REV-ERB激动剂SR9009通过上调SLC7A11/GPX4轴限制铁死亡减轻2型糖尿病大鼠心肌缺血再灌注损伤

REV-ERB Agonist SR9009 Attenuates Myocardial Ischemia-reperfusion Injury in Type 2 Diabetic Rats by Limiting Iron Death through Up-regulation of SLC7A11/GPX4 Axis

目的评价时钟基因REV-ERB激动剂SR9009在2型糖尿病大鼠心肌缺血再灌注损伤中的作用及其调控铁死亡的潜在机制。方法SPF级健康雄性SD大鼠24只,体质量为90~110g,采用随机数字表法分为糖尿病假手术组(DS组,n=6)、糖尿病心肌缺血再灌注组(DIR组,n=6)、糖尿病心肌缺血再灌注+REV-ERB激动剂组(DIR+SR9009组,n=6)、糖尿病心肌缺血再灌注+铁死亡激活剂+REV-ERB激动剂组(DIR+Erastin+SR9009组,n=6)。采用腹腔注射1%链脲佐菌素-枸橼酸盐缓冲液40mg/kg的方法制备大鼠2型糖尿病模型。DIR+SR9009组于缺血前6h腹腔注射REV-ERB激动剂SR9009100mg/kg,DIR+Erastin+SR9009组分别于缺血前24h和12h腹腔注射铁死亡激活剂Erastin 15mg/kg,缺血前6h腹腔注射REV-ERB激动剂SR9009100mg/kg。2型糖尿病建模成功继续高脂饲料喂养5周后,采用结扎左冠状动脉前降支30min后恢复血流灌注的方法制备大鼠心肌缺血再灌注损伤模型。于再灌注2h时颈动脉采血后处死大鼠,采用酶联免疫吸附试验(enzyme-linked immunoadsordent assay,ELISA)检测血清心肌肌钙蛋白I(cardiac troponin I,cTnI)浓度和超氧化物歧化酶(superoxide dismutase,SOD)活性,比色法测定心肌Fe^(2+)、线粒体丙二醛(malondialdehyde,MDA)含量,TTC法检测心肌梗死面积,苏木精-伊红染色法观察病理结果,Western blot法检测心肌REV-ERBα、胱氯酸/谷氨酸逆向转运蛋白溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)的表达水平。结果与DS组比较,DIR组血清cTnI浓度、心肌Fe^(2+)、MDA含量升高,SOD活性降低,REV-ERBα表达升高,SLC7A11和GPX4表达下调(P<0.05);与DIR组比较,DIR+SR9009组血清cTnI浓度、心肌Fe^(2+)、MDA含量降低,SOD活性升高,REV-ERBα表达下调,SLC7A11和GPX4表达升高,心肌梗死面积减少(P<0.05);与DIR+SR9009组比较,DIR+Erastin+SR9009组血清cTnI浓度、心肌Fe^(2+)、MDA含量升高,REV-ERBα表达升高,SLC7A11和GPX4表达下调,心肌梗死面积增加(P<0.05)。结论时钟基因REV-ERB激动剂SR9009预处理可减轻2型糖尿病大鼠心肌缺血再灌注损伤,且与SLC7A11/GPX4轴抗氧化系统异常诱导的铁死亡密切相关。

Objective To evaluate the role of clock gene REV-ERB agonist SR9009 in myocardial ischemia-reperfusion injury in type 2 diabetes mellitus rats and its potential mechanism of regulating iron death.Methods Twenty-four SPF-grade healthy male SD rats,weighing 90-110g,they were divided into 4groups by random number table method:diabetic sham-operated group(DS group,n=6),diabetic myocardial ischemia-reperfusion group(DIR group,n=6),diabetic myocardial ischemia-reperfusion+REV-ERB agonist group(DIR+SR9009 group,n=6),diabetic myocardial ischemia-reperfusion+iron death agonist+REV-ERB agonist group(DIR+Erastin+SR9009 group,n=6).The rat model of type 2 diabetes mellitus was prepared by intraperitoneal injection of 1%streptozotocin-citrate buffer 40mg/kg.DIR+SR9009 group was intraperitoneally injected with REV-ERB agonist SR9009100mg/kg at 6h before ischemia,DIR+Erastin+SR9009 group was intraperitoneally injected with iron death activator Erastin 15mg/kg at 24h and 12h before ischemia,respectively,and intraperitoneally injected with REV-ERB agonist SR9009100mg/kg at 6h before ischemia.After 5 weeks of successful modeling of type 2 diabetes mellitus,the myocardial ischemia-reperfusion injury model rat was prepared by ligating the anterior descending branch of the left coronary artery for 30min and restoring blood perfusion.The rats were executed after carotid artery blood collection at 2h of reperfusion,serum cardiac troponin I(cTnI)concentration and superoxide dismutase(SOD)activity were measured by enzyme-linked immunoadsordent assay(ELISA);myocardial Fe^(2+),mitochondrial malondialdehyde(MDA)content were measured by colorimetric method,myocardial infarct area was measured by TTC method,pathological results were observed by hematoxyn-eosin staining,and the expression levels of myocardial REV-ERBα,SLC7A11 and GPX4 were detected by Western blot.Results Compared with the DS group,serum cTnI concentration,myocardial Fe^(2+),MDA content were increased,SOD activity was decreased,REV-ERBαexpression level was increased,and SLC7A11 and GPX4 expression level were down-regulated in the DIR group(P<0.05);compared with the DIR group,serum cTnI concentration,myocardial Fe^(2+),MDA content were decreased,SOD activity was increased,REV-ERBαexpression level was down-regulated,SLC7A11 and GPX4 expression level was increased,and myocardial infarct area was decreased in the DIR+SR9009 group(P<0.05);compared with the DIR+SR9009 group,serum cTnI concentration,myocardial Fe^(2+),and MDA content were increased,REV-ERBαexpression level was increased,SLC7A11 and GPX4 expression was down-regulated,and myocardial infarct size was increased in the DIR+Erastin+SR9009 group(P<0.05).Conclusion Administration of the clock gene REV-ERB agonist SR9009 pretreatment attenuated myocardial ischemia-reperfusion injury in type 2 diabetic rats,which was closely associated with iron death induced by abnormalities in the antioxidant system represented by the SLC7A11/GPX4 axis.