Objective To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury.Methods The in vitro model of chronic ethanol-induced hepatocyte injury was established using primary hepatocytes isolated from Sprague-Dawley rats.Ethanol-induced changes (24,48 and 72 h; 50,100,200,400 and 800 mmol/L) in expression of the SOCs proteins stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Oria1) were detected by qualitative PCR analysis (mRNA) and western blotting (protein).The possible role of these two SOCs proteins in the ethanol-induced extmcellular calcium influx and related liver cell injury was determined by treating the cell system with various channel blockers (EGTA,La3+,and 2-APB).Cell viability was determined by MTT assay and cytosolic free calcium ion concentration was determined by flow cytometry.Results After 24 h of exposure to 0 (untreated) to 800 mM/L ethanol,the cell viability was reduced in a concentration-dependent manner.The 400 mmol/L concentration of ethanol decreased cell viability by 57.34％ ± 2.34％.and was chosen for use in subsequent experiments.Compared with the untreated control cells,the ahanol-treated cells showed significantly up-regulated mRNA and protein expression of both STIM1 and Omil at all times examined,suggesting that the ethanol-stimulated expression of STIM1 and Orai1 could persist for at least 72 h.The ethanol treatment induced increase in cytoplasmic calcium levels was significantly (and similarly) reduced by co-treatment with any of the three channel blockers.Conclusion Chronic ethanol exposure can increase the expression of STIM1 and Ora il in primary liver cells,suggesting that ethanol may increase extracelhlar calcium influx by up-regulating expression of these SOCs protein molecules,ultimately aggravating liver cell damage.%目的 研究钙池操纵的钙离子通道(SOCs)在乙醇诱导的原代肝细胞钙超载及损伤中的作用.方法 通过建立体外乙醇诱导原代肝细胞慢性损伤模型,检测SOCs通道蛋白分子:间质相互作用因子1 (STIM1)及钙释放激活钙通道蛋白1(Oria1)的表达量及胞质中游离钙离子浓度([Ca2+]cyt)的变化,并利用通道阻断剂鉴定SOCs在慢性乙醇诱导原代肝细胞钙超载及损伤中的作用.两组之间均数的比较采用t检验,多组之间的比较采用完全随机设计单因素方差分析.结果 0 ～ 800mmol/L乙醇(刺激24 h)浓度依赖性降低原代肝细胞的存活率；400 mmol/L乙醇刺激时细胞存活率为57.34％±2.34％ (MTT),因此选择400 mmol/L乙醇作为下一步的刺激实验浓度.与正常对照组相比,400 mmol/L乙醇刺激明显增加原代肝细胞STIM1及Orai1的基因及蛋白表达量,且其表达增加持续至少72 h; SOCs通道阻断剂EGTA、La3+、二氨基乙氧基双苯萘酯(2-APB)干预降低400mmol/L乙醇刺激原代肝细胞增高的[Ca2+]cyt水平、增高原代肝细胞存活率,三者之间差异无统计学意义.结论 乙醇可能通过增高原代肝细胞中SOCs通道蛋白分子表达,增加胞外钙离子内流而加重肝细胞钙超载及损伤.
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