased the INa,Late by alleviating the inactivation of the channels and increasing the window current of sodium channel. Conclusion Increased INa,Late in SHR atrial myocytes and the prolonged APD were inhibited by All coming from Chinese herb medicine.

Protective effect of sodium ferulate on cardiac hypertrophy in spontaneously hypertensive rats

OBJECTIVE To investigate the inhibitory effect and mechanism of sodium ferulate(SF)on myocardial hypertrophy in spontaneously hypertensive(SHR).METHODS Forty 14-week-old SHR male rats were randomly divided into model group(SHR,receive distilled water)and SF treatment groups(SF 20,40 and 80 mg·kg^-1 per day,respectively).Age-matched male Wistar-Kyoto(WKY)rats gavaged with distilled water served as controls.After 12 weeks of treatment,the effects of SF on cardiac hypertrophy were evaluated using echocardiographic measurement,pathological analysis and the expression of atrial natriuretic peptide(ANP),myosin heavy chainβ(β-MHC)-a gene related to myocardial hypertrophy.In order to explore the mechanism of SF on myocardial hypertrophy,the calcium-sensing receptor(CaSR),calcineurin(CaN),nuclear factor of activated T cell 3(NFAT3),phosphorylation NFAT3(p-NFAT3),zinc finger transcription factor(GATA4),phosphorylation GATA4(p-GATA4),protein kinase Cβ(PKC-β),Raf-1,extracellular regulated protein kinase 1/2(ERK 1/2),phosphorylation ERK1/2(p-ERK 1/2)and mitogen-activated protein kinase phosphatase-1(MKP-1)were detected.RESULTS The myocardial hypertrophy parameters,myocardial cell cross section area,left ventricular wall thickness and expression of ANP and β-MHC,CaSR,CaN,NFAT3,p-GATA4,PKC-β,Raf-1,and p-ERK 1/2 were significantly increased,while the left ventricular cavity was significantly smaller,expression of p-NFAT3 and MKP-1 were significantly decreased,meanwhile,the ultra⁃structure of cardiomyocytes was significantly damaged in 26-week-old SHR rats.Notably,SF significantly ameliorated myocardial hyper⁃trophy in 26-week-old SHR rats;suppressed the overexpression of ANP,β-MHC,CaSR,CaN,NFAT3,p-GATA4,PKC-β,Raf-1,and p-ERK 1/2 and increased the expression of p-NFAT3 and MKP-1.CONCLUSION SF can inhibit cardiac hypertrophy in SHR rats,and the mechanism may be related to the inhibition of CaSR mediated signaling pathway.

Mangiferin alleviates renal infammatory injury in spontaneously hypertensive rats by inhibiting MCP-1/CCR2 signaling pathway

Objective: Hypertension is a low-grade infammation state of the disease and was easily complicated by kidneys’ infammatory response. Mangiferin(MGF), a pharmacologically active compound in various plants including Mangifera indica, has a strong anti-infammatory activity. However, the effects of MGF on renal infammatory injury in spontaneously hypertensive rats(SHRs) remain unclear. The purpose of this study was to investigate the protective effects and mechanisms of MGF on renal infammatory injury in SHRs.Methods: MGF was used in SHRs at the doses of 10, 20, 40 mg/kg/d for 8 weeks consecutively. The blood and urine were collected for assessment of renal function. Renal tissues were collected for histological,immunohistochemistry, ELISA, Western blot and real time reverse transcription PCR(RT-PCR) analysis.Results: The results showed that the levels of interleukin 6(IL-6), tumor necrosis factor-a(TNF-a), monocyte chemoattractant protein-1(MCP-1) and recombinant chemokine C-C-Motif receptor 2(CCR2) were increased in SHRs, meanwhile, the level of IL-10 was decreased in SHR. Treatment of MGF inhibited the expression of IL-6, TNF-a, MCP-1 and CCR2, and promoted the expression of IL-10. Furthermore, the content of blood urea nitrogen(BUN) and serum uric acid(SUA) was significantly increased in the model group, and treatment of MGF had no obvious effects on these parameters at all dose levels.Conclusion: Our study proved that the kidneys of SHRs had significant infammatory injury, and MGF had the protective effects on renal infammatory injury in SHRs;The protective mechanism may be mediated partly by the MCP-1/CCR2 signaling pathway. Thus, it is a potential new drug for the treatment of hypertension.

