脂联素通过减轻内质网应激抑制缺氧复氧诱导的心肌细胞损伤

目的:通过培养SD大鼠乳鼠心室肌细胞建立缺氧/复氧(H/R)模型,观察脂联素(adiponectin,APN)对心肌细胞缺氧/复氧内质网应激所致心肌细胞损伤的影响,为心脏缺血/再灌注损伤的防治提供理论依据。方法: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,实验分为以下3组:正常对照组、单纯H/R组、APN+H/R(3 mg/L、10 mg/L、20 mg/L、30 mg/L)组。实验终止后,观察心肌细胞形态的变化,测定乳酸脱氢酶(LDH)的释放,通过流式细胞术来检测心肌细胞的凋亡。用RT-PCR和Western blotting测定内质网应激指标GRP78、caspase-12的表达。结果:与空白对照组相比,单纯H/R后,细胞凋亡率显著增加(68.20%±1.73%vs0.73%±0.21%,P0.05),乳酸脱氢酶(LDH)的活性增加,内质网应激蛋白GRP78、caspase-12在蛋白及mRNA水平表达明显增高,APN预处理后进行H/R,可较大程度地逆转上述指标变化,与H/R组相比均具有显著差异(P0.05),并且呈现一定的浓度依赖性。结论:H/R可以诱导心肌细胞的内质网应激;脂联素可以通过减轻内质网应激来减轻心肌细胞凋亡,对心肌细胞有保护作用。

Effects of adiponectin on reducing endoplasmic reticulum stress injury induced by hypoxia-reoxygenation in cultured rat neonatal cardiomyocytes

AIM: To investigate the effects of adiponectin (APN) on hypoxia-reoxygenation (H/R) induced endoplasmic reticulum stress injury in cultured cardiomyocytes. METHODS: Primary cultured cardiomyocytes were obtained from neonatal rats by enzymatic digestion method. The α-actin expression as molecular marker of the cardiomyocytes was observed by immunocytochemistry. The cells cultured for 72 h were used in the experiment and divided into groups randomly: control group, H/R group, APN+H/R (3 mg/L, 10 mg/L, 20 mg/L, 30 mg/L) groups. The morphological changes of the cardiomyocytes were observed under phase contracted microscope. The content of LDH was measured. The cardiomycocyte apoptosis was detected by flow cytometry. The expression levels of GRP78 and caspase-12 were examined by RT-PCR and Western blotting. RESULTS: The apoptotic rate was significantly increased in H/R group as compared to that in control group (68.20%±1.73% vs 0.73%±0.21%, P<0.05). The levels of LDH in H/R group were also significantly increased. Compared to untreated cells, the protein and mRNA levels of GRP78 and caspase-12 increased significantly in H/R cells. The APN preconditioning significantly reversed these changes. The indexes above improved obviously as compared to H/R group (P<0.05) in a dose-dependent manner. CONCLUSION: Hypoxia/reperfusion induces endoplasmic reticulum stress in rat cardiomyocytes. Adiponectin decreases the endoplasmic reticulum stress injury and plays a protective role by extenuation of cadiomyocyte apoptosis.