双参颗粒促进心肌梗死大鼠心肌血管生成的机制

Mechanism of Shuangshen Granules promoting myocardial angiogenesis in rats with myocardial infarction

目的探讨双参颗粒(SG)对心肌梗死大鼠心功能的保护作用及机制。方法将60只雄性无特定病原体(SPF)级SD大鼠随机分为心肌梗死模型组(MI组,n=10)、假手术组(Sham组,n=10)、SG低剂量组(SG-L组,n=10)、SG中等剂量组(SG-M组,n=10)、SG高剂量组(SG-H组,n=10)、复方丹参滴丸组(CDDP组,n=10);对大鼠进行血流动力学分析和超声心动图检查;测定血清中相关指标的含量;分离大鼠心肌组织,检测毛细血管密度,检测心肌组织中血小板内皮细胞黏附分子-1(PECAM-1/CD31)和血管内皮生长因子(VEGF)的表达水平,采用Western blot检测心肌转化生长因子-β1和Smad2/3蛋白的表达水平以及磷酸化磷脂酰肌醇3-激酶(p-P13K)、磷酸化蛋白激酶B(p-ATK)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的蛋白表达水平。结果①与Sham组比较,MI组血流动力学指标均有显著性差异(P<0.05),且经SG治疗可部分恢复;②与MI组相比,LDL-C含量只在SG-H组和CDDP组有所下降(P<0.05);③各用药组CK和CK-MB的水平与MI组相比均显著下降(P<0.05),且具有剂量依赖性;与药物治疗组相比,MI组心肌梗死面积最大,室壁更薄,心肌细胞计数更少,梗死区胶原纤维结构紊乱;与MI组(34.29%±3.86%)相比,SG-M和SG-H剂量依赖性地将心肌梗塞面积分别缩小至(15.07%±1.50%)和(14.58%±1.46%);④与MI组比较,SG-L、SG-M、SG-H、CDDP治疗组的毛细血管密度显著增加(P<0.05);⑤各用药组p-P13K、p-ATK、p-mTOR蛋白的表达水平与MI组相比明显上调(P<0.05);⑥免疫染色检测显示,相比MI组,各SG用药组和CDDP组的CD31阳性表达水平显著更高,呈剂量依赖关系(P<0.05);与MI组相比,SGM、SG-H和CDDP治疗组VEGF的IOD值显著增加(P<0.05);⑦与Sham组相比,MI组TGF-β1和Smad2/3蛋白的表达增强;与MI组相比,各用药组TGF-β1和SG-H组Smad2/3蛋白表达水平显著降低。结论双参颗粒具有促进血管生成和心肌保护作用,其可激活PI3k-Akt-mTOR信号和增加VEGF水平并具有抑制自噬等作用。

AIM To investigate the protective effect and mechanism of Shuangshen Granule(SG)on cardiac functions in rats with myocardial infarction.METHODS Sixty male SPF SD rats were randomly divided into six groups:myocardial infarction model group(MI group,n=10),sham operation group(sham group,n=10),SG low-dose group(SG-L group,n=10),SG medium-dose group(SG-M group,n=10),SG high-dose group(SG-H group,n=10)and compound salvia miltiorrhise dropping pill group(CDDP group,n=10).Hemodynamic analysis and echocardiogram examination were performed,the content of serum-related indicators was determined,rat myocardial tissue was separated,capillary density was detected and expression levels of adhesion molecule-1(PECAM-1/CD31)of platelet endothelial cells in myocardial tissue were also detected.Western blot was used to detect the expression levels of myocardial TGF-β1 and Smad2/3 proteins as well as phosphoryl inositin 3-kinase(p-P13K)and phosphorylated protein kinase B(p-A)protein expression levels of rapamycin target protein(p-mTOR)in TK and phosphorylated mammals.RESULTS Compared with Sham group,the hemodynamic indexes of MI group were significantly different(P<0.05),and could be partially recovered by SG treatment.Compared with that in MI group,the LDL-C content decreased only in SG-H and CDDP groups(P<0.05).The levels of CK and CK-MB in SG-L,SG-M,SG-H and CDDP groups decreased significantly compared with that in MI group(P<0.05)and they were dose-dependent.Compared with those in SG-L,SG-M,SG-H and CDDP groups,the myocardial infarction area was the largest in MI group,the ventricular wall was thinner,myocardial cell count was less and collagen fiber structure was disordered in the infarction area.Compared with that in MI group(34.29%±3.86%),SG-M and SG-H doses reduced the area of myocardial infarction respectively to(15.07%±1.50%)and(14.58%±1.46%).Compared with that in MI group,the capillary densities in SG-L,SG-M,SG-H and CDDP groups increased significantly(P<0.05).The expression levels of p-P13K,p-ATK and p-mTOR protein in SG-L,SG-M,SG-H and CDDP groups were significantly higher than that in MI group(P<0.05).Immune staining tests showed that CD31-positive expression levels in each SG group and CDDP group were significantly higher than that in MI group,showing a dose-dependent relationship(P<0.05).Compared with those in sham group,the expression levels of TGF-β1 and Smad2/3 proteins in MI group were enhanced,whereas the expression levels of Smad2/3 protein in SG-L,SG-M,SG-H and CDDP groups and the expression level of TGF-β1 in SG-H group were significantly reduced.CONCLUSION Shuangshen Granules can promote angiogenesis and protect myocardium,which can activate PI3k-Akt-mTOR signal,increase VEGF level and inhibit autophagy.