| Background: Alcoholic liver disease(ALD)is a major global health problem,which affects millions more people every year.Despite years of research,there is still a lack of effective clinical treatment and drugs.Recent studies have shown that bile acid accumulation and metabolic disorders are important factors of hepatocyte injury in ALD,which promote the further deterioration of ALD.Regulation of bile acid metabolism and its associated signaling can protect alcohol-induced liver injury,which is a new therapeutic target for ALD.Objective: To study the mechanism of the protective effect of Kaempferol(KAE)on the liver of mice with alcoholic liver disease based on the regulation of bile acid metabolism,and to provide experimental basis for further application and development of the drug.Methods: 1.In vivo experiment: Lieber-De Carli liquid diet was used to establish the ALD model in mice.During the modeling period,mice in the normal diet group were given liquid diet,and the other groups were given alcohol diet with an alcohol concentration of 5%.The diet was configured on the same day,the food intake of mice was recorded every day,and the weight of mice was weighed once every 7 days.Part 1: specific pathogen free(SPF)C57BL/6 male mice(8 weeks of age,20-22 g)were randomly divided into liquid diet control group,alcohol liquid diet model group,Kaempferol low-dose group and Kaempferol high-dose group with 12 mice in each group.The low-dose and high-dose groups were given kaempferol by gavage of50 mg/kg/d and 100 mg/kg/d,respectively,while the other groups were given the corresponding drug solvent 0.5 % sodium carboxymethyl cellulose(CMC-Na)once a day for 6 weeks.In the second part,C57BL/6 male mice were randomly divided into liquid diet control group,alcohol liquid diet model group,Kaempferol group and FXR inhibitor group,with each mouse weighing 20-22 g and 12 mice in each group.The Kaempferol group was given 100 mg/kg/d by gavage,and the FXR inhibitor group was given 10 mg/kg/d Z-Guggulsterone by gavage.The other groups were given the corresponding drug solvent 0.5 % sodium carboxymethyl cellulose(CMC-Na)once a day for 6 weeks.After the end of the last administration,the mice were fasted for 12 hours and weighed.After anesthesia,blood was taken from the eyeball to kill the mice.The following indexes were observed:(1)the body weight and liver index of the mice were calculated;Mouse liver function index was detected.Histological changes of liver and intestinal tract were observed by biopsy.(2)The expression levels of inflammation-related factors and lipid metabolism-related factors in mouse liver were detected.(3)The expression levels of intestinal inflammatory factors and tight junction protein were detected.(4)Detect farnesoid X receptor(FXR)in mouse intestine.2.In vitro experiments:(1)Alcohol stimulated MC38 cells(mouse colon cancer cells)and Caco-2 cells(human colorectal adenocarcinoma cells)to establish alcohol damage models in vitro,and detected the effects of kaempferol on FXR and FGF-15/19 protein expression levels in MC38 cells and Caco-2 cells under alcohol exposure.(2)kaempferol directly stimulated Caco-2 cells to establish FXR activation model in vitro.The expression levels of FXR and FGF-19 protein in Caco-2 cells were detected.(3)The interaction between kaempferol and FXR promoter was detected by luciferase assay.Results: 1.In vivo experiments: The effects of kaempferol on bile acid metabolism in alcoholic liver disease mice:(1)Compared with liquid diet control group,body weight of alcohol-fed mice decreased(p<0.01)and liver index increased(p<0.01);After kaempferol intervention,the body weight of kaempferol high-dose group was significantly increased(p<0.05)and liver index was decreased(p<0.05).(2)Liver H&E staining showed that,compared with the liquid diet control group,the liver sinusoids disappeared and vacuolar degeneration occurred in the alcohol diet group.After treatment with kaempferol,the liver sinuses of mice in the kaempferol high-dose group basically recovered to normal,and the vacuolar degeneration was significantly reduced.The results of liver oil red O staining showed that kaempferol significantly reduced the lipid deposition in the liver of mice in the alcohol diet group.(3)Kaempferol inhibited the contents of ALT and AST in serum and TC and TG in liver of mice(p<0.05).