PERK/eIF2α通路在酒精性肝损伤大鼠肝细胞凋亡中的作用

Role of PERK/eIF2α signaling pathway in hepatocyte apoptosis of alcoholic liver injury rats

目的 探讨内质网类似激酶（PERK）/真核生物翻译起始因子（eIF）2α信号通路在酒精性肝损伤大鼠肝细胞凋亡中的作用.方法 建立大鼠酒精性肝损伤模型.设4、6、10周和12周4个时间点,动态观察肝组织病理变化;流式细胞术检测肝细胞凋亡率;酶联免疫吸附法检测血清同型半胱氨酸（tHCY）水平;实时荧光定量聚合酶链反应和Western blot检测肝组织PERK/eIF2α通路信号分子mRNA和蛋白的表达水平.多组样本均数的两两比较采用One-Way ANOVA分析.结果 4周时造模大鼠发生急性肝损伤改变,12周时则出现慢性肝损伤改变;6周时造模大鼠肝细胞凋亡率较正常组显著增加（P＜0.05）,随着造模时间的延长,肝细胞凋亡程度逐渐加剧,12周时早期和总凋亡率分别达到26%和29%;自6周起,造模大鼠血清tHCY水平明显高于正常大鼠（P＜0.01）;自4周起,造模大鼠肝组织eIF-2α蛋白发生明显磷酸化,12周时peIF-2α蛋白表达量上升了2.81倍（P＜0.01）,葡萄糖调节蛋白（GRP） 78/Bip、GRP94、caspase 12和caspase-3则表现为过度活化,12周时基因和蛋白表达量分别为正常大鼠的4.70、12.95、3.83、4.05倍和3.93、6.93、9.88、3.31倍（P＜0.01）.结论 PERK/eIF2α通路的活化与酒精性肝损伤大鼠肝细胞凋亡的发生和持续发展密切相关.

Objective To investigate the role of PERK/eIF2α signaling pathway in hepatocyte apoptosis of alcoholic liver injury rats. Methods Rat models with ethanol-induced liver injury were successfully developed by gastric gavage with ethanol-corn oil mixtures for 12 weeks. At different time points （4, 6,10, 12 week）, liver pathology was dynamically observed. The hepatocyte apoptosis was quantitatively analyzed by Annexin V-FITC/PI double-labeled flow cytometry, the serum total homocysteine （tHCY） level was detected by ELISA and the expressions of eIF2α, p-eIF2α, GRP78/Bip, GRP94, caspase-3 and caspase-12 in liver were examined using Real-time PCR and Western blot. Results Typical acute liver injury and chronic liver injury were observed at week 4 and week 12 respectively. The hepatocyte apoptosis rates in 6-week model rats significantly increased compared with normal rats （P 〈 0.05）, and the degree of hepatocyte apoptosis continued to increase with the modeling time, and the percentages of early and total apoptosis reached 26% and 29% at week 12. From week 6 to week 12, the serum tHCY levels in model rats were obviously higher than in normal rats （P 〈 0.01）. Since week 4, eIF2α protein phosphorylation in model rat livers remarkly elevated compared with that in normal rat livers （P 〈 0.01）, and at week 12 the peIF2α protein expression in model rat livers increased by 2.81-fold. Since week 4 the expressions of GRP78/Bip,GRP94, caspase-12 and caspase-3 mRNA and protein in model rat livers showed a significant increase as compared to normal rat livers, and at week 12, these gene and protein levels increased 4.70, 12.95, 3.83, 4.05 fold and 3.93, 6.93, 9.88, 3.31 fold, respectively （P 〈 0.01）. Conclusion Activation of PERK/eIF2α signaling pathway contributes to the occurrence and development of hepatocyte apoptosis in alcoholic liver injury rats and it might be as a potential target for therapeutic applications in alcoholic liver diseases.