载脂蛋白J对缺氧/复氧诱导的乳鼠心肌细胞损伤的影响

Effect of apolipoprotein-J on hypoxia/reoxygenation induced myocardial cells injury in neonatal rat

目的观察载脂蛋白J(ApoJ)对缺氧/复氧(H/R)诱导的乳鼠心室肌细胞损伤的影响及其相关的信号传导通路。方法用携带ApoJ基因的重组腺病毒感染乳鼠心肌细胞使ApoJ高表达。利用三气培养箱建立H/R模型,SOD类似物Mn(Ⅲ)TBAP和真核细胞翻译起始因子2α(eIF2α)去磷酸化抑制剂Salubrinal提前预处理,将心肌细胞分成对照组、H/R组、ApoJ组、ApoJ+H/R组、Mn(Ⅲ)TBAP+H/R组、Salubrinal+H/R组。利用MTT法检测细胞的存活率;酶联免疫吸附试验(ELISA)测定细胞乳酸脱氢酶(LDH)的漏出量、凋亡蛋白酶半胱天冬酶-3/7(caspase-3/7)及细胞内超氧化物歧化酶(SOD)活性;Western blot检测ApoJ、氧化酶Nox2/gp91phox、内质网特异性凋亡蛋白caspase-12、CHOP的表达及eIF2α的磷酸化水平。结果 ApoJ基因重组腺病毒转染后,心肌细胞ApoJ蛋白高表达。与对照组相比,H/R组细胞存活率和SOD的活性明显下降,LDH的漏出量及caspase-3/7的活性升高,Nox2/gp91phox、caspase-12、CHOP蛋白的表达明显升高,eIF2α的磷酸化水平升高。与H/R组相比,ApoJ组、Mn(Ⅲ)TBAP组及Salubirnal组LDH的漏出量及caspase-3/7的活性明显降低,ApoJ高表达使细胞存活率和SOD的活性显著升高,Nox2/gp91phox、caspase-12、CHOP蛋白的表达明显下降,而eIF2α的磷酸化水平显著升高。结论 ApoJ通过抗氧化应激及内质网应激的作用,减轻H/R诱导的心肌细胞损伤。

Objective To evaluate the effects of apolipoprotein-J（ApoJ）on hypoxia/reoxygenation（H/R）induced neonatal rat ventricular cells（NRVCs）injury and its related signaling pathways.Methods ApoJ high expression was achieved by infection with recombinant adenovirus in NRVCs.The three gas incubator was used to establish H/R model,SOD mimetic Mn（Ⅲ）tetrakis（4-benzoic acid）porphyrin chloride and eIF2αdephosphory inhibitor Salubrinal were performed the pretreatment.The NRVCs were divided into four groups：normal control group,H/R,ApoJ group,ApoJ＋H/R group,Mn（Ⅲ）TBAP＋H/R group and Salubrinal＋H/R group.The cell viability was measured by MTT assay;the leakages of LDH,the expression of SOD and caspase-3/7activity were detected by ELISA.The protein expression levels of ApoJ,Nox2/gp91 phox,caspase-12,CHOP,and the phosphorylation level of eukaryotic initiation factor 2α（eIF2α）were determined by Western blot.Results ApoJ protein in myocardial cells was highly expressed after infection by recombinant adenoviru.Compared with the control group,the cell viability and the activity of SOD were significantly decreased,the leakages of LDH and the activity of caspase-3/7were increased in the H/R group,the protein expression level of Nox2/gp91 phox,caspase-12 and CHOP and the phosphorylation level of eIF2αwere increased.Compared with the H/R group,the leakages of LDH and the activity of caspase-3/7in the ApoJ overexpression group,Mn（Ⅲ）TBAP group and Salubirnal group were significantly decreased.ApoJ overexpression significantly increased the cell viability and the activity of SOD.Moreover,the protein expression level of Nox2/gp91 phox,caspase-12 and CHOP were significantly decreased,while the phosphorylation level of eIF2αwas markedly increased.Conclusion ApoJ alleviates H/R induced myocardial cellular injury by antioxidative stress and endoplasmic reticulum stress.