Antihypertension and anti-cardiovascular remodeling by phenylalanine in spontaneously hypertensive rats: effectiveness and mechanisms

odeling · mechanismObjective To investigate mechanisms of anti hypertension and anti cardiovascular remodeling by phenylalanine (phe) in spontaneously hypertensive rats (SHRs) Methods The comparison of blood pressure (BP) increment with the ages and cardiovascular changes of SHRs was made between the 3% phe intervented group (SHR phe) and the control SHRs group Detection of the structural changes with the VIDAS digital vedio frequency processing technique and light and electron microscopy were made The cell growth and proliferation of cultured smooth muscle cells (CSMCs) of the thoracic aortas or myocardial fibroblasts were evaluated by measuring the 3 H thymidine counts per minute (cpm) incorporated into the new synthesized desoxyribonucleic acid (DNA) and determining the cell number with the crystal violet stain technique The Ca 2+ influx was measured in counts/min of 45 CaCl 2 after incubating it with 5 different concentrations of phenylalanine and the intracellular [Ca 2+ ] i by Fura Ⅱ/Am indicator The total messenger ribonucleic acid (mRNA) of the myocardium was extracted and Northern blot analysis was performed with the probe collagen α 2(Ⅰ)cDNA The tyrosine hydroxylase (TH) activity was measured by high performance liquid chromatography (HPLC) with electrochemical detector after having reacted with its substrate tyrosine and other reagents The catecholamine contents in brain homogenat were detected by HPLC method The comparison of pharmacokinetics of phenylalanine among SHR phe, SHRs and control Wistar Kyoto (WKY) rats was made after intravenous injection of 3 H L phe (1?ml/kg) by PK GRAPH Program for kinetic calculation The 3H L phe uptake by CSMCs after incubating for difinite intervals was also detected and compared Results Phenylalanine could prevent the increase of BP with ages and the heart weight (heart/body weight index) The aortic media thickness and the collagen content in the myocardium were decreased significantly in SHR phe Whereas the dearranged cardiovascular structure was much improved The mechanisms might be direct and specific inhibition of the DNA synthesis and proliferation of cardiovascular cells which may be related to the inhibition of collagen α 2(Ⅰ)cDNA, c fos and c myc expression Other mechanisms may include decrease of intracellular [Ca 2+ ] i and an inhibition of central sympathetic activity due to the results of higher TH activity in the caudate nucleus and higher adrenaline content in the posterior hypothalamus Besides, partial recovery of phenylalanine metabolic aberrants existed in SHRs seems to be another possibility for its effectiveness Conclusions Phenylalanine intervention could exert a definite anti hypertension and anti cardiovascular remodeling effects on SHRs like seen in human essential hypertension Its mechanisms might be related to direct inhibition of growth in the cardiovascular cells, decrease of central sympathetic activity, the reverse of the exhibited phenylalanine metabolic aberrants in SHRs, and a decrement of intracellular [Ca 2+ ] i

Tong-xin-luo capsule inhibits left ventricular remodeling in spontaneously hypertensive rats by enhancing PPAR-γ expression and suppressing NF-κB activity

Background Tong-xin-luo capsule （TXL）, used as a traditional Chinese herb, offeres a therapeutic potential for treatment of cardiovascular diseases. It has been shown to exert a variety of pharmacological effects, including antihypertensive effects, and is able to improve ventricular remodeling. However, the mechanisms of its action are not completely understood. The aim of this study was to evaluate the molecular mechanisms of Tong-xin-luo capsule on left ventricular remodeling in spontaneously hypertensive rats （SHR）. Methods Sixteen eight-week-old SHRs were randomized into an SHR group （n=8） and a TXL group （n=8） that were given Tong-xin-luo capsule （1.5 mg·kg^-1·d^-1）. Eight Wistar Kyoto （WKY） rats fed with 0.9% NaCl served as the control group （WKY group）. Systolic blood pressure （BP）, body weight and heart rate were monitored once every two weeks. Ventricular remodeling was detected by histopathological examination. Nuclear factor kappa B P65 （NF-κB P65） and peroxisome proliferators activated receptor y （PPAR-γ） protein and phosphorylated inhibitor kappa a （IκBα） protein were detected by immunohistochemistry and western blot respectively. The physical interaction of the P65-P50 heterodimer with IκBα and NF-κB were measured by co-immunoprecipitation. PPAR-γ mRNA, collagen Ⅰ mRNA and collagen Ⅲ mHNA were measured by real-time PCR.Results TXL inhibited NF-κB P65 expression and ventricular remodeling and suppressed the activation of NF-κB compared with the SHR group （P〈0.01, P〈0.05）. TXL reduced IκBα phosphorylation, increased expression of PPAR-γ protein and enhanced the physical interaction of the P65-P50 heterodimer with IκBα. The mRNA expression of PPAR-γ was enhanced but the mRNA expression of collagen Ⅰ mRNA and collagen Ⅲ mRNA were suppressed by TXL. Conclusions In spontaneously hypertensive rats, TXL could inhibit ventricular remodeling induced by hypertension, and the inhibitory effect might be associated with the process of TXL increasing the expression of PPAR-γ that could result in the inhibition of the activation of NF-κB.

