Experimental Study On Shumai Capsule On Stimulating Angiogenesis, Attenuating Left Ventricular Remodeling In Rats With Myocardial Ischemia

Introduction:Ischemic heart disease is one of the leading causes of morbidity and afflicts more than hundreds of millions worldwide.It's necessary to improve regional myocardial blood flow in prevention and treatment of ischemic heart disease.Theraputic angiogenesis can stimulate the formation of bypass circuit,and improve ischemic injury and has been the hot issue of cardialvascular research.Herbal medicines have been shown to exhibit numerous biological activities;however, compared to synthesized chemical medicines,systematic scientific evidence and proof of efficacy and safety are generally lacking.The underlying mechanisms are largely unclear. Therefore,it is important to do systematic research to assess the actions of herb medicines. Ischemic heart disease is one of the leading causes of morbidity and mortality worldwide. Myocardial ischemia(MI) subsequently contributes to angina,myocardial infarction,heart failure and death.So it is important to improve myocardial perfusion and function after MI.Shu-Mai Capsule(SMC) is one of the most frequently used Chinese prescriptions in traditional Chinese medicine(TCM) clinical practice for the treatment of ischemic heart disease.According to the concepts of traditional Chinese medicine,SMC consists of 7 medicinal compositions.Astragaloside,Salvianolic acid,Sanchinoside,and Ginsenoside Rg1 were identified as the major effective constituents.SMC,a prepared compound of traditional Chinese medicine,has been used as an anti-ischemic agent for years in clinic.In the present study,the changes of the pathology of ischemic myocardium were observed in myocardial ischemic(MI) rats,and the variation of VEGF and PDGF-BB mRNA and protein expression was detected by real-time RT-PCR and Western blot,and the variation of PI3K,p-Akt was detected by Western blot.In order to discover novel agents on stimulating stable collaterogenesis and improving cardiac function,the possible mechanism of SMC on stimulating angiogenesis and arteriogenesis in the ischemic myocardium was investigated,which could provide scientific evidence for the treatment of ischemic heart disease in clinic.Aim1.To investigate the effects of SMC on angiogenesis,arteriogenesis through observing the expression von Willebrand factor(vWF) andα-smooth muscle actin(α-SMA),and detecting the cardiac function in rats with myocardial ischemia(MI)..2.To investigate the potential mechanism of SMC on stimulating angiogenesis, arteriogenesis in myocardial ischemia rat models through observing the phosphorylation of PI3K/Akt and detecting the expression of vascular endothelial growth factor(VEGF), platelet derived growth factor(PDGF-BB) in the ischemical myocardium.MethodsAll animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001) and the guidelines for the Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine,China.Wistar rats weighing 230-290 g were purchased from the Animal Center of Shandong University(Shandong,Jinan).Rats were housed in temperature-(22±2℃) and humidity-(55±5%) controlled rooms with a 12/12-hr light/dark cycle.Solid rodent chow and tap water were given freely.Animals were allowed to acclimatize for 1 week before entry into the experimental protocol.Then they were intraperitoneally anesthetized by use of 10%chloral hydrate(300 mg/kg) and ventilated with a VIP Bird ventilator(tidal volume,3.0 ml;respiratory rate,60 cycles/min).The MI model was created by ligation of the left anterior descending coronary artery approximately 3 mm distal from its origin with use of a 6-0 polypropylene suture.Twenty four rats were randomly chosen as the sham-operated group,which underwent a similar surgical procedure but without coronary artery ligation.The rats with MI were randomly divided into five groups of 24 rats,including high- and low-dose SMC(SMCH and SMCL),high-dose SMC and PI3K inhibitor LY294002(SMCH/LY),bFGF,and myocardial ischemia rat models(MIR).We also include the sham operated rats(Sham).SMCL rats received low doses of SMC(342mg/kg; equal to six fold the clinic dosage),SMCH rats received high doses(1.71 g mg/kg;equal to 30-fold the clinic dosage),and MIR rats