五参顺脉胶囊通过自噬改善心肌缺血再灌注损伤

Wushen-Shunmai capsule improves myocardial ischemia-reperfusion injury through autophagy

目的探讨自噬在五参顺脉胶囊改善大鼠心肌缺血再灌注损伤中的作用。方法建立心肌缺血再灌注损伤模型,随机法,将其分为假手术组、模型组、五参顺脉胶囊组、自噬抑制剂组(五参顺脉胶囊+LY294002)每组15只。酶联免疫吸附试验(ELISA)检测血清心肌肌钙蛋白I(cTnI)、肌酸激酶同工酶MB(CK-MB)浓度2,3,5-氯化三苯基四氮唑(TTC)染色测定心肌梗死体积,原位末端标记(TUNEL)法检测心肌细胞凋亡,电镜下计数大鼠心肌细胞自噬体数量,Western blot检测总PI3K、Akt、哺乳动物雷帕霉素靶蛋白(mTOR)及其磷酸化蛋白表达,自噬标记物微管相关蛋白1轻链3(LC3)蛋白表达。结果与假手术组比较,模型组大鼠血清cTnI、CK-MB浓度、心肌梗死区比例、凋亡指数、自噬体数量、心肌组织LC3-Ⅱ/LC3-Ⅰ、caspase3、Bax增加(P<0.05),心肌组织p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、Bcl2水平降低(P<0.05);与模型组比较,五参顺脉胶囊组大鼠血清cTnI、CK-MB浓度、心肌梗死区比例、凋亡指数、自噬体数量、心肌组织LC3-Ⅱ/LC3-Ⅰ、caspase3、Bax降低(P<0.05),心肌组织p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、Bcl2水平增加(P<0.05);与五参顺脉胶囊组比较,自噬抑制剂组大鼠血清cTnI、CK-MB浓度、心肌梗死区比例、凋亡指数、自噬体数量、心肌组织LC3-Ⅱ/LC3-Ⅰ、caspase3、Bax增加(P<0.05),心肌组织p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR、Bcl2水平降低(P<0.05)。结论五参顺脉胶囊抑制心肌细胞过度自噬对心肌缺血再灌注损伤大鼠发挥保护作用。

Objective To investigate the role of autophagy in the improvement of myocardial ischemia-reperfusion injury in rats by Wushen-Shunmai capsule.Methods Following establishment of a myocardial ischemia-reperfusion injury model,rats were randomly divided into sham,model,Wushen-Shunmai capsule,and autophagy inhibitor(WushenShunmai capsule+LY294002) groups,with 15 rats in each group.The concentrations of serum cardiac troponin I(cTnI)and creatine kinase isoenzyme MB(CK-MB) were detected by enzyme-linked immunosorbent assay(ELISA).Myocardial infarction volume was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining,and cardiomyocyte apoptosis was detected by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay.The number of autophagy events was counted under electron microscope,while the expression of total PI3K,Akt,mammalian rapamycin target protein(mTOR) and its phosphorylated protein,and autophagy marker microtubule associated protein 1 light chain 3(LC3) was detected by Western blot.Results Compared with the sham group,serum cTnI,CK-MB concentration,myocardial infarction area ratio,apoptosis index,autophagy number,myocardial LC3-Ⅱ/LC3-Ⅰ,caspase3,and Bax were significantly higher in the model group(P<0.05),and the levels of p-PI3K/PI3K,p-Akt/Akt,pmTOR/mTOR,and Bcl2 were significantly lower(P<0.05).Compared with the model group,serum cTnI,CK-MB concentration,myocardial infarction area ratio,apoptosis index,autophagy number,myocardial LC3-Ⅱ/LC3-Ⅰ,caspase3,and Bax were significantly lower in the Wushen-Shunmai capsule group(P<0.05),and the levels of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,and Bcl2 were significantly higher(P<0.05).Compared with the Wushen-Shunmai capsule group,serum cTnI,CK-MB concentration,myocardial infarction area ratio,apoptosis index,autophagy number,myocardial LC3-Ⅱ/LC3-Ⅰ,caspase 3,and Bax were significantly higher in the autophagy inhibitor group(P<0.05),and the levels of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR and Bcl2 were significantly lower(P<0.05).Conclusions Wushen-Shunmai capsule inhibits excessive autophagy of myocardial cells and plays a protective role in myocardial ischemia-reperfusion injury.