YAP对糖尿病心肌缺血再灌注损伤大鼠BNIP3及线粒体自噬的影响

Effects of YAP expression on BNIP3 and mitophagy in myocardial ischemia-reperfusion injury rats with diabetes

目的探究Yes相关蛋白(YAP)调控B细胞淋巴瘤-2(Bcl-2)/E1B-19k Da相互作用蛋白3(BNIP3)在糖尿病大鼠心肌缺血再灌注损伤(MIRI)中的作用。方法无特定病原体(SPF)级成年雄性SD大鼠,腹腔注射链脲佐菌素(SZT)法构建1型糖尿病模型,成功造模后继续饲养12周。取非糖尿病大鼠12只和糖尿病大鼠24只,分为6组:非糖尿病假手术组(NS组)、非糖尿病缺血再灌注组(NIR组)、糖尿病假手术组(DS组)、糖尿病缺血再灌注组(DIR组)、糖尿病缺血再灌注+YAP抑制剂Super-TDU组(DIR+ST组)、糖尿病缺血再灌注+YAP抑制剂Super-TDU+自噬抑制剂3-MA组(DIR+ST+3-MA组),每组6只。MIRI模型通过结扎左冠状动脉前降支30 min后再灌注120 min的方法建立。Super-TDU和3-MA分别于再灌注前1 d和1 h注射。再灌注结束后ELISA法检测各组大鼠血清肌酸激酶同工酶(CK-MB)和乳酸脱氢酶(LDH)水平;2,3,5-三苯基氯化四氮唑(TTC)法检测各组大鼠心肌梗死面积,苏木素-伊红(HE)染色法观察心肌组织病理变化,蛋白免疫印迹(Western blot)法检测各组大鼠心肌组织YAP、BNIP3和Beclin表达水平。结果与各自NS组、DS组比较,NIR组和DIR组大鼠血清CK-MB和LDH表达水平升高,心肌组织YAP、BNIP3和Beclin表达水平上升(均P<0.05);与NIR组比较,DIR组血清CK-MB和LDH表达水平升高,心肌梗死面积增加,心肌病理损伤加重,心肌组织YAP表达水平明显上升,且BNIP3和Beclin表达水平下降(均P<0.05);与DIR组比较,DIR+ST组血清CK-MB和LDH表达水平和心肌梗死面积明显降低,心肌病理损伤减轻,心肌组织YAP表达水平明显下调,BNIP3和Beclin表达水平降低(P<0.05);与DIR+ST组比较,DIR+ST+3-MA组血清CK-MB和LDH表达水平升高,心肌梗死面积增加,心肌病理损伤加重,BNIP3和Beclin表达水平降低(P<0.05)。结论YAP介导的BNIP3线粒体自噬可能是调控糖尿病大鼠MIRI的重要机制。

Objective To investigate the role of Yes-associated protein(YAP)in regulating Bcl-2/adenovirus E1B19k Da-interacting protein 3(BNIP3)in myocardial ischemia-reperfusion injury(MIRI)of diabetic rats.Methods Adult male SD rats of SPF grade were construct the type 1 diabetes model by intraperitoneal injection of SZT,and were continuously raised for 12 weeks after successful modeling.Twelve non-diabetic rats and 24 diabetic rats were taken and randomly divided into 6 groups:the non-diabetic sham operation group(NS group),the non-diabetic ischemia-reperfusion group(NIR group),the diabetic sham operation group(DS group),the diabetic ischemia-reperfusion group(DIR group),the diabetic ischemia-reperfusion+YAP inhibitor Super-TDU group(DIR+ST group),the diabetic ischemia-reperfusion+YAP inhibitor Super-TDU+autophagy inhibitor 3-MA group(DIR+ST+3-MA group),6 cases in each group.The MIRI model was established by ligating the left anterior descending coronary artery for 30 min and then perfusion for 120 min.The Super-TDU and 3-MA were injected before reperfusion,respectively.After reperfusion,ELISA was used to detect serum CK-MB and LDH levels,the TTC method was use d to detect the myocardial infarction area,the HE staining was used to observe the pathological changes of myocardial tissue,and the Western blot method was used to detect the expression levels of YAP,BNIP3 and Beclin proteins in myocardial tissue.Results Compared with each NS group and DS group,the serum CK-MB and LDH levels in the NIR group and DIR group were increased,and the expression levels of YAP,BNIP3 and Beclin in myocardial tissue were increased(P<0.05).Compared with the NIR group,the levels of serum CK-MB and LDH and the myocardial infarction area in the DIR group were significantly increased,the pathological damage was aggravated,the YAP expression level in myocardial tissue was significantly increased,while the BNIP3 and Beclin expression levels were decreased(P<0.05).Compared with the DIR group,the levels of serum CK-MB and LDH and the myocardial infarction area in the DIR+ST group were significantly decreased,the myocardial pathological injury was alleviated,the YAP expression level in the myocardial tissue was significantly down-regulated,the BNIP3 and Beclin expression levels were decreased(P<0.05).Compared with the DIR+ST group,the serum CK-MB and LDH expression levels and myocardial infarction area in the DIR+ST+3-MA group were increased,myocardial pathological damage was aggravated,and the expression levels of BNIP3 and Beclin were decreased(P<0.05).Conclusion YAP-mediated BNIP3 mitochondrial autophagy might be an important possible mechanism for regulating MIRI in diabetic rats.