淫羊藿总黄酮对大鼠急性心肌梗死后缺血心肌血管新生作用的影响

Effect of Epimedii Folium Total Flavonoids on Angiogenesis of Ischemic Myocardium in Rats After Acute Myocardial Infarction

目的:研究淫羊藿总黄酮在大鼠急性心肌梗死后对缺血心肌血管新生的促进作用,探讨其在抗心肌缺血,改善心功能方面的分子生物学机制。方法:采用大鼠冠状动脉左前降支结扎的方法建立大鼠急性心肌梗死模型。雄性SD大鼠随机分为假手术组、心肌梗死模型组,地尔硫卓组(10 mg·kg-1·d-1)和淫羊藿总黄酮高、低剂量组(200,100 mg·kg-1·d-1),每组8只。模型组、假手术组均给予同体积生理盐水干预。手术成功后开始每天灌胃1次,共7 d。给药结束后,采用小动物超声影像系统检测各组大鼠心脏结构与功能;苏木素-伊红(HE)染色法观察大鼠心肌缺血区病理改变;免疫组化法检测大鼠缺血心肌组织CD31与α-平滑肌肌动蛋白(α-SMA)表达变化;蛋白免疫印迹法(Western blot)检测大鼠心肌组织血管内皮生长因子受体2(VEGF-R2)表达和蛋白激酶B磷酸化(p-Akt)水平;实时荧光定量聚合酶链式反应(Real-time PCR)法检测心肌缺血部位血管内皮生长因子(VEGF)与碱性成纤维细胞生长因子(bFGF)mRNA的表达变化。结果:与假手术组比较,模型组大鼠心脏左室收缩末期内径(LVIDs),左室舒张末期内径(LVIDd),左室收缩末期容积(LVEVs)和左室舒张末期容积(LVEVd)均显著增加,而左室前壁收缩末期厚度(LVAWs),左室前壁舒张末期厚度(LVAWd),左室后壁收缩末期厚度(LVPWs),每搏输出量(SV),射血分数(EF),短轴缩短率(FS)和心输出量(CO)显著下降,心肌缺血组织出现显著病理性改变,而CD31与α-SMA表达显著降低(P<0.01);同时p-Akt水平,VEGF-R2蛋白表达,VEGF与bFGF mRNA表达也显著下降(P<0.01)。与模型组比较,淫羊藿总黄酮高剂量组大鼠心肌缺血病理变化明显改善;心脏LVIDs,LVIDd,LVEVs和LVEVd均明显下降,而LVAWs,LVAWd,LVPWs,SV,EF,FS和CO明显增加,心肌缺血组织CD31与α-SMA表达升高(P<0.05,P<0.01);同时p-Akt水平,VEGF-R2蛋白表达以及VEGF与bFGF mRNA表达也明显增加(P<0.05,P<0.01)。结论:淫羊藿总黄酮能够通过上调急性心肌梗死后缺血心肌bFGF,VEGF及其受体VEGF-R2表达,激活磷脂酰肌醇3-激酶(PI3K)/Akt/VEGF细胞信号传导通路促使缺血心肌血管新生,从而改善心肌梗死区周边心肌有效灌注不足,减缓急性心肌梗死后心室重构和心力衰竭的发展。

Objective: To observe the effect of total flavonoids from Epimedii Folium(TEF)on the angiogenesis of ischemic myocardium in rats after acute myocardial infarction(AMI)and discuss its molecular biological mechanism of attenuating myocardial ischemia and improving cardiac function. Method:AMI in rats was induced through the ligation of left anterior descending coronary artery. All male SD rats were randomized into sham-operated group,model group,diltiazem group(10 mg·kg-1·d-1),and TEF low-dose and high-dose groups(100 and 200 mg·kg-1·d-1),with 8 rats in each group. After modeling,rats in the diltiazem group and TEF groups were given corresponding doses of diltiazem and TEF,respectively,and those in the model group and sham-operated group received normal saline of equivalent volume, once a day for 7 days. After the administration,VisualSonics Vevo2100 imaging system was used to detect the cardiac structure and function and hematoxylin-eosin(HE) staining to observe the histomorphological changes in myocardial ischemic area.Immunohistochemistry was employed to analyze the expression of CD31 and α-smooth muscle actin(α-SMA)in ischemic myocardium and Western blot to detect the expression of vascular endothelial growth factor-receptor2(VEGF-R2)and phosphorylation of protein kinase B(Akt)in ischemic myocardium. Real-time PCR was applied to quantify the mRNA levels of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF). Result: Compared with the sham-operated group,the model group demonstrated significant increase in left ventricular systolic diameter(LVIDs), left ventricular internal diameter at end-diastole(LVIDd),left ventricular end-systolic volume(LVEVs),and left ventricular end-diastolic volume(LVEVd),significant decrease in End-systolic thickness of left ventricular anterior wall(LVAWs),end-diastolic thickness of left ventricle anterior wall(LVAWd),end systolic thickness of left ventricular posterior wall(LVPWs),stroke volume(SV),ejection fraction(EF),fractional shortening(FS),and cardiac output(CO),obvious pathological changes in the ischemic myocardium,and plummet of the expression of CD31 and α-SMA(P<0.01),Akt phosphorylation level,protein level of VEGF-R2,and mRNA levels of VEGF and bFGF(P<0.05,P<0.01). High-dose TEF significantly alleviated the pathological changes of ischemic myocardium as compared with the model group. Moreover,TEF high-dose group showed significantly lower levels of LVIDs,LVIDd,LVEVs,and LVEVd,significantly higher levels of LVAWs,LVAWd,LVPWs,SV,EF,FS,and CO,higher expression of CD31 and α-SMA(P<0.05,P<0.01),and higher levels of VEGF-R2 protein,phosphorylated Akt,and VEGF and bFGF mRNA than the model group(P<0.05,P<0.01). Conclusion: TEF can effectively improve myocardial perfusion in peri-myocardial infarction area and attenuate ventricular remodeling and heart failure after AMI by up-regulating the expression of bFGF,VEGF,and VEGF-R2 in ischemic myocardium following AMI and activating phosphatidylinositol 3-kinases(PI3 K)/Akt/VEGF signaling transduction pathway which can promote angiogenesis in ischemic myocardium.