温心方对心肌缺血再灌注损伤大鼠线粒体能量代谢的作用机制

Protective Effect of Wenxin Prescription on Mitochondrial Energy Metabolism in Rats with Myocardial Ischemia-reperfusion Injury

目的:揭示温心方对心肌缺血再灌注损伤大鼠线粒体能量代谢的改善作用及其对沉默信息调节因子1(SIRT1)/过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)/雌激素受体相关受体α(ERRα)能量信号通路的作用。方法:SPF级Wistar雄性大鼠90只,随机分为假手术组、模型组、温心方低、中、高剂量组;温心方低、中、高剂量组分别给予0.99、1.98、3.96 g·kg^(-1)的颗粒剂灌胃,假手术组和模型组均予以等体积生理盐水灌胃;预给药21 d后,模型组及温心方组采用冠状动脉左前降支结扎30 min,再灌注2 h复制大鼠心肌缺血再灌注损伤(MIRI)模型,假手术组只穿线,不结扎;TTC染色观察心肌梗死面积,苏木素-伊红(HE)染色观察心肌组织病理形态,试剂盒检测血清肌酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)活性、心肌组织ATP含量及线粒体复合体Ⅳ(CCO)、琥珀酸脱氢酶(SDH)活性,实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)检测心肌组织SIRT1、PGC-1α、ERRα、TFAM mRNA及蛋白表达。结果:与假手术组比较,模型组心肌纤维断裂,排列紊乱,细胞胞质水肿,细胞核出现固缩、偏移;CK-MB、LDH活性明显升高(P<0.05,P<0.01),ATP含量及CCO、SDH活性明显降低(P<0.05,P<0.01),心肌组织心肌组织SIRT1、PGC-1α、ERRα、mtTFA mRNA及蛋白表达均明显降低(P<0.05,P<0.01);与模型组比较,温心方预处理各组心肌梗死面积显著缩小,且以高剂量组最显著(P<0.01),心肌组织病理形态有所改善,CK-MB、LDH活性明显降低(P<0.05,P<0.01),ATP含量及CCO、SDH活性均有提高,以高剂量组最显著(P<0.01),心肌组织SIRT1、PGC-1α、ERRα、TFAM mRNA及蛋白表达均有不同程度提高(P<0.05,P<0.01)。结论:温心方可通过调控SIRT1/PGC-1α/ERRα信号通路,改善心肌线粒体能量代谢,保护心肌缺血再灌注损伤。

Objective:To reveal the effect of Wenxin prescription on mitochondrial energy metabolism and silent information regulator 1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α)/recombinant estrogen-related receptor α(ERRα) signaling pathway in rats with myocardial ischemiareperfusion injury.Method:Totally 90 male Wistar rats of SPF grade were randomly assigned into a sham operation group,a model group,and low-,medium-,and high-dose Wenxin prescription groups,with 18 rats in each group.The rats in low-,medium-,and high-dose Wenxin prescription groups were administrated with0.99,1.98,and 3.96 g·kg^(-1)granules by gavage,respectively,and those in the sham operation group and model group with the same amount of normal saline.Twenty-one days after pre-administration,the rat model of myocardial ischemia-reperfusion injury was established by ligation of the left anterior descending coronary artery for 30 min and reperfusion for 2 h,and the rats in the sham operation group were only threaded without ligation.Myocardial infarction area was observed through 2,3,5-triphenyl-2h-tetrazolium chloride(TTC)staining,and the myocardial histopathology through hematoxylin-eosin(HE)staining.The levels of creatine kinase-MB(CK-MB) and lactate dehydrogenase(LDH) in serum,cytochrome C oxidase(CCO) and succinate dehydrogenase(SDH) in mitochondrion,and ATP in myocardial tissue were detected according to kit instructions.The mRNA and protein levels of SIRT1,PGC-1α,ERRα,and mitochondrial transcription factor A(TFAM)in myocardial tissue were determined by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot,respectively.Result:Compared with the sham operation group,the model group showed broken and disordered myocardial fibers,cytoplasmic edema,and pyknosis and deviation of nuclei.Moreover,the modeling increased the levels of CK-MB and LDH(P<0.05,P<0.01),lowered the levels of ATP,CCO,and SDH(P<0.05,P<0.01),and down-regulated the mRNA and protein levels of SIRT1,PGC-1α,ERRα,and TFAM in myocardial tissue(P<0.05,P<0.01).Compared with the model group,Wenxin prescription reduced the myocardial infarction area(especially in the high-dose group,P<0.01),restored the pathological changes,lowered the levels of CK-MB and LDH(P<0.05,P<0.01),increased the levels of ATP,CCO,and SDH(especially in the high-dose group,P<0.01),and up-regulated the mRNA and protein levels of SIRT1,PGC-1α,ERRα,and TFAM in myocardial tissue(P<0.05,P<0.01).Conclusion:Wenxin prescription can protect rats from myocardial ischemia-reperfusion injury by regulating myocardial mitochondrial energy metabolism via the SIRT1/PGC-1α/ERRα signaling pathway.