南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (2): 226-235.doi: 10.12122/j.issn.1673-4254.2024.02.04

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秋水仙碱通过激活AMPK减轻小鼠心肌缺血再灌注损伤

陈国栋，罗素新   

  1. 重庆医科大学附属第一医院心血管内科，重庆 400016
  • 发布日期:2024-03-13

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

CHEN Guodong, LUO Suxin   

  1. Department of Cardiology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
  • Published:2024-03-13

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摘要： 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。

关键词: 秋水仙碱；心肌缺血再灌注损伤；AMPK；氧化应激；凋亡

Abstract: 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.

Key words: colchicine; myocardial ischemia-reperfusion injury; AMPK; oxidative stress; apoptosis