received an equal volume of distilled water as did Sham rats for 2(n=12 rats/group) and 4(n=12 rats/group) weeks.LY294002 (phosphatidylinositol 3-kinase(PI3K) inhibitor) was dissolved in sterile double H₂O and given at the indicated dose(0.3mg/kg).The rats received the LY294002 via intraperitoneal administration once every three days for 2 and 4 weeks.After the last administration,all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 10%chloral hydrate(300 mg/kg) which was followed by other experiments.The detection contents listed below:(1) Echocardiographic Analysis;(2) Determination of the levels of VEGF,PDGF-BB,PI3K, p-Akt in the ischemic myocardium with western blot;(3) Analysis of VEGF and PDGF-BB mRNA expression by real-time RT-PCR;(4) Determination of the myocardial microvessel density(MVD)(angiogenesis:expression of vWF;arteriogenesis:expression ofα-SMA); (5) Determination of the area of fibrosis in the ischemic myocardium by Masson stainning.Statistical AnalysisAll values are expressed as means±SD.Data analysis involved use of SPSS v11.5 (SPSS;Chicago,IL).Intergroup differences were analyzed by one-way ANOVA followed by the Bonferroni-corrected post-hoc analysis for multiple comparisons.Data with non-normal distribution were analyzed by nonparametric statistics.A P＜0.05 was considered significant.Results1.Echocardiographic AnalysisAt week 2 after treatment,a significant improvement of LVEF was observed in the SMC-treated groups(SMCH and SMCL) and bFGF group as compared with the MIR group(P＜0.05).There was no significant difference between SMCH and SMCL or bFGF group(P＞0.05).LVEDD and LVESD did not differ significantly between the SMCH/LY group and MIR group(P＞0.05),and were higher than those in the Sham group(P＜0.05). Taken together,the early improvement indicated that SMC stimulation rescued cardiac structure and function after injury.To determine whether SMC treatment has a long-term effect,we performed an additional and separate blinded and randomized study and treated the rats for 4 weeks.SMC therapy increased LVEF and decreased LVEDD in a dose dependent manner as compared to the MIR(P＜0.05).There was no significant difference between the SMCH and bFGF(P＞0.05).These findings demonstrate that SMC therapy remarkably stimulates myocardial collaterogenesis,leading to a significant improvement of regional as well as global myocardial contractile functions.2.Western blot analysesVEGF plays important role in angiogenesis because of its ability to stimulate vessel hyperpermeability,endothelial-cell proliferation,migration and capillary formation. PDGF-BB,are important arteriogenic factors for stabilization of the newly formed vasculature.To investigate whether SMC on stimulates stable collaterogenesis through high induction of VEGF and PDGF-BB via PI3K/Akt pathway,we determine the expression phosphorylation of Akt(p-Akt),PI3K,VEGF,and PDGF-BB in the myocardium.At week 2,p-Akt,PI3K,VEGF,and PDGF-BB protein expression were significantly increased in the SMCH,SMCL,and bFGF as compared with the MIR(P＜0.05),and with little detectable p-Akt,PI3K,VEGF,and PDGF-BB levels in the Sham rats.Administration of PI3K inhibitor L294002 blocked SMC-induced VEGF and PDGF-BB production,and inhibited the phosphorylation of Akt.At week 4,p-Akt,PI3K, VEGF,and PDGF-BB protein expression were significantly increased in the SMC-treated groups as dose dependence as compared with the MIR(P＜0.05),and with little detectable p-Akt,PI3K,VEGF,and PDGF-BB levels in the Sham rats.There was no significant difference between the SMCH group and bFGF group(P＞0.05).Administration of PI3K inhibitor L294002 blocked SMC-induced VEGF and PDGF-BB production,and inhibited the phosphorylation of Akt.These data showed that SMC could promote VEGF and PDGF-BB biosynthesis as dose dependence in the ischemic myocardium via PI3K/Akt signal pathway.3.Comparison of expression of VEGF and PDGF-BB in the myocardium by real time RT-PCR At week 2,the expression of VEGF and PDGF-BB mRNA was increased in the MIR group than that in the Sham group(P＜0.05).The expression of VEGF and PDGF-BB mRNA was increased significantly in the SMCH group,the SMCL group and the bFGF group as compared with that in the MIR group or SMCH/LY group(all P＜0.05).There was no