摘要目的 探讨内质网应激和有丝分裂原活化蛋白激酶(MAPK)信号在蛋氨酸一胆碱缺乏饮食( MCDD)诱导的非酒精性脂肪性肝纤维化肝细胞凋亡中的作用.方法 (1)MCDD喂养10周诱导非酒精性脂肪性肝纤维化大鼠模型(M组),恢复组(R组)于第9周开始将MCDD转变为蛋氨酸一胆碱对照饮食(MCCD)喂养2周；(2)评价肝细胞脂肪变性、纤维化和炎性反应程度,采用免疫组织化学、蛋白印迹或real time-PCR(RT-PCR)方法评价肝星状细胞活化；(3)采用4一羟基壬烯酸(4-HNE)、HO-1/2免疫组织化学、蛋白印迹或RT-PCR方法评价肝细胞氧化损伤；(4)肝细胞凋亡采用TUNEL染色法评价；(5)采用RT-PCR及蛋白印迹方法评价内质网应激相关细胞因子ERP78、caspase-12、caspase-7、cleaved caspase-7、caspase-9、caspase-3、cleaved caspase-3,以及MAPK信号相关细胞因子c-Jun、ERKl/2、p-ERKl/2.结果 MCDD转换为MCCD后,肝细胞脂肪变性、炎性反应、氧化损伤及肝纤维化程度明显减轻.与正常组比,M组ERP78、caspase-12、caspase-7、cleaved caspase-7蛋白表达均显著增加(P ＜0.05或＜0.01),RT-PCR分析显示ERP78、caspase-12、caspase-7 mRNA表达水平亦显著增加(灰度值分别为3.03 ±0.41比2.12±0.37,1.86±0.36比0.78 ±0.20,2.38 ±0.19比1.84±0.13,P＜0.05或＜0.01).但caspase-3、cleaved caspase-3蛋白表达以及caspase-3、caspase-9mRNA表达在正常组、M组和R组之间差异无统计学意义(P＞0.05)；M组与正常组比c-Jun mRNA表达升高(0.19 ±0.03比0.11 ±0.05,P＜0.05),R组降低(与M组相比,0.13±0.04比0.19±0.03,P＜0.05)；ERK1和p-ERK1蛋白表达在M组均升高(P＜0.01),R组均降低(与M组比,P＜0.01).结论 内质网应激相关的caspase-12凋亡途径可能不是非酒精性脂肪性肝纤维化肝细胞凋亡的主要机制,而MAPK信号可能在该模型肝细胞凋亡中发挥重要作用.

abstractsObjective To clarify the effects of endoplasmic reticulum stress (ER stress ) and mitogen-activated protein kinase (MAPK) on hepatocyte apoptosis in rats with non-alcoholic fatty liver fibrosis induced by methionine-choline-deficient diet (MCDD).Methods Nonalcoholic steatohepatitis with advanced fibrosis was induced in rats by giving a MCDD for l0 weeks(group M). A methionine-cholinecontrol diet (MCCD) instead of MCDD was given for the last 2 weeks to the experimental group( group R).Steatosis,fibrosis and inflammation were determined by tissue staining. The activation of hepatic stellate cells and oxidative stress were determined by immunostaining,immunoblotting or real time-PCR (RT-PCR),respectively. Hepatocyte apoptosis was determined by TUNEL staining. Expressions of glucose-regulated protein 78 (GRP78),caspase-12,caspase-7,cleaved caspase-7,caspase-3,cleaved caspase-3,and caspase-9 were evaluated to clarify the presence of ER stress. Expressions of c-Jun,ERK1/2,p-ERK1/2were evaluated to clarify the states of MAPK signaling.Results Changing the diet from MCDD to MCCD triggered the reduction of fat in hepatocytes,a decrease in inflammatory response,oxidative stress,and fibrosis.The protein expressions of ERP78,caspase-12,caspase-7,and cleaved caspase-7 were increased significantly in group M compared with normal control group( group N,P ＜ 0.05 or P ＜ 0.01 ),the mRNA expressions of ERP78,caspase-12,and caspase-7 were also increased significantly in group M compared with groupN (3.03±0.41 vs 2.12 ±0.37,1.86±0.36 vs0.78±0.20,and 2.38 ±0.19 vs 1.84±0.13,respectively,P ＜ 0.05 or P ＜ 0.01 ),while they recovered immediately in group R.In contrast,the protein levels of caspase-3,cleaved caspase-3 and mRNA expressions of caspase-3 and easpase-9 revealed no significant differences in three groups (P ＞ 0.05).The mRNA expressions of c-Jun and protein levels of ERK1 and p-ERK1 were increased significantly in group M compared with group N (P ＜0.01 ),while they recovered immediately after changing the diet from MCDD to MCCD.Conclusions ER stress plays a role in the development and regression of non-alcoholic fatty liver fibrosis induced by MCDD,however,ER stressrelated caspase-12 pathway may not be the main mechanism of hepatic apoptosis,and MAPK signaling may play an important role in hepatic apoptosis in the model.