基于PI3K/Akt/mTOR信号通路探讨炙甘草汤抗大鼠MIRI致室速和室颤的作用机制  

Based on PI3K/Akt/mTOR Signaling Pathway to Explore Mechanism of Zhigancao Tang Against MIRI-induced Ventricular Tachycardia and Ventricular Fibrillation in Rats

作　　者：郑旭颖 麻春杰[1] 陈永真 任存霞[1] 牛仁秀[1] 莲花[1] 陈琨[1] 谭晓慧[1] 闫清支 张星[1] 吴昊[1] ZHENG Xu-ying;MA Chun-jie;CHEN Yong-zhen;REN Cun-xia;NIU Ren-xiu;LIAN Hua;CHEN Kun;TAN Xiao-hui;YAN Qing-zhi;ZHANG Xing;WU Hao(Inner Mongolia Medical University,Hohhot 010110,China)

机构地区：[1]内蒙古医科大学,呼和浩特010110

出　　处：《中国实验方剂学杂志》2020年第17期1-8,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：内蒙古自然科学基金项目(2017MS0801)。

摘　　要：目的:通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路,探讨炙甘草汤干预心肌缺血再灌注损伤(MIRI)致心律失常(室速和室颤)的作用及其机制。方法:将72只SD大鼠随机分为假手术组,模型组,炙甘草汤低、中、高剂量组(11.43,22.86,45.72 g·kg^-1),稳心颗粒组(2.43 g·kg^-1),连续给药干预10 d。末次给药2 h,采用结扎冠状动脉左前降支法制备大鼠MIRI模型,记录心电图的变化。造模成功后采集血液及心脏组织,检测血清中肌酸肌酶(CK),乳酸脱氢酶(LDH)及丙氨酸氨基转移酶(AST)的含量;酶联免疫吸附测定(ELISA)检测心肌肌钙蛋白(CtnI)的含量;免疫组化法检测心肌PI3K,Akt,mTOR的表达;采用蛋白免疫印迹法(Western blot)检测心肌自噬相关蛋白微管相关蛋白1轻链3(LC-3),自噬标志物Beclin1和PI3K/Akt/mTOR信号通路相关蛋白表达及磷酸化p-PI3K,p-Akt,p-mTOR的水平。结果:模型组大鼠100%发生室速,91.67%发生室颤;与假手术组比较,模型组大鼠血清CK,LDH,AST以及CtnI的含量显著升高(P<0.01),心肌组织中PI3K,Akt,mTOR表达显著升高(P<0.01),LC3-Ⅱ/LC3-Ⅰ与Beclin1的相对表达量显著升高(P<0.01),p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR显著降低(P<0.01);与模型组比较,炙甘草汤高剂量组室速、室颤发生率显著降低(P<0.01),较其他给药组持续时间短(P<0.01);炙甘草汤高剂量组CK,LDH,AST水平及CtnI含量显著降低(P<0.01);炙甘草汤给药组的PI3K,Akt,mTOR的表达随剂量的增加而显著降低(P<0.01);LC-3,Beclin1的表达随炙甘草汤各剂量组的增加有不同程度地下降(P<0.01),而PI3K/Akt/mTOR相关蛋白表达比值明显升高(P<0.05,P<0.01)。结论:炙甘草汤预处理能使异常升高的心肌酶CK,LDH,AST,CtnI降低,抑制细胞的过度自噬,上调PI3K,Akt和mTOR的表达,说明炙甘草汤抗MIRI致心律失常的作用可能与PI3K/Akt/mTOR信号通路有关。Objective:Through phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian rapamycin target protein(mTOR)signaling pathway,explore the effect of Zhigancao Tang on myocardial ischemia-reperfusion injury(MIRI)The role and mechanism of arrhythmia(ventricular tachycardia and ventricular fibrillation).Method:The 72 SD rats were randomly divided into sham operation group,model group,Zhigancao Tang low,medium and high dose group(11.43,22.86,45.72 g·kg^-1),Wenxin granule group(2.43 g·kg^-1),continuous drug intervention for 10 days.Two hours after the last administration,the MIRI model of rat was prepared by ligating the left anterior descending coronary artery,and the changes of electrocardiogram were recorded.After successful modeling,blood and heart tissue were collected to detect the content of creatine creatine(CK),lactate dehydrogenase(LDH)and aspartate aminotransferase(AST)in the serum,the enzyme-linked immunoassay(ELISA)method was used to detect cardiac troponin(CtnI)content,immunohistochemical detection of myocardial PI3K,Akt,mTOR expression.Western blot was used to detect the myocardial autophagy-related protein microtubule-associated protein 1 light chain 3(LC-3),autophagy markers Beclin1 and PI3K/Akt/mTOR signaling pathway related protein expression and phosphorylated p-PI3K,p-Akt,p-mTOR levels.Result:In model group,100%of ventricular tachycardia and 91.67%of ventricular fibrillation occurred.Compared with sham operation group,the serum levels of CK,LDH,AST,and CtnI in the model group were significantly increased(P<0.01),PI3K,Akt,mTOR AOD values in myocardial tissue were significantly increased(P<0.01),the relative expression of the ratio of LC3-Ⅱ/LC3-Ⅰand Beclin1 was significantly increased(P<0.01),p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR were significantly reduced(P<0.01).Compared with model group,the incidence of ventricular tachycardia and ventricular fibrillation in the high-dose Zhigancao Tang group was significantly reduced(P<0.01),and the duration was the shortest compared with other admi

关 键 词：炙甘草汤 心肌缺血再灌注损伤 心律失常 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路 

分 类 号：R2-0[医药卫生—中医学] R289