significant difference between the SMCH group and the bFGF group or the SMCL group(P＞0.05).At week 4,the expression of VEGF and PDGF-BB mRNA in the SMCL group was lower than that in the SMCH group(P＜0.05).There was no significant difference between the SMCH group and the bFGF group(P＞0.05).The expression of VEGF and PDGF-BB mRNA in the SMCH at week 4 was slightly downregulated as compared with that at week 2.4.Determination of the myocardial capillaries and arterioles densityQuantitative analysis confirmed that capillary density stained for the presence of vWF in the ischemic border zone was significantly higher in the SMCH(36.93±5.49),SMCL, and bFGF groups than that in the SMCH/LY together treated group(15.13±5.11),MIR (17.76±5.19) and Sham groups(9.44±2.68) at week 2(P＜0.05).Capillary density was higher in the SMCH,SMCL,and bFGF at week 4 than that at week 2 but was significantly lower in the MIR and SMCH/LY.Additionally,the sections were stained for the presence ofα-SMA,a marker expressed in both the pericytes and the smooth muscle cells of mature vessels(arterioles).Of course, vascular smooth muscle cells(VSMCs) are necessary for vascular maturation and arteriogenesis,which is the process by which capillaries acquire a VSMC coat to become arterioles with the ability to regulate blood flow.The formation of mature vessels was thought to be more important than capillary formation in restoring functional blood flow. The mature vessels index was significantly higher in the SMC-treated groups and bFGF group as compared with the SMCH/LY together treated group.Long-term follow-up analysis(week 4) showed that the collateral index continued to increase and remained stable in the SMC-treated groups(dose dependent) and bFGF group. Administration of PI3K inhibitor LY294002 blocked SMC-mediated angiogenesis and arteriogegesis.It suggested that high production of VEGF and PDGF-BB mediated by SMC promotes formation of capillaries and mature vessels via the PI3K/Akt pathway. 5.Myocardial collagen depositionAt week 2,the MIR group showed many newly formed collagen deposits(Red) between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the Sham group.Myocardial collagen deposition was greater in the SMCH, SMCL and bFGF than in the Sham group(P＜0.05),but was less than that in the MIR group (P＜0.05).There was no significant difference between the SMCH/LY group and MIR group.The Sham myocardium showed a normal array of myocardial fibers and very little interstitial collagen in myocardium.At week 4,the area of fibrosis in the SMCL was higher than the SMCH and lower than the MIR.There was no significant difference between the SMCH and bFGF.Conclusion1.The findings of the current study illustrate for the first time that treatment with SMC induces significantly increase of capillaries and arterioles in a dose-dependent manner in the ischemic myocardium,which lead to functional improvement such as promoting LVEF,decresing LVESD and LVEDD.To improve the cardiac function is the aim for SMC to protect the ischemic myocardium.2.SMC can stimulate the expression of VEGF and PDGF-BB,and enhance the phosphorylation of PI3K and Akt as compared with the MIR group,which indicated that SMC stimulated angiogenesis,arteriogenesis through upregulating the phosphorylation of PI3K,Akt,and the expression of VEGF and PDGF-BB.3.PI3K inhibition(LY294002) significant inhibits the activity of PI3K/Akt signaling pathway,and also decrease the effect of SMC on stimulating angiogenesis and arteriogenesis.The mechanism is the therapeutic effect of SMC on promoting VEGF and PDGF-BB-mediated angiogenesis,arteriogenesis,and stabilization of myocardial collateral networks through the PI3K/Akt signal pathway. IntroductionPersistent remodeling contributes to functional decompensation and,eventually,heart failure and even death.Cardiac fibrosis is an important factor in the process of LV remodeling after MI.Thus,it is crucial to improve regional myocardial blood flow and attenuate LV myocardial fibrosis in ischemic tissues.Pro-inflammatory cytokines play important roles in the pathogenesis and pathophysiology of ischemic heart diseases.In both patients and experimental animal models,the induction of proinflammatory cytokines is closely associated with