Electrical remodeling of membrane ionic channels of hypertrophied ventricular myocytes from spontaneously hypertensive rats

To study the difference in membrane ionic currents between hypertrophied and normal myocytes and to explore the electrical remodeling of hypertrophied myocytes Methods Membrane ionic channels were studied in enzymatically dispersed spontaneously hypertensive rats (SHRs) left ventricular myocytes using the whole cell configuration of patch clamp technique, with normal Wistar rats ventricular myocytes as controls We observed depolarizing currents (sodium current, I Na ; L type calcium current, L I Ca ) and repolarizing currents (inward rectifier potassium current, I K1 ; delayed rectifier potassium current, I K; transient outward potassium current, I to ) and compared the differences between normal and hypertrophied myocytes Results The heart to body weight ratio of Wistar rats and SHRs was 3 70±0 29?mg/g and 5 66±0 46?mg/g, respectively ( P <0 001), and the mean cell membrane capacitances were 189 94±56 59?pF and 280 68±67 98?pF, respectively ( P <0 05) These differences suggest that SHRs have heart hypertrophy and hypertrophied myocytes The amplitude of L I ca of SHRs (1944±466 8?pA) was significantly greater than that of Wistar rats (1136±383 3?pA) ( P <0 001), and the current density was 6 93±1 71?pA/pF and 6 19±2 85?pA/pF respectively when normalized to cell capacitance, and the slow inactivation time constant of SHRs was significantly prolonged (56 01±13 36?ms vs 43 63±17 89?ms, P <0 001) The amplitude of I Na of SHRs (6132 5±1162 9?pA) was significantly greater than that of Wistar rats (3613 9±794 44?pA) ( P <0 001), but there was no difference when normalized to cell capacitance (24 61±6 72?pA/pF vs 24 95±6 99?pA/pF) Channel activation and inactivation time constants were also the same The amplitude of I K of SHRs (3461 5±1967 10?pA) was greater than that of Wistar rats (2302 4±893 72?pA) ( P <0 05), but there was no difference when normalized to cell capacitance (12 38±5 46 ?pA/pF vs 11 86±3 59?pA/pF) The inward portion of I K1 of SHRs was significantly lower than that of Wistar rats (11 3±2 26?pA/pF vs 14 3±3 00?pA/pF, P <0 05), but there was no difference in the outward portion (2 360±0 86?pA/pF vs 2 957±1 27? pA/pF) The current density of I to of SHRs (8 21±6 64?pA/pF) was significantly lower than that of Wistar rats (19 16±6 17?pA/pF) ( P <0 001), but channel kinetics were similar, suggesting that the reduction of I to may result from the decrease in channel number Conclusions Membrane ionic current changes of hypertrophied left ventricular myocytes in SHRs include: 1 there was an increase of L I ca , I Na and I k, but the current density was similar to that in normal myocytes, indicating that channel numbers increase as the myocytes become hypertrophied; 2 I to was small in hypertrophied ventricular myocytes and its current density was even smaller, indicating that channel numbers decrease as the myocytes enlarge The former is recognized as a physiologically compensatory change which does not lead to electrophysiological disturbance; the latter is viewed as pathological change, where the reduction of I to may lead to a repolarizing delay in myocytes, prolongation of the action potential and the occurrence of arrhythmias because of repolarizing heterogeneity Therefore, the reduction of I to in hypertrophied myocytes should be recognized as a significant or substantial change of electrical remodeling

Effect of Huanglian Jiedu Decoction on vascular endothelium factor and expression of RhoA protein factor in young spontaneously hypertensive rats

To investigate the mechanism of effect of Huanglian Jiedu Decoction(HLJD) on vascular endothelium, we assessed the protective effect of HLJD on vascular endothelium in spontaneously hypertensive rats(SHR) and the expression of RhoA in thoracic aorta. A total of 40 SHR rats were randomly and evenly divided into model group(SHR group), positive group(captopril group), HLJD high-dose group, and HLJD low-dose group. Simultaneously, 10 Wistar Kyoto rats were used in the blank group. All groups were treated by gavage for 6 weeks. The changes of nitric oxide synthase(NOS), von Willebrand factor(vWF), endothelin(ET-1) and calmodulin(CAM) in rat serum were tested by enzyme linked immunosorbent assay(ELISA) method. The expression of RhoA at the protein and mRNA levels in thoracic aorta was determined by Western blotting(WB) and quantitative real-time PCR, respectively. Compare with the blank group after 6 weeks, the levels of ET-l and VWF in serum of the model group were significantly increased(P<0.01), and the levels of NOS and CAM were significantly decreased(P<0.01). Conversely, the levels of ET-1 and vWF in the positive and HLJD groups were significantly decreased(P<0.01 or P<0.05), and the levels of NOS and CAM were significantly increased(P<0.01 or P<0.05) compared with the model group. The expression of Rho A at the mRNA and protein levels was decreased obviously(P<0.05) in HLJD high-dose group. The results shown that HLJD increased diastolic factor(CAM and NOS) in the vascular endothelial of rats, leading to reduced contraction factor(ET-1 and vWF). HLJD revealed the preventive function in the vascular endothelial dysfunction of the early stage hypertension through adjusting secretion of blood vessel endothelium(BVE) relaxing factor and improving vascular endothelial function. The mechanism might be associated with the inhibition of the activity of RhoA protein factor.

