心肌缺血再灌注损伤中白介素-17A诱发心肌细胞凋亡的作用机制

Effects of IL-17A on cardiac myocytes apoptosis induced by myocardial ischemia-reperfusion injury in mice

目的探讨心肌缺血再灌注损伤小鼠心肌组织中白细胞介素-17A(IL-17A)的表达及其对心肌细胞凋亡的影响机制。方法采用结扎小鼠左冠状动脉前降支(LAD)法进行心肌缺血再灌注损伤模型构建;伊文思蓝(Evan’s blue)和2,3,5-三苯基氯化四氮唑(TTC)染色与心肌损伤标记物肌酸激酶(CK)、肌钙蛋白T(c TnT)血清含量检测小鼠心肌损伤情况;Western blot检测心肌缺血再灌注(I/R)后3 h、24 h、72 h不同时间点心肌组织IL-17A的表达水平,TUNEL染色和Caspase 3活力检测进一步反映细胞凋亡情况;原代分离培养心肌细胞,不同质量浓度(10、20、40、80 ng/ml)IL-17A处理细胞24 h,TUNEL染色检测细胞凋亡率、Western blot检测凋亡相关蛋白Caspase 3、Bax、Bcl-2及PI3K/Akt通路蛋白表达变化。结果 I/R后心肌梗死区与缺血区(I/AAR)比率显著增加(P<0.05),而心肌缺血区与左心室的比率(AAR/LV)无明显变化,且心肌损伤标记物肌酸激酶(CK)、肌钙蛋白T(cTnT)血清含量也明显增加;与Sham组(假手术组)相比,I/R后3 h、24 h、72 h时间点心肌组织IL-17A表达显著增加,心肌细胞凋亡率和Caspase 3活性亦明显增加;不同浓度IL-17A能够促进原代心肌细胞凋亡,诱导Caspase 3、Bax表达,抑制Bcl-2及PI3K/Akt通路蛋白表达。结论心肌缺血再灌注损伤小鼠IL-17A明显增加,高表达的IL-17A可能通过抑制PI3K/Akt通路来诱导心肌细胞凋亡。

This study was performed to investigate the expression of interleukin-17 A(IL-17 A)and its effect on cardiomyocyte apoptosis in myocardial tissue of myocardial ischemia-reperfusion injury mice.Myocardial ischemia-reperfusion injury model was established by ligation of the left anterior descending coronary artery(LAD)in mice.The myocardial injury in mice was evaluated by Evan′s blue,TTC staining and serum levels of myocardial injury markers creatine kinase(CK)and troponin T(cTnT).The level of IL-17 A in myocardium was detected by Western blotting at 3 h,24 h and 72 h after I/R.The apoptosis of myocardial tissue was detected by TUNEL staining and Caspase 3 activity assay.Primary cultured cells were isolated and treated with different concentration of IL-17 A(10,20,40,80 ng/ml)for 24 h,then the apoptosis rate and the expression of Caspase 3,Bax,Bcl-2 and PI3 K/Akt pathway proteins were measured by TUNEL staining and Western blotting,respectively.Data showed that the ratio of myocardial infarct area to ischemic area(I/AAR)significantly increased after I/R(P<0.05),while the ratio of myocardial ischemia to left ventricle(AAR/LV)had no significant changes.The serum levels of myocardial injury markers creatine kinase(CK)and troponin T(cTnT)were also increased significantly.Compared with sham group,the expression of IL-17 A in myocardial tissue increased significantly at 3 h,24 h and 72 h after I/R,and the apoptosis rate and Caspase 3 activity of cardiomyocytes were also significantly increased.Different concentrations of IL-17 A could promote primary cardiomyocyte apoptosis,induce Caspase 3 and Bax expression,inhibit Bcl-2 and PI3 K/Akt pathway protein expression.Taken together,IL-17 A is significantly increased in mice with myocardial ischemia-reperfusion injury,and high expression of IL-17 A may induce cardiomyocyte apoptosis by inhibiting the PI3 K/Akt pathway.