(4)Kaempferol decreased the m RNA expression levels of TNF-α,IL-1β,Mcp-1 and F4/80 in liver of alcohol-fed mice(p<0.05)and the m RNA expression levels of Fasn,Srebp1,Lipin1 and Nr1d1(p<0.05).(5)ileum H&E staining showed that,compared with liquid diet control group,alcohol diet group had serious ileum villi damage;After treatment with kaempferol,the ileum villi damage of mice in high-dose kaempferol group was significantly reduced.Kaempferol increased the expression level of Occludin m RNA and protein in ileum of alcohol-fed mice,and inhibited the expression level of TNF-α,IL-1β and i NOS m RNA in intestinal tissue.(6)Compared with liquid diet control group,the m RNA and protein expression levels of FXR,FGF-15 and SHP in ileum of mice in alcohol diet group were significantly decreased(p<0.05),while the protein expression level of ASBT was increased(p<0.05);After kaempferol treatment,m RNA and protein expression levels of FXR,FGF-15 and SHP in ileum of mice in kaempferol high-dose group were increased(p<0.05),while the expression level of ASBT protein was decreased(p<0.05).(7)Compared with liquid diet control group,the expression levels of CYP7A1 and CYP27A1 proteins in liver of mice in alcohol diet group were significantly increased(p<0.01),while the expression levels of NTCP and BSEP proteins were decreased(p<0.01).After kaempferol treatment,the expression levels of CYP7A1 and CYP27A1 proteins in liver of mice in kaempferol high-dose group were decreased(p<0.01),while the expression levels of NTCP and BSEP proteins were increased(p<0.05).(8)Kaempferol significantly decreased the content of TBA in serum(p<0.01)and liver of alcohol-fed mice(p<0.05);Kaempferol significantly inhibited the ratio of bound to unbound bile acids in serum of mice induced by alcohol(p<0.05),and decreased the content of hydrophobic bile acids in serum and liver(p<0.05).2.The mechanism of inhibiting bile acid synthesis in alcoholic liver disease mice with kaempferol:(1)The results of liver H&E and oil red O staining showed that compared with the alcohol diet group,after treatment with KAE,the liver sinuses of the medication group mice basically returned to normal,vacuolar degeneration was significantly reduced,and lipid deposition was significantly reduced;Mice that were also treated with FXR inhibitors still showed disappearance of hepatic sinusoids,vacuolar degeneration,and significant lipid deposition in the liver.(2)Compared with the alcohol diet group,the levels of serum ALT,AST,and TBA in the KAE intervention group mice decreased(p<0.01);Compared with the medication group,the levels of serum ALT,AST,and TBA in the FXR inhibitor group mice increased(p<0.05).(3)Compared with the alcohol diet group,after intervention with KAE,the expression levels of FXR,FGF-15 m RNA and protein in the ileum of the medication group mice increased(p<0.05),while the expression levels of SHP m RNA increased(p<0.01).The expression levels of FXR,SHP m RNA in the liver increased(p<0.05),while the expression levels of CYP7A1 m RNA and protein decreased(p<0.05);Compared with the medication group,the expression levels of FXR,FGF-15 m RNA and protein in the ileum of the FXR inhibitor group mice decreased(p<0.05),while the expression levels of SHP m RNA decreased(p<0.01).The expression levels of FXR,SHP m RNA in the liver decreased(p<0.01),and the expression levels of CYP7A1 m RNA and protein increased(p<0.05).3.In vitro experiments:(1)Compared with the control group,the expression levels of FXR,FGF-15,and FGF-19 proteins in MC38 and Caco-2 cells in the alcohol model group decreased;Compared with the alcohol group,the expression levels of FXR,FGF-15,and FGF-19 proteins in MC38 and Caco-2 cells in the kaempferol treated group increased(p<0.05).(2)Direct stimulation of Caco-2 cells with kaempferol for 24 hours significantly increased the expression levels of FXR,FGF-19,SHP m RNA and protein(p<0.05).(3)The results of Luciferase report experiment showed that kaempferol directly regulated the activity of FXR promoter and enhanced the expression and activity of its Luciferase.Conclusion:(1)Kaempferol can significantly improve liver inflammation and lipid metabolism disorder in alcohol-fed mice,and has liver protection effect on ALD mice.(2)Kaempferol inhibits bile acid synthesis and regulates bile acid metabolism in ALD mice by activating FXR-FGF15 signals in the gut.(3)Kaempferol may directly regulate FXR promoter and enhance FXR protein expression to improve FXR activity in cells. |
