The Ameliorative Effect And Mechanism Of Oleanolic Acid Derivative DKS26 On Nonalcoholic Fatty Liver Disease In Mice

Objective:To observe whether oleanolic acid derivative DKS26 has any ameliorative effect on Nonalcoholic Fatty Liver Disease in mice,and to explore its possible mechanism of action.Methods:Forty 5-week-old C57/BL mice were randomly selected and divided into c ontrol group（CON）,Nonalcoholic fatty liver disease model group（MOD）,Met formin positive control treatment group（MET）and DKS26 treatment group（D KS26）groups,with 10 mice in each group.Except for the normal control gro up,mice in the three groups were fed by high-fat diet plus subcutaneous inject ions of 40%CCl₄ vegetable oil（1 time/3d）for 4 times,in total 8 weeks,to e stablish the mouse NAFLD model.After modeling,mice in the MET and DKS26 groups were given Metformin and DKS26（100mg/kg/d）by gavage,while mice in the CON and MOD groups were given the same volume of sodium c arboxymethylcellulose by gavage for a total of 6 weeks.At the end of the exp eriment,blood and liver were collected from the rats,and the serum biochemic al indexes ALT,AST and TG content of liver tissues were measured by micro plate method;oil red O staining was performed to observe the deposition of li pid droplets in liver tissues;HE staining was performed to detect the morpholo gical changes of liver tissue pathology;Western Blot method was used to dete ct the liver tissue Fndc5/Irisin,PI3K,p-PI3K,Akt,p-Akt,Fox O1 and PEPCK protein expression changes were detected by Western Blot;RT-q PCR was perfo rmed to detect Fox O1 and PEPCK gene expression.Results:（1）Compared with the CON group,the serum ALT and AST levels and liver tissue TG content in the MOD group were significantly increased（P<0.01）,and the pathological histomorphology showed liver vacuole degeneration,and there were more oily red stained lipid deposits in the liver.WB assay found that Fndc5/Irisin（P<0.05）,PI3K,p-PI3K and p-Akt expressions were significantly down-regulated（P<0.01）in the liver tissues of mice in the MOD group.Compared with the CON group,Akt protein expression was not significantly changed in the liver tissues of mice in the MOD group;total Fox O1 and Fox O1 of nucleus protein expression were significantly up-regulated（P<0.05）in the liver tissues of mice in the MOD group,while the expression of Fox O1 protein in the cytoplasm of liver tissues was significantly decreased（P<0.01）in the liver tissues of mice in the MOD group;the expression of PEPCK protein in the liver of mice in the MOD group was significantly up-regulated compared with that in the control group（P<0.01）.RT-q PCR revealed that Fox O1 and PEPCK m RNA expression was significantly upregulated in the liver tissues of mice in the MOD group compared with the CON group（P<0.01）.（2）Compared with the MOD group,the serum ALT and AST levels and the TG content of liver tissues in the MET and DKS26 groups decreased significantly（P<0.01）,and the decrease in serum AST level in the DKS26 group was more obvious than that in the MET group（P<0.01）,which was close to the normal level;pathological histomorphology showed that the fatty vacuole-like degeneration and oil-red stained lipid in the liver of mice in both treatment groups were significantly improved compared with the MOD group.The results of WB assay showed that compared with the MOD group,the expression of Fndc5/Irisin,PI3K,p-PI3K,p-Akt（P<0.01）in liver tissues of mice in MET and DKS26 groups recovered significantly（P<0.05）and approached normal levels,and the expression of Fndc5/Irisin,PI3K,p-PI3K,p-Akt（P<0.01）in liver tissues of mice in DKS26 group decreased significantly（P<0.05）compared with the MET group,while the expression of Akt did not differ significantly.Total Fox O1（P<0.01）and Fox O1 protein in nuclei in the MET and DKS26 groups were significantly lower（P<0.05）,and there was no significant difference between the two groups.And the intracytoplasmic Fox O1protein expression was significantly upregulated（P<0.05）.Compared with the MOD group,the hepatic PEPCK protein expression was significantly down-regulated in the MET and DKS26 groups（P<0.05）;RT-q PCR detected that the hepatic PEPCK m RNA expression was significantly down-regulated in the MET and DKS26 groups compared with the MOD group（P<0.01）;the hepatic Fox O1 m RNA expression was significantly down-regulated in the MET group compared with the MOD group.The liver Fox O1 m RNA expression of mice in the MET group was down-regulated compared with that in the MOD group,but the difference was not significant,and the liver Fox O1 m RNA expression of mice in the DKS26 group was significantly down-regulated compared with that in the MOD group（P<0.01）,which was not significantly different from that in the MET group.Conclusions:DKS26 can improve Nonalcoholic fatty liver disease in mice induced by high-fat diet combined with CCl₄,which can reduce hepatic lipid deposition an d improve liver function.The mechanism of action may be related to the regul ation of（Fndc5/Irisin）/PI3K/Akt signaling pathway and its downstream regulator s Fox O1 and glucolipid metabolizing enzyme PEPCK.