| Objective: To investigate the intervention effects of silibinin(SIL)on lipid deposition and miR-122 expression in high-fat induced NAFLD model in vivo and in vitro,and to explore its possible mechanism.Methods:In vivo experiment: 36 C57BL/6JC mice(7 weeks old)were randomly divided into 2 groups after 1 week of adaptive feeding: 12 mice in the control group(normal diet,ND)and 24 mice in the high fat group(high fat diet,HFD).The ND group was fed with normal diet,while the HFD group was fed with high-fat diet.After 4 weeks,the HFD group was randomly divided into the HFD group and the HFD+SIL group with 12 mice each.The HFD+SIL group was intragastrically administered with 54 mg/kg body weight of SIL every day,and the ND group and the HFD group were intragastrically administered with the same volume of normal saline.After 4 weeks of drug intervention,the blood glucose values at each time point were measured by IPGTT and the area under the glucose curve was calculated.Serum samples were collected to detect fasting blood glucose.Fasting serum insulin was detected by ELISA.Liver tissue samples were collected to detect triglyceride content.Hepatic histopathological changes were observed by HE staining and oil red O staining of liver tissue.The miRNA and total RNA were extracted from mice liver tissue.The expression of miR-122 in mice liver tissue was detected by Real-time fluorescence quantitative PCR(RT-PCR).The mRNA and protein expression of fatty acid synthase(FAS),acetyl-CoA carboxylase(ACC)and carnitine palmitoyltransferase 1A(CPT1A)in mice liver tissue were detected by RT-PCR and Western Blotting.In vitro experiment: HepG2 cells were randomly divided into control group(Con),palmitic acid intervention group(PA,containing 0.25mmol/L palmitic acid medium)and SIL intervention group(PA+SIL,SIL concentration 25?M).The cells were collected after 48 hours of intervention,and the triglyceride content of each group of HepG2 cells was determined.The degree of intracellular lipid deposition was observed by oil red O staining.The miRNA and total RNA were extracted from HepG2 cells.The expression of miR-122 in HepG2 cells was detected by RT-PCR.The mRNA and protein expressions of FAS,ACC and CPT1 A were detected by RT-PCR and Western Blotting.In addition,palmitic acid cultured HepG2 cells were transfected with miR-122 mimic,and then subjected to SIL intervention.The mRNA and protein expressions of FAS,ACC and CPT1 A were detected by RT-PCR and Western Blotting.Results:In vivo experiment: 1 Comparison of experimental results of fasting blood glucose,fasting insulin and IPGTT in mice of each group: There were no significant differences in fasting blood glucose and fasting insulin between the three groups(P>0.05).The blood glucose levels in the HFD group at 15 min,30 min,60min and 120 min were significantly higher than those in the ND group,and the area under the blood glucose curve(AUC)was significantly increased(P <0.05).The blood glucose levels in the HFD+SIL group at 15 min,30min,60 min and 120 min were significantly lower than those in the HFD group,and the area under the blood glucose curve(AUC)was significantly reduced(P< 0.05).2 Effects of silibinin on liver lipid deposition in high-fat fed mice 2.1 Comparison of liver TG content in mice of each group: The liver TG content was significantly increased in the HFD group Compared with the ND group(P<0.01),and the liver TG content in the HFD+SIL group was reduced significantly in comparison with that in the HFD group(P<0.01).2.2 Comparison of liver histopathological test results 2.2.1 HE staining: in the ND group,the liver tissue structure was intact,the hepatocytes were neatly arranged,the hepatic lobules were regular,the cytoplasm was uniform,and no obvious inflammatory cells and steatosis were observed.In the HFD group,the hepatic lobule structure was disordered,the hepatocytes were swollen,and a large number of lipid droplets and vacuoles were observed in the cytoplasm of the hepatocytes,and the balloon-like changes were obvious.There were a large number of inflammatory cells infiltration in the portal area and lobules.The inflammatory cells infiltration of hepatocytes in the HFD+SIL group were reduced,and the degree of steatosis was significantly reduced.2.2.2 Oil red O staining: No significant lipid droplets were observed in the ND group,a large number of lipid droplets in the HFD group,and a significant decrease in lipid droplets in the HFD+SIL group.3 Expression of miR-122 in mice liver of each group: Compared with the ND group,the expression of miR-122 was significantly increased in the HFD group(P<0.01).Compared with the HFD group,the expression of miR-122 was significantly decreased in the HFD+SIL group(P<0.01).4 Comparison of mRNA and protein levels of FAS,ACC and CPT1 A in mice liver of each group: Compared with the ND group,the mRNA and protein expressions of FAS and ACC were increased in HFD group(P<0.01),and the mRNA and protein expressions of CPT1 A were decreased(P <0.01).Compared with the HFD group,the mRNA and protein expressions of FAS and ACC were decreased in the HFD+SIL group(P<0.01),and the mRNA and protein expressions of CPT1 A were increased(P<0.01).In vitro experiment 1 Effect of silibinin on lipid deposition in HepG2 cells cultured with palmitic acid.1.1 Changes in TG content in HepG2 cells: Compared with the Con group: the TG content in HepG2 cells increased significantly after PA intervention(P<0.01).Compared with the PA group,the TG content in the PA+SIL group was significantly reduced(P<0.01).1.2 Results of oil red O staining: There were no significant lipid droplets in the Con group,a large amount of red stained lipid droplets in the PA group,and the lipid droplets in the PA+SIL group were significantly reduced.2 Effect of silibinin on the expression of miR-122 in HepG2 cells: Compared with the Con group,the expression of miR-122 was increased in the PA intervention group(P<0.01).Compared with the PA group,the expression of miR-122 was decreased in the PA+SIL group(P<0.01).3 Effects of silibinin intervention on the mRNA and protein levels of FAS,ACC and CPT1 A in palmitate cultured HepG2 cells: Compared with the Con group,the mRNA and protein expressions of FAS and ACC were increased in the PA group(P<0.01),and the mRNA and protein expressions of CPT1 A were decreased(P<0.01).Compared with the PA group,the mRNA and protein expressions of FAS and ACC were decreased in the PA+SIL group(P<0.01),and the mRNA and protein expressions of CPT1 A were increased(P <0.01).4 Effects of silibinin on the mRNA and protein expressions of FAS,ACC and CPT1 A after transfection with miR-122 mimic in HepG2 cells: Compared with the Con group,the mRNA and protein expressions of FAS and ACC were increased in the PA group(P<0.01),and the mRNA and protein expressions of CPT1 A were decreased(P<0.01).Compared with the PA group,the mRNA and protein expressions of FAS and ACC were decreased in the PA+SIL group(P<0.01),and the mRNA and protein expressions of CPT1 A were increased(P<0.01).After transfection with miR-122 mimic,the mRNA and protein expressions of FAS and ACC were increased in PA+SIL+ miR-122 mimic group(P<0.01),and the mRNA and protein expressions of CPT1 A were decreased(P<0.01).Conclusion:1.Silibinin can improve lipid deposition in high-fat induced NAFLD model in vitro and in vivo.2.High-fat induces activation of hepatocyte lipid de novo synthesis pathway and inhibits fatty acid oxidation,while Silibinin inhibits lipid de novo synthesis and increases fatty acid oxidation.3.High-fat can increase the expression of miR-122 in mice liver and HepG2 cells,and Silibinin can down-regulate the expression of miR-122.4.Silibinin reduces lipid de novo synthesis by down-regulating the expression of miR-122 and increases fatty acid oxidation to improve lipid deposition in high-fat induced NAFLD model in vitro and in vivo. |
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