秋水仙碱通过激活AMPK减轻小鼠心肌缺血再灌注损伤

Colchicine alleviates myocardial ischemia-reperfusion injury in mice by activating AMPK

目的探究秋水仙碱(CCC)对心肌缺血再灌注损伤(I/R)的影响与相关机制。方法采用缺氧复氧(H/R)的方式在H9C2细胞上模拟缺血再灌注损伤,使用3 nmol/L秋水仙碱对行H/R处理的细胞进行干预;采用手术的方式在雄性C57/BL6小鼠上建立心肌缺血再灌注模型,将32只小鼠随机分为:假手术(SO)组,I/R组、I/R+CCC组和I/R+CCC+Dorsomorphin(DSMP)组,每组8只。使用CCK-8检测细胞活力,使用Western blot检测AMPK及其磷酸化水平、氧化应激指标(NOX4、NRF2、SOD2)和凋亡指标(BAX、Bcl-2、cleaved caspase-3),使用商用试剂盒检测心肌组织ATP含量,使用免疫荧光染色检测心肌组织8-OHdG和cleaved caspase-3阳性率,分离心肌线粒体和胞浆后分别检测线粒体BAX(Mito-BAX)和胞浆细胞色素C(Cyt-Cyto C),使用心脏超声评估心脏功能,使用2,3,5-氯化三苯基四氮唑(TTC)溶液染色评估梗死面积,使用商用试剂盒检测血清乳酸脱氢酶(LDH)和心肌肌钙蛋白T(cTnT)含量,为明确AMPK在其中的作用,使用DSMP抑制小鼠体内AMPK活性。结果体外实验中,H/R可以导致AMPK磷酸化水平、NRF2、SOD2和Bcl-2表达水平和细胞活力显著降低(P<0.05),并提高NOX4、BAX、cleaved caspase-3表达水平(P<0.05);秋水仙碱处理可以减轻H/R的损伤效应(P<0.05)。体内实验中,与SO组相比,I/R组AMPK磷酸化水平、ATP含量、NRF2、SOD2和Bcl-2表达水平、心脏功能均显著降低(P<0.05),NOX4、Mito-BAX、Cyt-Cyto C、BAX、cleaved caspase-3表达水平,心肌切片8-OHdG和cleaved caspase-3阳性染色率,心肌梗死面积,血清LDH和cTnT含量显著升高(P<0.05),秋水仙碱治疗可以逆转I/R带来的损伤效应(P<0.05),但DSMP可以抵消秋水仙碱的保护作用(P<0.05)。结论秋水仙碱可以通过激活AMPK减轻氧化应激和凋亡来减轻小鼠心肌缺血再灌注损伤,保护心功能。

Objective To investigate the protective effect of colchicine against myocardial ischemia-reperfusion injury(I/R)and explore the underlying mechanism.Methods H9C2 cells exposed to hypoxia/reoxygenation(H/R)were treated with 3 nmol/L colchicine,after which the changes in cell viability were assessed using MTT assay,and AMPK phosphorylation,the expressions of NOX4,NRF2,SOD2,BAX,Bcl-2,and cleaved caspase-3 were detected with Western blotting.Male C57BL/6 mice were randomized into sham operation,I/R,I/R+colchicine,and I/R+colchicine+dorsomorphin(DSMP)groups.After the treatments,myocardial expressions of p-AMPK/AMPK,8-OHdG,cleaved caspase-3,mitochondrial BAX(Mito-BAX),and cytoplasmic cytochrome C(Cyt-Cyto C)were examined and cardiac functions,infarct area,ATP content,and serum levels of lactic dehydrogenase(LDH)and cardiac troponin T(cTnT)levels were assessed.Results In H9C2 cells,H/R exposure significantly reduced AMPK phosphorylation and expressions of NRF2,SOD2,and Bcl-2,lowered cell viability,and up-regulated the expressions of NOX4,BAX,and cleaved caspase-3(P<0.05),and these changes were obviously alleviated by colchicine treatment(P<0.05).In the mouse models,myocardial I/R injury significantly reduced myocardial AMPK phosphorylation level,ATP content,and expressions of NRF2,SOD2 and Bcl-2,caused cardiac function impairment,enhanced NOX4,Mito-BAX,Cyt-Cyto C,BAX,8-OHdG,and cleaved caspase-3 expressions,and increased infarct area and serum LDH and cTnT levels(P<0.05).Colchicine treatment significantly reversed the damaging effects of I/R(P<0.05),but its protective effects was obviously antagonized by DSMP(P<0.05).Conclusion Colchicine alleviates myocardial I/R injury and protects cardiac function in mice by reducing myocardial oxidative stress and apoptosis via activating AMPK.