myocardial fibrosis and the onset and progression of cardiac remodeling.Among the pro-inflammatory cytokines,tumor necrosis factor-alpha(TNF-α) is considered to be the predominant cytokine to induce interstitial fibrosis.Findings in TNFR null mice suggest that TNF-αnormally account for the up-regulation of tissue inhibitor of matrix metalloproteinase (TIMP)-1 and may promote fibrosis by inhibiting collagen degradation.TNF-αalso is responsible for the proliferation of fibroblasts.Given the key role of TNFαas a mediator of myocardial fibrosis and the fact that fibrosis is a serious problem in left ventricular(LV) remodeling,inhibiting the induction of TNF-αis important on attenuation of myocardial fibrosis.In clinical and animal studies,elevated plasma and cardiac levels of TNF-αwere linked to interstitial fibrosis and ventricular remodeling.Inflammatory stimuli activate many intracellular signaling pathways,including the nuclear factor-κB pathway and three MAPK pathways mediated through extracellular-signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK) and p38 MAPK,with p38 MAPK considered a central regulator of inflammation.Treatment with the selective p38 inhibitor(SB203580[SB])reduced p38 MAPK activity in transgenic hearts,blocked TNF-αsecretion,and attenuated extracellular matrix(ECM) remodeling.Some studies have provided direct in vivo and in vitro evidence that a stress-activated mitogen-activated protein kinase(p38 MAPK) is activated in hearts under ischemia and other pathological conditions.The activation of p38 MAPK is a critical regulator of inflammatory response and is sufficient to induce the release of TNF-αin cardiomyocytes with significant contribution to marked myocardial fibrosis and pathological remodeling. Administration of the selective p38 MAPK inhibitor blocks the secretion of TNF-α,which reduces myocardial fibrosis and attenuates cardiac remodeling by reversing the balance between matrix metalloproteinases(MMPs) activity and tissue inhibitor of metalloproteinase(TIMP)-1 expression.This leads us to propose that p38-mediated TNF-αinduction represents a potential signaling mechanism that contributes to myocardial fibrosis and progressive LV remodeling.Evidence gathered from a systematic review shows that herbal medicines,which seem to be relatively safe and convenient,may offer a much-needed alternative.Some herbal medicines have been proved to be effective on anti-inflammation.However,limited data are available about the efficacy and cost-effectiveness of complex prescriptions of herbal medicine on anti-inflammatory cytokines-induced myocardial fibrosis and LV remodeling. SMC-an extract of traditional Chinese medicine(TCM),having been used for symptomatic treatment of MI in clinic for years,is a modified herbal drug based on a traditional Chinese hepatotherapeutic formula.The aim of this study was to investigate the efficacy of SMC on anti-inflammatory cytokine TNF-α-induced myocardial fibrosis and LV remodeling in MI rats,as well as the potential mechanism.Aim1.To investigate the effects of Shu-Mai Capsule(SMC) on attenuating left ventricular (LV) remodeling and myocardial fibrosis through detecting the cardiac function and observing the proliferation of CFs(through detecting the expression ofα-SMA ) and the expression collagenâ… in the ischemical myocardium in rats with myocardial ischemia (MI). 2.To investigate the potential mechanism of SMC on attenuating LV remodeling in myocardial ischemia rat models through observing the changes of the morphology and the ultrastructure,the phosphorylation of a stress-activatedmitogen-activated protein kinase (p-p38 MAPK),and detecting the expression of tumor necrosis factor-alpha(TNF-α), tissue inhibitor of metalloproteinase(TIMP)-1 in the ischemical myocardium.MethodsAll animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001) and the guidelines for the Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine,China.Wistar rats weighing 230-290 g were purchased from the Animal Center of Shandong University(Shandong,Jinan).Rats were housed in temperature-(22±2℃) and humidity-(55±5%) controlled rooms with a 12/12-hr light/dark cycle.Solid rodent chow and tap water were given freely.Animals were allowed to acclimatize for 1 week before