王家玉?周浩雄?田轩?郭云蔚

【摘要】目的 探究內质网应激抑制剂GSK2606414在预防酒精联合棕榈酸诱导的AML12细胞凋亡的作用及机制。方法 选取C57BL/6雌性小鼠10只，随机分为对照组（n = 5）及模型组（n = 5）。采用Lieber-DeCarli液体饮食诱导小鼠酒精性脂肪肝模型。免疫组织化学法、蛋白免疫印迹法及实时定量PCR法检测内质网应激相关蛋白活化转录因子4（ATF4）和CCAAT/增强子结合蛋白同源蛋白（CHOP）的表达水平。使用酒精加棕榈酸处理AML12鼠正常肝细胞24 h后检测ATF4及CHOP的改变情况。利用GSK2606414抑制剂预处理AML12细胞，随后使用酒精加棕榈酸处理，蛋白免疫印迹法检测ATF4及CHOP的表达，TUNEL法检测细胞凋亡情况。结果 血清ALT、AST水平及病理结果均证实酒精性脂肪肝小鼠模型建立成功，模型组ATF4和CHOP蛋白的表达高于对照组（P均< 0.001）。酒精加棕榈酸刺激AML12肝细胞后ATF4和CHOP的蛋白表达水平升高（P均< 0.001）。使用GSK2606414抑制剂预处理AML12细胞与未预处理组相比，ATF4和CHOP的蛋白表达受到抑制（P均< 0.05），细胞凋亡减少。结论 GSK2606414抑制剂可改善酒精联合棕榈酸诱导的AML12细胞凋亡，其机制可能与其能抑制内质网应激有关。

【关键词】酒精性脂肪肝;内质网应激;GSK2606414;细胞凋亡

Effect and mechanism of GSK2606414 on the apoptosis of AML12 cells induced by alcohol combined with plamitic acid Wang Jiayu， Zhou Haoxiong， Tian Xuan， Guo Yunwei. Department of Gastroenterology， the Third Affiliated Hospital of Sun Yat-sen University， Guangzhou 510630， China

Corresponding author， Guo Yunwei， E-mail： guoyw1973@ hotmail. com

【Abstract】Objective To explore the role and mechanism of endoplasmic reticulum stress inhibitor GSK2606414 in preventing the apoptosis of AML12 cells induced by alcohol combined with palmitic acid. Methods C57BL/6 female mice were randomly divided into the control group （n = 5） and model group（n = 5）. The mouse models with alcoholic steatohepatitis were established by Lieber-DeCarli liquid diet. Immunohistochemistry， Western blot and RT-PCR were adopted to detect the expression levels of endoplasmic reticulum stress-related proteins ATF4 and CHOP. After normal liver cells of AML12 mice were treated with alcohol and palmitic acid for 24 h， the changes in the expression levels of Activating transcription factor-4（ATF4） and CCAAT/Enhancer binding protein homologous protein （CHOP） were detected. AML12 cells were pretreated with endoplasmic reticulum stress inhibitor GSK2606414， and then treated with alcohol and palmitic acid. Western blot was employed to detect the expression levels of ATF4 and CHOP， and TUNEL assay was utilized to determine the cell apoptosis. Results Serum ALT and AST levels and pathological results confirmed that the alcoholic steatohepatitis mouse model was successfully established. The expression levels of ATF4 and CHOP proteins in the model group were significantly higher than those in the control group （both P < 0.001）. The expression levels of ATF4 and CHOP protein were significantly up-regulated after AML12 hepatocytes were stimulated with alcohol and palmitic acid （both P < 0.001）. Compared with the non-pretreatment group， the expression levels of ATF4 and CHOP proteins were significantly inhibited and cell apoptosis was remarkably reduced in the AML12 cells pretreated with GSK2606414 inhibitor （both P < 0.05）. Conclusion GSK2606414 can mitigate the AML12 cell apoptosis induced by alcohol combined with palmitic acid， probably due to the inhibitory effect upon the endoplasmic reticulum stress.

【Key words】Alcoholic steatohepatitis;Endoplasmic reticulum stress;GSK2606414;Apoptosis

酒精性肝病是由于长期饮酒引起的肝损伤，是世界范围内慢性肝病的常见原因之一，可由单纯性酒精性脂肪肝发展为酒精性肝炎、肝纤维化、肝硬化甚至肝癌[1-3]。内质网是真核细胞的重要细胞器，在分泌蛋白的合成和加工，脂质合成以及钙的存储与释放中起重要作用[4]。它对内外环境刺激极其敏感，当其功能紊乱时导致错误折叠与未折叠蛋白在腔内聚集。近年来，酒精诱导的内质网应激和细胞蛋白质稳态的破坏已被认为是导致肝脏疾病的重要机制[5-7]。在本研究中，我们应用GSK2606414作为内质网应激抑制剂，探讨其在酒精性脂肪肝的作用及机制，这为预防治疗酒精性脂肪肝损伤提供了新的线索。……