CAJ | 학술논문

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.
목적 탐토내질망유사격매(PERK)/진핵생물번역기시인자(eIF)2α신호통로재주정성간손상대서간세포조망중적작용.방법 건립대서주정성간손상모형.설4、6、10주화12주4개시간점,동태관찰간조직병리변화;류식세포술검측간세포조망솔;매련면역흡부법검측혈청동형반광안산(tHCY)수평;실시형광정량취합매련반응화Western blot검측간조직PERK/eIF2α통로신호분자mRNA화단백적표체수평.다조양본균수적량량비교채용One-Way ANOVA분석.결과 4주시조모대서발생급성간손상개변,12주시칙출현만성간손상개변;6주시조모대서간세포조망솔교정상조현저증가(P＜0.05),수착조모시간적연장,간세포조망정도축점가극,12주시조기화총조망솔분별체도26%화29%;자6주기,조모대서혈청tHCY수평명현고우정상대서(P＜0.01);자4주기,조모대서간조직eIF-2α단백발생명현린산화,12주시peIF-2α단백표체량상승료2.81배(P＜0.01),포도당조절단백(GRP) 78/Bip、GRP94、caspase 12화caspase-3칙표현위과도활화,12주시기인화단백표체량분별위정상대서적4.70、12.95、3.83、4.05배화3.93、6.93、9.88、3.31배(P＜0.01).결론 PERK/eIF2α통로적활화여주정성간손상대서간세포조망적발생화지속발전밀절상관.
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.