齐墩果酸衍生物DKS26对非酒精性脂肪肝病小鼠的作用及分子机制

Effect of oleanolic acid derivative DKS26 on mice with non-alcoholic fatty liver disease and its molecular mechanism

目的探讨齐墩果酸衍生物DKS26对非酒精性脂肪肝病(NAFLD)小鼠的作用及分子机制。方法40只健康5周龄C57/BL小鼠随机均分为对照(CON)组、模型(MOD)组、二甲双胍(MET)组及DKS26组,后3组小鼠用40%CCl 4油溶液皮下注射加高脂饮食喂养、建立小鼠NAFLD模型,MET组和DKS26组小鼠分别给予100 mg/(kg·d)的MET和DKS26灌胃干预、CON组和MOD组小鼠给予100 mg/(kg·d)羧甲基纤维素钠(CMC-Na)溶液灌胃干预,1次/d,共6周;末次干预后麻醉各组小鼠,眼球取血,采用微板法检测血清丙氨酸氨基转移酶(ALT)和天门冬氨酸转移酶(AST)水平;取血后处死各组小鼠,取肝右叶组织,制作匀浆采用微板法检测各组小鼠肝组织甘油三脂(TG)含量,制作切片采用油红O染色和苏木素伊红(HE)染色观察各组小鼠肝组织中脂滴沉积情况及组织形态学特征,采用Western blot法检测各组小鼠肝组织中叉头转录因子O1(FoxO1)和磷酸烯醇式丙酮酸激酶(PEPCK)蛋白的表达。结果与CON组相比,MOD组小鼠血清ALT和AST水平及肝组织TG含量明显上升(P<0.01),肝细胞空泡样变明显、胞质脂质沉积过多,FoxO1与PEPCK蛋白表达上调(P<0.05);与MOD组比较,MET组和DKS26组小鼠血清ALT、AST水平及肝组织TG含量下降(P<0.01),且DKS26组小鼠血清ALT水平较MET组降低(P<0.05);病理形态学显示,MET组和DKS26组小鼠肝脏空泡变性及脂质沉积较MOD组改善,肝组织FoxO1和PEPCK蛋白表达下调(P<0.05),但组间无差异(P>0.05)。结论口服DKS26可改善NAFLD小鼠肝功能损害与肝细胞脂质沉积,其机制可能与调控FoxO1信号通路介导的下游糖脂代谢关键酶PEPCK有关。

Objective To investigate the effect of oleanolic acid derivative DKS26 on mice with non-alcoholic fatty liver disease(NAFLD)and its molecular mechanism.Methods Forty healthy 5-week-old C57/BL mice were randomly divided into control(CON),model(MOD),metformin control(MET)and DKS26 groups.The mice in the last 3 groups were fed with high-fat diet and subcutaneously injected with 40%CCl4 oil solution to establish mouse NAFLD model.MET and DKS26 groups were given with 100 mg/(kg·d)MET and DKS26 by gavage administration,respectively.CON and MOD groups were given with 100 mg/(kg·d)sodium carboxymethyl cellulose(CMC-Na)solution by gavage administration,once a day for a total of 6 weeks.After the last administration,the mice in each group were anesthetized,and blood was collected from retro-orbital venous sinus.The serum levels of alanine aminotransferase(ALT)and aspartate transferase(AST)were detected by microplate method.After blood collection,the mice in each group were sacrificed.The right lobe tissues of mouse livers were collected,homogenized for measuring triglyceride(TG)contents by microplate method,sectioned for red O staining and hematoxylin eosin(HE)staining to observe lipid droplet deposition and histopathological characteristics.Western blot was applied to detect the protein expressions of liver FoxO1 transcription factor and phosphoenol pyruvate carboxykinase(PEPCK).Results When compared with CON group,MOD group had significantly increased serum ALT,AST,and liver TG(P<0.01),obvious vacuole-like hepatic cells,excessive cytoplasmic lipid deposition and upregulated FoxO1 and PEPCK proteins(P<0.05).When compared with MOD group,the serum ALT and AST levels and liver TG content in MET and DKS26 groups were decreased(P<0.01).Serum ALT level in DKS26 group was decreased when compared with MET group(P<0.05).The histopathology results showed that vacuolar-like heaptic cells and lipid deposition were improved in MET and DKS26 group when compared with MOD group,and the expressions of FoxO1 and PEPCK in liver tissue were downregulated(P<0.05),but there were no differences between the groups(P>0.05).Conclusion Oral administration of DKS26 can improve liver function impairment and lipid deposition in the mice with NAFLD,and its mechanism might be related to the regulation of PEPCK,a key enzyme in downstream of glucolipid metabolism mediated by FoxO1 signaling pathway.