生物钟基因在非酒精性脂肪肝发病中的作用

The role of circadian clock genes in the pathogenesis of non-alcoholic fatty liver

目的观察生物钟基因在非酒精性脂肪肝大鼠发病中的作用以及利拉鲁肽的影响。方法构建SD大鼠(湖南斯莱克景达实验动物有限公司)非酒精性脂肪肝模型,分为对照组、脂肪肝模型组、利拉鲁肽干预组;于给药后的第2周和第4周进行取样,每次取样按各时点(0、12、24 h)分成3组;苏木精-伊红(HE)染色观察肝脏变化;检测肝脏组织甘油三酯含量;检测各组时钟昼夜调节因子(Clock)、隐花色素抑制因子(Cry1)、Fas的相对表达及Wnt/β-连环蛋白(β-catenin)的相对表达。多组资料间的比较采用单因素方差分析(ANOVA),两两比较采用LSD法。结果高脂饮食2周后大鼠肝细胞中出现大量脂滴,利拉鲁肽可减少脂滴;模型组肝脏甘油三酯含量升高,利拉鲁肽干预后可使其含量下降;模型组肝脏Clock基因表达最高为第2周12 h,为3.0947±0.4848,经干预后下降至1.5786±0.3688(F=28.385,P<0.05),Cry1基因在第2周24 h表达最高,为5.5084±0.8518,而干预后进一步升高,达20.7757±1.9391(F=215.516,P<0.05),Fas表达第2周24 h在3.2881±0.2994,干预组有所下降,为1.5019±0.2812(F=77.128,P<0.05)。高脂饮食诱导大鼠肝脏中Clock和Cry1显著升高,增大Clock和Cry1以及Fas节律变化振幅范围,利拉鲁肽可抑制该变化。高脂饮食诱导大鼠肝脏Wnt/β-catenin蛋白高表达,可被利拉鲁肽抑制。结论利拉鲁肽或可通过调控生物钟基因以及Wnt/β-catenin信号通路来抑制非酒精性脂肪肝进展。

Objective To investigate the role of circadian clock gene in the pathogenesis of nonalcoholic fatty liver in rats and the mechanism of liraglutide blocking disease progression.Methods A non-alcoholic fatty liver model of SD rats was constructed.The animals were divided into control group,fatty liver model group,and liraglutide group.The samples were taken in the second and fourth weeks after administration,and each sample was taken at each time point(0,12,24 h).The changes of liver tissue were observed by hematoxylin and eosin(HE)staining.The triglyceride content of liver tissue was tested in each group.The relative expression levels of circadian locomotor output cycles kaput(Clock),Cryptochrome(Cry1)and Fas in each group were detected by polymerase chain reaction(PCR),and the relative expression levels of Wnt/β-catenin in each group were detected by Western blotting.Results A large number of lipid droplets appeared in rat hepatocytes after 2 weeks of high-fat diet feeding,and liraglutide could reduce lipid droplet formation.High-fat diet raised the contents of triglycerides in the liver,and liraglutide could decrease them.The highest expression of Clock gene in the liver of the model group was 3.0947±0.4848,and decreased to 1.5786±0.3688 after intervention(F=28.385,P<0.05).The expression of Cry1 gene was the highest at 24 h of the 2nd week,which was 5.5084±0.8518,and it further increased after intervention,reaching 20.7757±1.9391(F=215.516,P<0.05).The Fas expression at 24 h of the 2nd week was 3.2881±0.2994,and decreased to 1.5019±0.2812 in the intervention group(F=77.128,P<0.05).High-fat diet could induce significant increase of Clock,Cry1 and Fas levels and the amplitude range of Clock,Cry1 and Fas rhythm changes in rat liver,but this increase could be inhibited by liraglutide.High-fat diet could induce the high expression of Wnt/β-catenin protein in rat liver,but liraglutide could inhibit the high expression.Conclusion Liraglutide may block the progress of NAFLD by regulating Clock genes and Wnt/β-catenin signaling pathway.