基于AMPK介导的线粒体自噬分析温阳益气方改善大鼠梗死后心衰的药理机制

To ameriorate the post-infarction heart failure in rats with a traditional Chinese medicine,Wenyang-Yiqi recipe,and to analyze its pharmacological mechanism by AMPK-mediated mitochondrial autophagy

目的观察温阳、益气方药对梗死后心力衰竭大鼠模型腺苷酸活化蛋白激酶(AMPK)介导的线粒体自噬的影响,完善其药理机制提供数据支撑。方法将100只健康SPF级大鼠随机分为5组,各组20只。除对照组和假手术组以外,均采用冠状动脉结扎法制备梗死后心力衰竭大鼠模型。模型制备成功后,中药干预组采用温阳益气方灌胃,AMPK激动组在温阳益气方灌胃的基础上肌肉注射AMPK激动剂EX229。干预4周,测定左室射血分数(LEVF)与心脏/体重比;采用线粒体呼吸机测定线粒体呼吸控制比(RCR),采用WB实验测定磷酸化AMPK/非磷酸化AMPK(p-AMPK/AMPK)及自噬标志物微管相关蛋白1轻链3(LC3II/LC3I)、PINK1、Parkin的蛋白表达。结果与对照组比较,模型组和AMPK激动组心脏/体重比明显升高,state3呼吸速率与RCR明显降低;与模型组比较,中药组心脏/体重比降低,state3呼吸速率与RCR升高;差异有统计学意义(P<0.05)。与对照组比较,模型组、中药组和AMPK激动组心肌组织p-AMPK/AMPK、LC3II/LC3I、PINK1及Parkin蛋白表达水平升高;与模型组相比,中药组p-AMPK/AMPK、LC3II/LC3I、PINK1及Parkin蛋白表达水平降低;组间差异有统计学意义(P<0.05)。结论温阳、益气方药能够改善梗死后心衰大鼠的心肌功能,其作用机制可能与抑制AMPK介导的线粒体自噬有关。

Objective To observe the effects of the traditional Chinese medicine(TCM),Wenyang-Yiqi recipe,on post-infarction heart failure in rats,and to investigate its pharmacological mechanism by AMPK-mediated mitochondrial autophagy.Methods A total of 100 healthy SPF rats were randomly divided into five groups(n=20 per group):control group,sham-operated group,model group,TCM intervention group,and AMPK agonist group.The rat model of post-infarction heart failure was established by coronary artery ligation except for the control group and the sham operation group.Then,the TCM intervention group was given Wenyang-Yiqi recipe,0.10 mL/kg by intragastric gavage once a day for four weeks,while the AMPK agonist group received the same dose of Wenyang-Yiqi recipe plus an intramuscular injection of EX229 at the same time.After 4 weeks of intervention,the left ventricular ejection fraction(LEVF)and heart/body weight ratio of the rats were measured.The mitochondrial respiratory control ratio(RCR)was determined by a mitochondrial ventilator,and the protein expressions of p-AMPK/AMPK,LC3II/LC3I,PINK1 and Parkin were determined by western blotting assay.Results Compared with the control group,the heart/body weight ratio of the model group and AMPK agonist group was increased,while the state3 respiratory rate and RCR were decreased.Compared with the model group,the heart/body weight ratio of the TCM intervention group was decreased,while the state3 respiratory rate and RCR were significantly increased(P<0.05).Compared with the control group,the expression levels of p-AMPK/AMPK,LC3II/LC3I,PINK1 and Parkin in the myocardial tissue of the model group,TCM intervention group and AMPK agonist group were significantly increased,and compared with the model group,the expression levels of p-AMPK/AMPK,LC3II/LC3I,PINK1 and Parkin proteins in the TCM intervention group were significantly decreased(P<0.05).Conclusions The traditional Chinese medicine Wenyang-Yiqi recipe improves the myocardial function of rats with post-infarction heart failure,and the mechanism might be related to the inhibition of AMPK-mediated mitochondrial autophagy.