entry into the experimental protocol.Then they were intraperitoneally anesthetized by use of 10%chloral hydrate(300 mg/kg) and ventilated with a VIP Bird ventilator(Bird Products Corp.;Palm Springs,CA)(tidal volume,3.0 ml; respiratory rate,60 cycles/min).The MI model was created by ligation of the left anterior descending coronary artery approximately 3 mm distal from its origin with use of a 6-0 polypropylene suture.Twenty rats were randomly chosen as the sham-operated group, which underwent a similar surgical procedure but without coronary artery ligation.The rats with MI were randomly divided into four groups of 24 rats,including highand low-dose SMC(SMCH and SMCL),p38 MAPK inhibitor SB203580(SB),and myocardial ischemia rat models(MIR).We also include the sham operated rats(Sham). SMCL rats received low doses of SMC(342mg/kg;equal to six fold the clinic dosage), SMCH rats received high doses(1.71 g/kg;equal to 30-fold the clinic dosage),and MIR rats received an equal volume of distilled water as did Sham rats.SB203580[p38 mitogen-activated protein kinase(p38 MAPK) inhibitor]was dissolved in sterile double H₂O and given at the indicated dose(2 mg/kg body weight).The rats received the SB203580 via intraperitoneal administration once every three days for 1 and 4 weeks. These five groups were further divided into 2 groups each(n=12 each) for 1- and 6-week treatment,respectively.After the last administration,all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 10%chloral hydrate(300 mg/kg) which was followed by other experiments.The detection contents listed below:(1) Echocardiographic analysis;(2) Determination of the levels of TNF-αby radioimmunoassay(RIA) assay;(3) Determination of the levels of p38 MAPK,TNF-α,TIMP-1 protein with western blot;(4) Analysis of collagenâ… mRNA expression by real-time RT-PCR;(5) Determination of the protein expression of collagenâ… ,α-SMA with immunohistochemistry assay;(6) Determination of the area of fibrosis in the ischemic myocardium;(7) Ultrastructural observation and Transmission Electron Microscope(TCM) study.Statistical AnalysisAll values are expressed as means±SD.Data analysis involved use of SPSS v11.5 (SPSS;Chicago,IL).Intergroup differences were analyzed by one-way ANOVA followed by the Bonferroni-corrected post-hoc analysis for multiple comparisons.Data with non-normal distribution were analyzed by nonparametric statistics.A P＜0.05 was considered significant.Results1.Eehocardiographie analysisAt week 1 after treatment,a significant improvement of LVEF was observed in the SMC-treated groups(SMCH and SMCL) and SB group as compared with the MIR group. There was no significant difference between SMCH and SMCL or SB group.LVEDD and LVESD were higher in the MIR group than those in the Sham group.Taken together,the early improvement indicated that SMC stimulation rescued cardiac structure and function after injury.To determine whether SMC treatment has a long-term effect,we performed an additional and separate blinded and randomized study and treated the rats for 6 weeks. SMC therapy increased LVEF and decreased LVEDD in a dose-dependent manner as compared to the MIR(P＜0.05).There was no significant difference between the SMCH and SB.These findings demonstrate that SMC therapy remarkably attenuated left ventricular remodeling,leading to a significant improvement of regional as well as global myocardial contractile functions.2.Inhibition effects of SMC on serum TNF-αlevelAnti-inflammation activity of SMC was investigated by radioimmunoassay.At week 1 and 6 week,the level of TNF-αin the MIR,SMCL,SMCH,and SB group was higher than that in the Sham group(P＜0.05).However,the serum level of TNF-αin the SMC-treated groups dropped gradually and a significant decrease was found at both 1 and 6 weeks in a dose-dependent manner as compared with the MIR(P＜0.05).No significant difference was shown between the SMCH and the SB group at both week 1 and 6.3.Western blot analysisThe p38 mitogen-activated protein kinase(MAPK) has been paid much attention, which mediates inflammatory cytokines induction and is associated with cardiac remodeling and contractile dysfunction.In TNFα,acting primarily via TNFR2,promotes fibrosis by stimulating myofibroblast proliferation and reducing