恩格列净诱导的线粒体稳态显著减轻小鼠心脏微血管缺血/再灌注损伤

Empagliflozin alleviates cardiac microvascular ischemia/reperfusion injury by maintaining myocardial mitochondrial homeostasis

目的探究恩格列净(EMPA)在缓解心脏缺血再灌注(I/R)损伤期间的微血管和内皮损伤的作用。方法将小鼠随机分为3组,每组5只,一组小鼠按照每只10 mg·kg^(-1)·d^(-1) EMPA辅以饲料和蒸馏水连续喂养7 d,作为I/R+EMPA组备用。其余两组(I/R组和sham组)以普通饲料和蒸馏水连续喂养7 d后进行造模。I/R组和I/R+EMPA组小鼠开胸手术以暴露心脏,并用丝线结扎左前降支冠状动脉45 min以诱导缺血,随后松开再灌注2 h;假手术(Sham)组小鼠仅开胸后将线穿过前降支、不结扎。在体外诱导sI/R损伤前12 h,将10μmol/L的EMPA添加到HCAEC中作为sI/R+EMPA组备用,然后将sI/R组和sI/R+EMPA组细胞置于缺氧孵箱中孵育45 min,随后在正常培养条件下恢复2 h,以模拟缺氧后复氧的培养环境改变。对照组HCAECs在正常条件下进行培养。通过电子显微镜检测心脏微血管的结构改变,蛋白质印迹和免疫荧光检测微血管中的纤维蛋白积累和内皮细胞相应蛋白表达情况、实时荧光定量聚合酶链式反应(qPCR)检测促炎细胞因子等方法,检测EMPA是否可以减轻心脏I/R损伤期间的微血管损伤和内皮损伤。结果电子显微镜检测I/R组小鼠的微血管壁显著增厚,管腔变窄;蛋白质印迹法测定纤维蛋白的表达I/R组中的纤维蛋白积聚情况比Sham组和I/R+EMPA组更明显(P<0.05)。I/R组小鼠微血管表面ICAM1的表达以及IL-6、TNF-α和MCP1的mRNA表达水平相比于Sham组和I/R+EMPA组明显增加(P<0.05),恩格列净减轻了I/R损伤诱导的微血管闭塞和微血栓形成。在细胞层面,sI/R组除了ET-1相对蛋白表达水平显著高于对照组和sI/R+EMPA组外,其余p-eNOS、Fak和Src激酶的蛋白水平均显著低于对照组和sI/R+EMPA组(P<0.05)。相比于对照组和sI/R+EMPA组,在sI/R组HCAEC中剩余的FITC-葡聚糖含量升高,而TER值降低(P<0.05)。sI/R损伤降低了HCAEC中的线粒体DNA复制数和转录,而EMPA治疗提高了线粒体DNA复制和转录情况(P<0.05)。sI/R组Cox-Ⅰ和Cox-Ⅱ的表达水平相比于对照组显著降低,但在sI/R+EMPA有明显回升(P<0.05)。结论EMPA可以在心肌I/R损伤期间通过维持线粒体稳态来减轻心脏微血管缺血/再灌注损伤,保护微血管系统。

Objective To evaluate the effect of empagliflozin(EMPA)in mitigating microvascular and endothelial damage induced by myocardial ischemia-reperfusion(I/R)injury.Methods Fifteen male C57BL/6J mice were randomized into sham operated group,I/R group and I/R+EMPA group,and in the latter two groups,myocardial I/R injury was induced by ligating the left anterior descending coronary artery followed by reperfusion for 2 h.EMPA treatment was administered at the daily dose of 10 mg/kg for 7 days.After the treatment,the changes in myocardial microvascular structure of the mice were observed under electron microscopy.In the cell experiment,cultured human coronary artery endothelial cells(HCAECs)were treated with 10μmol/L EMPA before exposure to hypoxia for 45 min followed normoxic culture for 2 h.Western blotting and immunofluorescence assay were performed to observe fibrin accumulation and endothelial cell protein expressions in the myocardial tissues of the mice and in HCAECs,and RT-qPCR was used to detect the expressions of pro-inflammatory cytokines.Results Electron microscopy revealed significant myocardial microvascular wall thickening and lumen narrowing in mice with myocardial I/R injury.Fibrin accumulation and ICAM1 expression in the microvessels were more pronounced in I/R group than in the sham-operated and I/R+EMPA groups(P<0.05).EMPA treatment obviously alleviated microvascular occlusion and microthrombus formation induced by I/R injury.At the cellular level,the protein levels of p-eNOS,Fak,and Src kinases in hypoxic exposure group were significantly lower than those in the control and EMPA treatment groups(P<0.05).Hypoxic exposure significantly reduced mitochondrial DNA replication and transcription and lowered the expression levels of Cox-I and Cox-II in HCAECs,and these changes were obviously improved by EMPA treatment(P<0.05).Conclusion EMPA can alleviate myocardial I/R injury by maintaining mitochondrial homeostasis to protect the microvascular system.