钙池操纵的钙通道在乙醇诱导HepG2细胞钙超载中的作用

Role of store-operated Ca2＋ channels in ethanol-induced intracellular Ca2＋ increase in HepG2 cells

目的研究乙醇诱导肝细胞钙超载的机制及钙池操纵的钙离子通道（SOes）在其中的作用。方法使用浓度梯度的乙醇处理HepG2细胞，并观察细胞外钙离子螯合剂乙二醇二乙醚二胺四乙酸（EGTA）及SOCs抑制剂2-APB对200umol／L乙醇刺激的干预效应。实验分为：（1）正常对照组：磷酸盐缓冲液刺激；（2）乙醇慢性刺激组：分别给予25、50、100、200、400、800μmol／L乙醇，刺激24h或200μmol／L乙醇，刺激24、48、72h；（3）EGTA处理组：200μmol／L乙醇＋0．5μmol／LEGTA，刺激24h；（4）2-APB处理组：200μmol／L乙醇＋50μmol／L2-APB，刺激24h。细胞计数CCK8试剂盒检测细胞存活率；全自动生物化学分析仪检测HepG32细胞培养上清液ALT、AST漏出量；流式细胞仪检测HepG2细胞胞浆Ca2＋浓度。荧光定量聚合酶链反应及蛋白质印迹法检测HepG2细胞中SOCs通道蛋白分子间质相互作用因子1及钙释放激活钙通道蛋白1的基因及蛋白表达。各组间均数的比较采用单因素方差分析或独立样本t检验。结果50、100、200、400、800μmol／L乙醇刺激24h使HepG2细胞的存活率分别降低为97．3％±2．9％、83．4%±3．3％、67．8％±4．4％、37．5％±3．o％、14．1％±4．9％，组间比较，F=99．945，P〈0．01，细胞存活率呈浓度依浓度依赖性；细胞培养基上清液ALT漏出量分别增加为（14．2±2．8）、（17．7±3．2）、（20．0±2．6）、（21．6±1．3）、（24．9±1．7）U／L，组间比较，F=15．286，P〈0．01；AST漏出量分别增加为（72．0±4．7）、（91．6±7．4）、（107．3±11．4）、（116．5±13．4）、（128．9±7．1）u／L，组间比较，F=39．674，P〈0．01;使HepG2细胞的[Ca2＋]i水平分别增高为正常对照组的（1．3±0．4）、（1．3±0．2）、（1．4±0．2）、（2．3±0．3）、（2．6±0．2）倍，F=56．978，P〈0．01。EGTA、2-APB干预使200μmol／L乙醇刺激后的HEN32细胞[Ca2＋]i水平分别降至正常对照组的（1．8±0．2）倍和（1．9±0．1）倍，t值分别为7．201和8．183，P值均〈0．01;同时将乙醇刺激后的细胞存活率分别增高至82．2％±3．9%和78．6%±1．5％，t值分别为6．692和6．124，P值均〈0．01）；使细胞培养基上清液ALT漏出量分别降低至（16．4±2．0）U／L和（17．2±1．9）U／L，t值分别为7．145和6．352，P值均〈0．01;AST漏出量分别降低至（86．4±8．8）U／L和（88．3±6．3）U／L，t值分别为9．337和8．704，P值均〈0．01。200μmol／L乙醇刺激明显增加HepG32细胞STIM1及Orail的基因及蛋白表达量，且其表达增加持续至少72h。结论乙醇增高SOCs通道蛋白分子表达，增加SOCs介导的Ca2＋内流，提示此可能是乙醇导致肝细胞[Ca2＋]增高及相关肝细胞损伤的重要分子机制。

Objective To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels （SOCs）. Methods HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate （2-APB）. Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2＋ concentration （[Ca2＋]i） were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 （STIM1）, and calcium release-activated calcium channel protein 1 （Orail） were evaluated by qPCR and western blotting. Results The ethanol treatment produced dose-dependent reduction in cell viability （1- = -0.985, P 〈 0.01） and increases in leakage ofALT （F = 15.286, P 〈 0.01） and AST （F = 39.674, P 〈 0.01）. Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2＋]i level （1.25 ±0.36, 1.31 ± 0.15, 1.41 ±0.18, 2.29 ± 0.25, 2,58 ± 0.19; F = 15.286, P 〈 0.01）. Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2＋]i increase （2.32±0.08 reduced to 1.79 ± 0.15 （t = 7.201, P 〈 0.01） and 1.86 ±0.09 （t = 8.183, P 〈 0.01） respectively）. EGTA and 2-APB also increased the ethanol-treated cells＇ viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orail, and the up- regulation effect lasted at least 72 h after treatment. Conclusion Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2＋]i rise in hepatocytes and the related liver cell injury.