Fas/FasL信号传导通路对NAFLD大鼠肝细胞凋亡的影响

摘要

Objective To investigate the roles of death receptor-mediated pathways in hepatocye apoptosis on nonalcoholic fatty liver discase(NAFLD) rats and to probe into the pathogcncsis of NAFLD. Methods Hepatocyte apoptosis index( AI) was detected by flow cytometry(FCM). The exprcssions of Fas and FasL were detected by immunohistochemistry method.Meanwhile, the ex prcssions of Caspase-8 was detected by real-time flurescent quantitive polymerase chain reaction. Results HE staining revealcd that fatty degeneration at 4 week, mild fatty liver at 8 w, moderate to severe fatty liver at 12 week in high-fat diet group , and electron mi croscopy showed ultrastructural changes in hepatocytes high-fat dict group (12 week) ,including swelling of hepatic mitochondria, shortened or even disappearance of crests.decrease of rough surfaced endoplasmic reticulum,and nuclear shape was irregular. AI of nonalcoholic stcatosis rat model of 4-12 weeks was significantly higher than that of normal. The percentage of hepatocyte apopto sis increased significantly as the time going. Immunohistochcmical staining showed that Fas and FasL expression in high-fat diet group , the degree of fatty changes and inflammatory staining depened and expanded , the number of positive cells increased , showed a diffuse positive. Real-time quantitative PCR assay showed that the relative expression quantity of Caspase-8 mRNA in model group was significant higher than the control group at the same time. There was also a significant differcnce in model group of different time point. Conclusion In rat model of NAFLD,the degree of hepatocyte apoptosis is closely related to the degree of liver injury. Pathological hepatocyte apoptosis promotes the progress of NAFLD. The severity of hepatocyte apoptosis is consistent with the Fas, FasL protein expression,the increase degree of Caspasc-8 mRNA expression in the development of NAFLD.%目的 通过研究肝细胞凋亡及死亡受体信号转导通路在大鼠非酒精性脂肪性肝病(NAFLD)形成中的作用,探讨NAFLD的发病机制.方法 将30只雄性SD大鼠随机分为对照组和高脂饲料(4、8、12周)组;HE染色观察肝组织光镜下的病理改变;电镜观察肝细胞超微结构变化;流式细胞仪检测肝细胞凋亡百分数;免疫组化法检测Fas、FasL在肝组织中的蛋白表达;实时荧光定量PCR法检测半胱天冬酶-8(Caspase-8)mRNA表达.结果 HE染色结果显示,对照组大鼠肝脏无异常发现,高脂组大鼠肝脏4周出现脂肪变,8周出现轻度脂肪肝,12周出现中、重度脂肪肝;电镜下观察高脂组大鼠肝细胞线粒体肿胀,嵴变短、减少甚至消失,粗面内质网减少,核型欠规则;流式细胞仪检测显示,与对照组比较,高脂组大鼠肝细胞凋亡百分数增加,随造模时间延长凋亡率增加更明显;免疫组化染色显示随着肝脏脂肪变加重,Fas、FasL蛋白染色加深,阳性细胞数增加;实时荧光定量PCR法显示Caspase-8 mRNA表达量在高脂组中显著高于对照组,且随肝脏脂肪变及炎症加重呈进行性上升.结论 NAFLD大鼠模型中,肝细胞凋亡促进NAFLD大鼠病情进展;肝细胞凋亡以及Fas、FasL、Caspase-8 mRNA相关调控蛋白的活化是引起NAFLD脂肪变性、炎症及纤维化的重要因素.