乌头赤石脂丸对大鼠心肌缺血再灌注损伤自噬及PI3K/Akt/GSK-3β信号通路的影响

Wutou Chishizhi Wan Regulates Autophagy and PI3K/Akt/GSK-3β Signaling Pathway in Rats with Myocardial Ischemia-reperfusion Injury

目的:探究乌头赤石脂丸对大鼠心肌缺血再灌注损伤(MIRI)自噬及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/糖原合成酶激酶-3β(GSK-3β)信号通路的影响及其作用机制。方法:60只雄性SD大鼠随机分为正常组、模型组、阳性药物组(曲美他嗪组)、乌头赤石脂丸低、中、高剂量组,每组10只。正常组和模型组给予等体积生理盐水灌胃,曲美他嗪组给予5.4 mg·kg^(-1)剂量灌胃,中药乌头赤石脂丸低、中、高剂量组分别给予1.63、4.9、14.7 g·kg^(-1)剂量灌胃。除正常组外,其他组均采用冠状动脉左前降支结扎法制备模型。心电图检测ST段以评价造模情况;苏木素-伊红(HE)染色观察心肌组织损伤情况;比色法测定天冬氨酸氨基转移酶(AST)、肌酸激酶(CK)含量,酶联免疫吸附测定法(ELISA)检测肌钙蛋白T(cTnT)、肌红蛋白(MYO)含量;蛋白免疫印迹法(Western blot)检测微管相关蛋白1轻链3(LC3)、自噬相关基因(Beclin-1)、PI3K、Akt、GSK-3β、磷酸化(p)-GSK-3β、p-Akt的蛋白表达。结果:与正常组比较,模型组血清AST、CK、cTnT、MYO水平均明显升高(P<0.01),HE显示大鼠心肌细胞排列紊乱、细胞核散乱无序、心肌纤维扭曲断裂,LC3Ⅱ/Ⅰ及Beclin-1蛋白表达显著升高(P<0.01),PI3K、p-Akt及p-GSK-3β蛋白表达均显著降低(P<0.01)。与模型组比较,乌头赤石脂丸低、中、高剂量组与曲美他嗪组血清中AST、CK、cTnT、MYO活性均显著降低(P<0.01);乌头赤石脂丸低、中、高剂量组与曲美他嗪组心肌细胞结构完整、坏死程度减轻、肌纤维排列整齐;乌头赤石脂丸低、中、高剂量组LC3Ⅱ/Ⅰ蛋白表达降低(P<0.05,P<0.01),乌头赤石脂丸低、中、高剂量组及曲美他嗪组Beclin-1表达显著降低(P<0.01),乌头赤石脂丸低、中、高剂量组及曲美他嗪组PI3K、p-Akt及p-GSK-3β蛋白表达均显著增高(P<0.01)。与曲美他嗪组比较,乌头赤石脂丸低剂量组血清AST含量明显升高(P<0.05),乌头赤石脂丸高剂量组血清AST含量显著降低(P<0.01),乌头赤石脂丸中剂量组Beclin-1蛋白表达显著降低(P<0.01),乌头赤石脂丸中、高剂量组LC3Ⅱ/Ⅰ蛋白表达明显降低(P<0.05,P<0.01),乌头赤石脂丸低、中、高剂量组PI3K蛋白表达显著降低(P<0.01),乌头赤石脂丸低、中剂量组p-Akt蛋白表达显著升高(P<0.01),乌头赤石脂丸中剂量组p-GSK-3β蛋白表达显著降低(P<0.01)。结论:该研究表明,不同剂量的乌头赤石脂丸皆可干预MIRI,且低、中剂量效果低于高剂量组,高剂量效果最佳。乌头赤石脂丸可以降低心肌损伤标志物AST、CK、cTnT、MYO的水平,减轻心肌组织病理性损伤,下调Beclin-1、LC3Ⅱ/Ⅰ的蛋白表达,上调PI3K、p-Akt及p-GSK-3β的表达,可能通过抑制过度自噬的发生,调控PI3K/Akt/GSK-3β信号通路,对MIRI大鼠起到保护作用。

Objective:To explore the mechanism of Wutou Chishizhi Wan in regulating autophagy and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/glycogen synthase kinase-3β(GSK-3β)signaling pathway in rats with myocardial ischemia-reperfusion injury(MIRI).Method:Sixty male SD rats were randomly assigned into the normal group(normal saline),model group(normal saline),positive control(trimetazidine,5.4 mg·kg^(-1))group,and low-,medium-,and high-dose(1.63,4.9,14.7 g·kg^(-1),respectively)Wutou Chishizhi Wan groups,with 10 rats in each group.The rats in other groups except the normal group underwent left anterior descending coronary artery ligation for modeling.Electrocardiogram was employed to detect the ST-segment elevation to evaluate the modeling.Hematoxylin-eosin(HE)staining was performed to reveal the damage of myocardial tissue.The levels of aspartate aminotransferase(AST)and creatine kinase(CK)were determined by colorimetry,and those of cardiac troponin T(cTnT)and myoglobin(MYO)by enzyme-linked immunosorbent assay(ELISA).Western blot was carried out to determine the protein levels of microtubule-associated proteins 1 light chain 3(LC3),autophagy-related gene Beclin-1,PI3K,Akt,GSK-3β,p-GSK-3β,and p-Akt.Result:Compared with the normal group,the modeling elevated the serum levels of AST,CK,cTnT,and MYO(P<0.01),destroyed the arrangement of myocardial cells abd nucle,twisted and broken myocardial fibers,up-regulated the protein levels of LC3Ⅱ/Ⅰand Beclin-1(P<0.01),and downregulated the protein levels of PI3K,p-Akt,and p-GSK-3β(P<0.01).Compared with the model group,trimetazidine and Wutou Chishizhi Wan(all the doses)lowered the levels of AST,CK,cTnT,and MYO in serum(P<0.01),restored the arrangement of myocardial cells and muscle fibers,reduced necrosis,downregulated the protein level of Beclin-1(P<0.01),and up-regulated the protein levels of PI3K,p-Akt,and p-GSK-3β(P<0.01).Additionally,Wutou Chishizhi Wan(all the doses)down-regulated the protein level of LC3Ⅱ/Ⅰ(P<0.05,P<0.01).Compared with those in the trimetazidine group,the serum AST level rose in the low-dose Wutou Chishizhi Wan group(P<0.05)and declined in the high-dose group(P<0.01),and the protein level of Beclin-1 was down-regulated in the medium-dose group(P<0.01).Additionally,the trimetazidine group had higher protein level of LC3Ⅱ/Ⅰthan medium-and high-dose Wutou Chishizhi Wan groups(P<0.05,P<0.01),higher protein level of PI3K than low-,medium-,and high-dose groups(P<0.01),lower protein level of p-Akt than low-and medium-dose groups(P<0.01),and higher p-GSK-3βprotein level than the medium-dose group(P<0.01).Conclusion:Different doses of Wutou Chishizhi Wan can ameliorate MIRI,and the high dose has the best effect.Wutou Chishizhi Wan can reduce the activity of myocardial injury markers AST,CK,cTnT,and MYO,and alleviate the pathological damage of myocardial tissue.It can down-regulate the protein levels of beclin-1,LC3Ⅱ/Ⅰ,and up-regulate those of PI3K,p-Akt,and p-GSK-3β.In summary,Wutou Chishizhi Wan may inhibit excessive autophagy and regulate the PI3K/Akt/GSK-3βsignaling pathway to exert protective effect on MIRI rats.