不同浓度脂联素对心肌细胞氧化应激损伤后GRP78及Caspase-12表达的影响及意义

目的 通过原代培养SD大鼠的乳鼠心肌细胞建立H_2O_2心肌细胞氧化应激损伤模型,观察脂联素对心肌细胞氧化应激所致内质网应激的保护作用.方法 采用酶消化法原代培养乳鼠心肌细胞,倒置相差显微镜下观察细胞生长状态,通过α-肌动蛋白免疫荧光法对培养的心肌细胞进行鉴定.选用原代培养3～4天的心肌细胞,随机分为对照组、H_2O_2组、H_2O_2+10 mg/L脂联素组、H_2O_2+20 mg/L脂联素组和H_2O_2+30 mg/L脂联素组.实验终止后,在倒置相差显微镜下观察心肌细胞形态的变化,采用化学比色法测定乳酸脱氢酶的释放,通过流式细胞术来检测心肌细胞的凋亡,用RT-PCR与western Blotting方法检测内质网应激指标GRP78和Caspase-12的表达.结果 与对照组相比,给予H_2O_2后,细胞凋亡率显著增加(70.7%±6.4%比1.0%±0.6%,P<0.05),LDH释放增加(1411.5 ±189.7 U/L比353.3 ±50.3 U/L,P<0.05),内质网伴侣蛋白GRP78以及Caspage-12在mRNA(分别为1.25±0.50比0.18 ±0.10和1.32±0.15比0.26±0.06)及蛋白水平(分别为0.92±0.50比0.37±0.10和1.24 ±0.50比0.51±0.01)表达增加(P<0.05),30 mg/L脂联素预处理后给予H_2O_2,可较大程度地逆转上述指标变化,细胞凋亡率显著下降(43.6%±3.8%),LDH释放减少(686.7±61.1 U/L),内质网伴侣蛋白GRP78以及Cagpase-12在mRNA(分别为0.56±0.03和0.83±0.04)及蛋白水平(分别为0.66±0.03和0.64±0.03)表达减少(P<0.05).结论 氧化应激使GRP78和Caspase-12表达增强,启动内质网应激,脂联素可以通过减轻内质网应激逆转H_2O_2所致的心肌细胞损伤及凋亡作用,对心肌细胞有保护作用.

The Effects of Different Concentrations of Adiponectin on the Expressions of GRP78 and Caspase-12 in Cardiomyocytes After Oxidative Stress

Aim To investigate the effects of adiponectin on endoplasmic reticulum stress injury of the 3 ～ 4 days SD rat cardiomyocytes, which was induced by the intervention of H_2O_2. Methods Primary cardiomyocytes were ob-tained from neonatal rat and cultured by enzymatic digestion methods. The morphology of neonatal rat cardiomyocytes was studied by phase contramicroscope. Its molecular markers were observed by α-actin immunocytochemistry. Primary 3 ～ 4 days cells were used in experiment, and they were randomly divided into control group, H_2O_2 group (200 μmol/L H_2O_2, 2 h) , H_2O_2 + 10 mg/L APN group, H_2O_2 + 20 mg/L APN group, and H_2O_2+ 30 mg,/L APN group. The change of mor-phology of cardiomyocytes was observed by electron microscope. The content of lactate dehydrogenase (LDH) was detec-ted by chemistry chromatometry; the cardiomycocytes apoptosis was detected by agarose gel electrophoresislevels and An-nexin V-FITC/PI flow cytometry. The expressions of GRP78 and caspase-12 were detected by Western Blotting and RT-PCR. Results Adiponectin pretreatment significantly decreased the apoptosis rate (43.6% ± 3.8% vs 70.7% ± 6.4%), and the release Of LDH (686.7±61.1 U/L vs 1411.5±189.7 U/L,P<0.05). The expression of GRP78 and caspase-12 attenuated(mRNA: 0.56±0.03 vs 1.25±0.50; 0.83±0.04 vs 1.32±0.15), (protein: 0.66 ±0.03 vs 0.92 ± 0.50; 0.64 ± 0.03 vs 1.24 ± 0.50). Conclusion Oxidative stress enhances the expressions of GRF78 and caspase-12, and makes endoplasmic reticttlum stress to start. Adiponectin which can attenuate endoplasmic reticulum stress, have a protective effect on myocardial cells.