参莲方对超细颗粒物促发心肌I/R损伤的保护作用研究

Protective Effect of Shenlian Formula(参莲方)on Myocardial Injury Induced by Ischemia-Reperfusion(I/R)and Exposure to Ultrafine Particulate Matter

目的研究参莲方对超细颗粒物促发大鼠心肌缺血再灌注(ischemia-reperfusion,I/R)损伤的保护作用及机制。方法采用超细颗粒物(DPM 0.1 mg·kg^(-1))染毒基础上结扎冠状动脉左前降支建立超细颗粒物促发I/R大鼠模型,参莲方73、146、292 mg·kg^(-1)灌胃给药,TTC法检测心肌梗死面积,HE染色光镜下观察心肌组织形态学改变,透射电镜观察心肌及微血管超微结构,比色法检测血清中肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)、穿透素3(pentraxin-3,PTX-3)的含量变化。H9c2细胞经超细颗粒物100μg·mL^(-1)染毒24 h,观察参莲方5、10、20μg·mL^(-1)对其保护作用,比色法检测乳酸脱氢酶(LDH)、CK-MB水平,流式细胞仪检测细胞凋亡率,荧光探针法检测细胞内活性氧(ROS)、细胞线粒体含量、线粒体膜电位,化学发光法检测线粒体Na^(+)-K^(+)-ATP酶活性,Western Blot检测凋亡相关蛋白的表达。结果参莲方可显著减少模型大鼠心肌梗死面积,降低血清中心肌损伤标志物CK-MB和PTX3的含量,减轻心肌组织损伤和炎性细胞浸润,对心肌细胞线粒体结构异常改善明显。体外实验表明,参莲方可降低H9c2细胞LDH、CK-MB、ROS水平,增加线粒体含量,逆转线粒体膜电位丢失,同时显著降低H9c2细胞凋亡率及Caspase-3、Caspase-9蛋白表达,上调Bcl-2蛋白表达。结论参莲方对超细颗粒物促发的心肌I/R损伤保护作用明显,可能与减轻线粒体途径细胞凋亡有关。

Objective To evaluate the protective effect of Shenlian Formula(参莲方,SL)on myocardial injury induced by ischemia-reperfusion(I/R)and ultrafine particulate matter.Methods The rats were exposed to Ultrafine Particulate Matter(UFPM)by tail vein injection at the dose of 0.1 mg·kg^(-1).The myocardial ischemia-reperfusion was generated by ligating the left anterior descending coronary artery(LAD)within 24 h after the infected.The changes of myocardial infarction area was determined by 2,3,5-triphenyltetrazolium chloride(TTC)stain.The myocardial tissue morphology was determined by HE stain,and myocardial tissue morphology and ultrastructure changes were observed with transmission electronic microscopy.After drug intervention,serum levels of creatine kinase isoenzyme(CK-MB)and pentraxin-3(PTX-3)contents were detected with colorimetric analysis.H9c2 cells were stimulated with 100μg·mL^(-1).The protecting effect on UFPM for 24 h after incubation with SL 5,10 and 20μg·mL^(-1) was observed,and the levels of lactate dehydrogenase(LDH)and CK-MB in the cell supernatant were detected by colorimetric assay.The apoptotic rate was examined by flow cytometry and the intracellular reactive oxygen species(ROS),cell mitochondria content and mitochondrial membrane potential were detected by probe method.Besides,the activity of mitochondrial Na+-K+-ATPase was detected by chemiluminescence,and the apoptosis-related protein expression was detected by Western Blot.Results After the intervention of SL extract,compared with that of the model group,the myocardial infarction area of rats in each dose group was significantly reduced.Rat myocardial injury markers CK-MB and PTX-3 were decreased significantly,myocardial damage and the infiltration of inflammatory cells were reduced and the mitochondrial structure of cardiomyocytes was significantly improved.In vitro experiments showed that SL increased the contents of mitochondria,reverse dmitochondrial membrane potential loss,decreased the apoptosis rate of H9c2 cells,decreased the expressions of Caspase-3 and Caspase-9 protein and increased the expression of Bcl-2 protein at the same time.Conclusion It is suggested that SL has a significant protective effect on myocardial I/R induced by UFPM,which may be related to the reduction of mitochondrial apoptosis.