Catestatin could ameliorate proliferating changes of target organs in spontaneously hypertensive rats

Background Catestatin, a chromogranin A-derived peptide, is a potent antagonist of nicotine-evoked catecholamine release. We know that catecholamine plays an important role in cardiovascular remodeling induced by hypertension, therefore we hypothesized that catestatin would affect target-organ structure during hypertension. Methods Twelve spontaneously hypertensive rats （SHRs） were randomized to SHR control group and catestatin group, the normal control group was comprised of six healthy Wistar-Kyoto rats of the same age. Tail-cuff blood pressure and pulse rate were obtained at weeks 1, 4 and 8. At the end of the eight-week period, the heart, abdominal aorta and left kidney were excised and weighed, VG staining was done and the intima-media thickness of vessels and the collagen volume fraction were assessed by an image acquisition and analysis system. The proliferating cell nuclear antigen （PCNA） was observed by immunohistochemistry, and real time reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the mRNA levels of proliferative genes including cyclin A, ki67 and PCNA in the abdominal aorta. Results All the parameters in SHR observed in the present study increased significantly compared to Wistar Kyoto rats （P 〈0.01 ）. With intervention with catestatin, the systolic blood pressure decreased slightly but it was not significantly different from the SHR control, the cardiac mass index and left ventricular mass index both decreased significantly, the collagen volume fraction decreased by nearly 30% in the heart, by 25% in vessels and by 10% in the kidney, and the intima-media thickness and expression of proliferative genes, including cyclin A, ki67 and PCNA, in the abdominal aorta also decreased significantly. Conclusions The present study indicated that catestatin could ameliorate proliferating changes of heart, kidney and vessels during hypertension, especially to the deposition of interstitial collagen. Blood pressure was not the main factor to mediate this effect, which suggested that catestatin could become a novel protective factor for hypertensive target organs.

Inflammatory reaction versus endogenous peroxisome proliferator- activated receptors expression, re-exploring secondary organ complications of spontaneously hypertensive rats

Background The chronic pathological changes in vascular walls of hypertension may exert destructive effects on multiple organ systems. Accumulating evidence indicates that inflammatory reactions are involved in the pathological changes of hypertension. Three peroxisome proliferator-activated receptors （PPARs） have been identified： PPARa, PPARβ/δ, and PPARγ, all of which have multiple biological effects, especially the inhibition of inflammation. The aim of this study was to evaluate PPAR isoforms expression profile in important organs of spontaneously hypertensive rats （SHR） and to understand the modulation of endogenous PPAR isoforms under inflammatory condition. Methods l]ssues （kidney, liver, heart, and brain） were dissected from SHR and age-matched control Wistar-Kyoto rats （WKY） to investigate the abundance of PPAR isoforms and PPAR-responsive genes （acyI-CoA oxidase and CD36）. The expression of CCAAT/enhancer-binding protein 6 （C/EBP6）, which can trans-activate PPARγ expression, was also observed. The inflammatory response was analyzed by the expression of inflammatory mediators inducible nitric oxide synthase （iNOS）, intercellular adhesion molecule-1 （ICAM-1）, vascular cell adhesion molecule-1 （VCAM-1）, E-selectin, interleukin-1 beta （IL-1β）, and tumor necrosis factor alpha （TNFα）, and formation of carbonyl and nitrated proteins. Results The expressions of 3 PPAR isoforms and PPAR-responsive genes were markedly upregulated in SHR compared with those of WKY. Specifically, the expression of PPARa protein in the kidney, liver, heart and brain increased by 130.76 %, 91.48%, 306.24%, and 90.70%; PPARβ/δ upregulated by 109.34%, 161.98%, 137.04%, and 131.66%; PPARγ increased by 393.76%, 193.17%, 559.29%, and 591.18%. In consistent with the changes in PPARy, the expression of C/EBPδ was also dramatically elevated in SHR. Inflammatory mediators expressions were significantly increased in the most organs of SHR than WKY. As a consequence, increased formation of carbonyl and nitrated proteins were also observed in the most organs of SHR. Conclusions These findings suggest an enhanced inflammatory response in the organs of SHR, which might play a key role in pathogenesis of hypertension and secondary organ complications. Changes （increases） in PPARs expression may reflect a compensatory mechanism to the inflammatory status of hypertensive rats.