collagen degradation by inducing TIMP-1.To investigate whether SMC on attenuating left ventricular remodeling through downregulation of TNFαand TIMP-1 via p38 MAPK pathway,we determine the expression of phosphorylation of p38 MAPK(p-p38),TNF-α,and TIMP-1 in the myocardium at both week 1 and 6.p-p38 MAPK,TNF-α,and TIMP-1 protein expression were significantly decreased in the SMC-treated rats in a dose dependent manner and SB group as compared with the MIR group,and with little detectable p-p38 MAPK,TNF-α, and TIMP-1 levels in the Sham rats at both week 1 and 6.Administration of SMC significant inhibited the phosphorylation of p-38 MAPK as the p38 MAPK inhibitor SB203580.These data showed that SMC could reduce TNF-αand TIMP-1 biosynthesis in ischemic myocardium via the inhibiting the p38 MAPK signal pathway.4.Analysis of collagenâ… mRNA expression by real-time RT-PCRAt week 1,the expression of collagenâ… was increased in the MIR group than that in the Sham group(P＜0.05).The expression of collagenâ… mRNA was decreased significantly in the SMCH group,the SMCL group and the SB group as compared with that in the MIR group(all P＜0.05).There was no significant difference between the SMCH group and the SB group or the SMCL group(P＞0.05).At week 6,the level of collagenâ… was higher in the MIR group than all the other groups.The expression of collagenâ… in the SMCL group was higher in the SMCL than that in the SMCH group.There was no significant difference between the SMCH group and the SB group(all P＞0.05).The expression of collagenâ… mRNA in the all groups except for the Sham group at week 6 was slightly downregulated as compared with that at week 1.5.Expression ofα-SMA and and Collagenâ… protein by immunohistochemistryα-SMA was detected within the media of the arteries and arterioles in the normal myocardium of the Sham group.An increased accumulation ofα-SMA-positive cells was also observed in the ischemic myocardium of the MIR group.SMC and SB treatment decreased the accumulation ofα-SMA positive cells in the myocardium,which indicated that number of fibroblasts in the MIR were higher than the SMC-treated groups(in a dose dependent manner),and SB at both week 1 and 6.No significant difference was shown between the SMCH and SB(p38 MAPK inhibitor SB203580) group.Collagenâ… was detected widely in extracellular matrix in the MIR group at week 6.However,the expression of collagenâ… was attenuated in the SMC-treated groups in a dose dependent manner and the SB group.6.Myocardial fibrosisThe MIR group showed many newly formed collagen deposits(Red) between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the Sham group.The Sham myocardium showed a normal array of myocardial fibers and very little interstitial collagen in myocardium.Myocardial collagen deposition was greater in the SMC-treated group and the SB group than in the Sham group(P＜0.05),but was less than that in the MIR group at both week 1 and 6(P＜0.05).7.Ultrastruetural observation and transmission electron microscope studyStructural studies using transmission electron microscope(TEM) were performed. Cardiac muscle fibers in the Sham group were abundant,with regular arrays of myofibrils closely arranged within the sarcomere and normal size of the mitochondria with normal numbers in myocardium.In the MIR group,the structure of mitochondria was damaged seriously.There were apparent cellular and tissue swelling.Most myofibrils were either disappeared or disorganized.Mitochondria were swollen obviously and loosely arranged. Mitochondrial membranes were vague or partly ruptured and cristae were obviously loose and dissolved,a lot of vacuoluses were formed.A significant ultrastructural organization change in mitochondria and myofibrils were ameliorated in the SMC-treated groups and the SB group.ConclusionIn summary,the findings of the current study illustrate for the first time that SMC therapy showed benefits in preservation of cardiac structure and function in the MI rat model.The mechanism is that SMC improves cardiac function through attenuating inflammatory-induced LV myocardial fibrosis after MI.It attenuates inflammatory cytokine TNF-α-induced myocardial fibrosis via inhibiting the p38 MAPK pathway and downregulated the expression of TIMP-1 andα-SMA.