Nrf2及其相关因子在非酒精性脂肪肝炎形成过程中的作用

Effect of Nrf2 and related factors on the progression of nonalcoholic steatohepatitis

目的:通过观察大鼠非酒精性脂肪肝炎(NASH)形成过程中脂质代谢、肝组织病理学改变、核因子E2相关因子2(Nrf2)及相关关键因子的转录和蛋白水平的变化,探讨Nrf2及其相关因子在NASH形成过程中的作用。方法:SD大鼠分为正常组和模型组,以饲喂高脂饲料建立非酒精性脂肪肝炎模型,分别于4、12周末处死,检测血清和肝组织中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白胆固醇(LDL-C)含量;油红O染色法检测肝组织内脂肪沉积变化,常规HE染色观察肝组织病理学改变,计算NAFLD活动度积分(NAS),免疫组化检测肝组织Nrf2表达;Real-time PCR和Western blot技术检测肝组织Nrf2及其相关因子mRNA和蛋白表达水平。结果:14周模型组大鼠血清ALT、AST、TC和肝组织TC、TG、LDL-C等指标较同期正常组均显著增高(P<0.01,P<0.05),12周模型组大鼠血清、肝组织脂质含量持续增高(P<0.01,P<0.05),肝组织HDL-C较正常组显著降低(P<0.05),比4周变化明显。24周和12周模型组大鼠肝细胞内沉积大量脂肪滴,肝细胞脂肪变严重,伴有肝细胞气球样变;且随着高脂饮食喂养时间的延长,肝组织内脂肪沉积以及肝细胞脂肪变程度明显加重,NAFLD评分、Nrf2表达强度均显著增高(P<0.01)。34周、12周模型组大鼠Nrf2、HO1、NQO1、rGCS、GST的mRNA和蛋白表达均有不同程度的上调或抑制,且12周比4周变化明显(P<0.01,P<0.05)。结论:Nrf2及相关因子可能参与了非酒精性脂肪性肝病的发生发展过程,在NASH形成过程中起着重要的作用。

Objective： To explore the role of NF-E2-related factor 2（Nrf2） and its related factors in the progression of nonalcoholi steato- hepatitis （NASH） by investigating the alterations of lipid metabolism and liver histopathology as well as the changes of mRNA and protein ex- pression levels of Nrf2 and its related factors in rats during NASH progression. Methods： Male SD rats were randomly divided into normal group and model group, which were administrated with high fat diet to establish nonalcoholic steatohepatitis model. The rats from both groups were randomly killed at the end of 4, 12 weeks respectively. The levels of alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, total cholesterol （TC）, triglyceride （TG）, high density lipoprotein cholesterol （HDL-C）, low density lipoprotein cholesterol （LDL-C） were detected in the serum and liver tissue; Changes in fat deposition in liver tissue were determined by oil red O staining. HE staining were used to observe the pathological changes of liver tissue and to calculate nonalcoholic fatty hver disease （NAFLD） activity score （hepatic steatosis, inflammation and ballooning degeneration of liver cells）. The expression of Nrf2 in liver was detected by staining. The mRNA and protein levels of Nrf2 and related factors in liver were determined by Realtime PCR and Western blot, respectively. Results： After 4 weeks of high fat diet, the levels of ALT, AST, TC in rat serum and TC, TG, LDL-C in liver were significantly increased compared with that of the normal group （ P 〈 0.01, P 〈 0.05）. After 4 weeks of high fat diet, the levels of ALT, AST, TC, TG in serum and TC, TG, LDL- C in liver increased further （P 〈 0.01, P 〈 0.05）. Until the 12th week, the content of HDL-C in liver was significantly lower than that of the normal group （ P 〈 0.05）. At the end of the 4th or the 12th week, lipid droplets in the model rat liver cells were heavily dyed red and hepatic steatesis increased severely, with ballooning degeneration of hver cells. With the extension of high fat diet feeding time, fat deposition in the liver tissue, hepatic steatesis, NAFLD score, Nrf2 expression were significantly increased （ P 〈 0.01）. Expression levels of mRNA and protein of Nrf2, heme oxyenase 1 （ HO1 ）, NADPH quinone oxidoreductase 1 （NQO1）, y-glutamylcysteine synthethase （T-GCS）, glutathione S-trans- ferase （GST） in the model rats increased or decreased at the end of the 4th or the 12th week differentially, （ P 〈0.01, P 〈0.05） with the more significant changes at the end of the 4th week than the 12th week. Conclusion： Nrf2 and its related factors may be involved in the occur- rence and development of nonalcoholic fatty liver disease, which may play an important role in the process of NASH formation.