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脂联素对乳鼠心肌细胞缺氧/ 复氧损伤的保护作用

边云飞, 郭晓夏, 肖传实^*

山西医科大学第二医院心内科，太原 030001

摘要

本研究通过在大鼠乳鼠心室肌细胞上建立缺氧/ 复氧(hypoxia/reoxygenation, H/R)模型，模拟在体心肌缺血/ 再灌注损伤，观察脂联素(adiponectin, APN)对心肌细胞H/R 损伤的影响，并探讨其作用机制。采用胰蛋白酶消化法原代培养乳鼠心室肌细胞，α- 肌动蛋白免疫荧光法进行鉴定。选用培养72 h 的单层心肌细胞进行实验，随机分为5 组：对照组、单纯H/R 组、H/R+APN 组、H/R+APN+ 腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)特异性抑制剂阿糖胞苷(AraA)组、H/R+AraA 组。观察各组心肌细胞形态及自发搏动频率，用琼脂糖凝胶电泳和流式细胞术检测各组心肌细胞凋亡情况，并测定细胞丙二醛(MDA)含量及培养液中超氧化物歧化酶(SOD)活性，激光共聚焦显微镜观察心肌细胞内钙荧光强度，Western blot 检测各组心肌细胞AMPK 磷酸化水平。结果显示，与对照组相比，单纯H/R 组细胞生长状态较差，搏动频率减慢甚至消失，DNA 电泳呈凋亡特征性的梯状条带，细胞凋亡率显著增加，胞浆MDA 水平增高，上清液中SOD活性下降，胞内钙荧光强度明显增高，AMPK 磷酸化水平升高(P<0.05)。与H/R 组细胞相比，APN 预处理后再进行H/R的心肌细胞搏动频率较快，凋亡率明显减少，MDA 水平明显下降，SOD 活性明显升高，心肌细胞AMPK 磷酸化水平明显增高(P<0.05)。AraA 可以阻断APN 的上述保护作用。以上结果表明，APN 可减轻H/R 导致的心肌细胞凋亡，减轻脂质过氧化及细胞内钙超载，这一保护作用可能与AMPK 途径激活有关。

关键词： 脂联素; 乳鼠心肌细胞; 缺氧/ 复氧; 腺苷酸活化蛋白激酶; 阿糖胞苷

分类号：R363

[Protective effects of adiponectin against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes.] [Ariticle in Chinese]

BIAN Yun-Fei, GUO Xiao-Xia, XIAO Chuan-Shi^*

Department of Cardiology, the Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, China

Abstract

The aim of the present study is to investigate the effects of adiponectin (APN) on hypoxia/reoxygenation (H/R) injury incultured cardiomyocytes. Primary cardiomyocytes were obtained from neonatal rats by enzymatic digestion method and identified byimmunofluorescent technique. Primary cells cultured for 72 h were used in experiment and divided into 5 groups randomly: Controlgroup, H/R group, H/R+APN group, H/R+APN+adenine 9-β-D-arabinfuranoside (AraA, AMPK inhibitor) group, and H/R + AraAgroup. The cardiocyte morphology and beating rate were observed under inverted microscope. The DNA ladder was examined byagarose gel electrophoresis, and the cell apoptosis was determined by flow cytometry. Moreover, the malondialchehyche (MDA)content in myocardial cells and the superoxide dismutase (SOD) activity in the supernatant were measured using kits, the fluorescenceintensity of intracellular Ca2+ was observed by laser scanning confocal microscope, and the phosphorylation of AMPK was determinedby Western blotting. Compared with control group, H/R group showed increased apoptotic rate, oxidative stress level, intracellular Ca2+concentration and phosphorylation level of AMPK (P<0.05), while significant ameliorations in the above indices were seen inH/R+APN group. On the contrast, AraA attenuated the protective effect of APN and decreased the phosphorylation of AMPK. Theseresults suggest that adiponectin can protect cardiomyocytes from H/R-induced oxidative stress and apoptosis through AMPK pathway.

Key words: adiponectin; cardiomyocyte; hypoxia/reoxygenation; AMP-activated protein kinase; adenine 9-β-D-arabinfuranoside

收稿日期：2009-09-01　　录用日期：2010-01-25

通讯作者：肖传实　　E-mail: ganxibaozhongxin@sina.com

引用本文：

边云飞, 郭晓夏, 肖传实. 脂联素对乳鼠心肌细胞缺氧/ 复氧损伤的保护作用[J]. 生理学报 2010; 62 (2): 149-155.

BIAN Yun-Fei, GUO Xiao-Xia, XIAO Chuan-Shi. [Protective effects of adiponectin against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (2): 149-155 (in Chinese with English abstract).