| Objective Non-alcoholic fatty liver disease(NAFLD)has become one of the most common chronic liver diseases worldwide with the increasing prevalence of the disease.Currently,lifestyle interventions are still the main treatment strategy for NAFLD.Drugs such as metformin,pioglitazone and vitamin E have been shown to have certain therapeutic effects on NAFLD,but their safety and side effects still need to be further evaluated.Therefore,research and development for NAFLD therapeutic drugs is urgently needed.Ginsenoside Rg1(G-Rg1)is one of the main active components in ginseng,which has anti-tumor,anti-oxidation,anti-diabetes,and other pharmacological effects.Our previous study found that liver lipid deposition,triglycerides(TG)and free fatty acid(FFA)levels could be significantly ameliorated in NAFLD model mice,suggesting that G-Rg1 has a positive effect on NAFLD treatment.Therefore,this study aims to further explore the possible mechanism and target of G-Rg1 in ameliorating lipid metabolism in NAFLD on the basis of previous studies,so as to provide new idea for the treatment of NAFLD.Methods1.Cell model and grouping: When the cell density in the 6-well plate reached 70%-80%,they were divided into 3 groups and treated differently.Normal group: ordinary medium culture(containing solution medium of equal concentration);Model group:1 mmol/L FFA medium [FFA was a mixture of oleic acid(OA)and palmitic acid(PA)(OA:PA=2:1)] was cultured for 24 h,followed by ordinary medium for 24 h;G-Rg1 group: 1mmol/L FFA medium was cultured for 24 h,followed by 25μM and 50μM G-Rg1 medium for 24 h.2.The cytotoxic effect of G-Rg1 was determined by CCK8 assay.The accumulation of lipid droplets was observed by Oil Red O staining.The levels of TG,TC,MDA and SOD was detected by micro method.Western blot and RT-q PCR were used for detecting the expression levels of m RNA and proteins of genes involved in lipid synthesis,lipid uptake,lipid oxidation and lipid export.3.SPSS 20.0 software was used for statistical analysis.Results were expressed as means ± standard deviation(SD).The difference between two groups was analyzed by one-way ANOVA followed by the SNK-q test.P<0.05 was considered statistically significant.Results1.Cytotoxic effects of G-Rg1: The results showed that Hep G2 cell activity decreased with the increase of G-Rg1 concentration.The concentration of G-Rg1 had no significant effect on the viability of Hep G2 cells at 25μM,but the cell survival rate was significantly reduced while the concentration of G-Rg1 reached 75μM(P<0.05).Therefore,G-Rg1 concentrations of 25μM and 50μM were selected for follow-up testing to avoid inhibition due to the cytotoxicity of G-Rg1.2.G-Rg1 ameliorate FFA-induced lipid accumulation in Hep G2 cells:Compared with the control group,the absorbance(485nm)value of the cells in the model group was increased,and intracellular red lipid droplet aggregation was significantly increased,presenting a large round particle shape.It indicated that NAFLD model construction is successful(P<0.01).Compared with the model group,the absorbance(485nm)of the 25μM and50μM G-Rg1 groups decreased to different degrees,and the intracellular red lipid droplets decreased(P<0.01).The determination of TG and TC content in Hep G2 cells by micrometer method showed that the TG and TC content in the model group increased significantly compared with that in the control group(P<0.01).After treatment with different concentrations of G-Rg1,the intracellular TG and TC contents were significantly decreased(P<0.01).3.Effect of G-Rg1 on FFA-induced Hep G2 cells MDA and SOD: The experimental results showed that SOD content decreased and MDA content increased in the model group compared with the control group(P<0.01).Compared with the model group,SOD content could be increased and MDA content could be decreased in the G-Rg1 group at different concentrations.The effect was most significant at a concentration of 50μM(P<0.01).4.Effect of G-Rg1 on expression of genes related to lipid synthesis in Hep G2 cells induced by FFA: Western blot results showed that SREBP1 c and FASN were both increased in the model group compared with the control group(P<0.01).The expression levels of SREBP1 c and FASN in25μM and 50μM G-Rg1 groups were decreased(P<0.01).In addition,there was no significant change in ACCα expression in the model group(P>0.05),but the expression of both p-ACCα and p-ACCα to ACCα ratio were significantly decreased(P<0.05).After treatment with G-Rg1,the expression of both p-ACCα and p-ACCα to ACCα ratio in the G-Rg1 group with the concentration of 50μM were significantly increased(P<0.05).RT-q PCR results showed that the expression levels of SREBP1 c,FASN,and ACCα were increased in the model group(P<0.05).Compared with the model group,the expression of SREBP1 c,FASN,and ACCα decreased in the Rg1 group with different concentrations,and the effect was most significant at the concentration of 50μM(P<0.05).5.Effect of G-Rg1 on expression of genes related to lipid uptake in Hep G2 cells induced by FFA: Western blot results showed that the expression of CD36,FATP2,FATP5,and FABP1 in the model group were all increased(P<0.05).The expression of CD36 and FATP5 was decreased in the 25μM and 50μM G-Rg1 groups(P<0.05).The protein expression of FATP2 and FABP1 decreased in the 50μM G-Rg1 group,but the decrease was not significant at 25μM(P>0.05).Then,the expression of genes related to lipid uptake at m RNA level was detected by RT-q PCR.Compared with the control group,the expression of CD36,FATP2,FATP5,and FABP1 were increased in the model group(P<0.01).After G-Rg1 treatment,CD36,FATP2,FATP5,and FABP1 m RNA expressions were all decreased(P<0.01).6.Effect of G-Rg1 on expression of genes related to lipid oxidation in Hep G2 cells induced by FFA: Western blot results showed that compared with the control group,CPT1 and ACOX1 expression in the model group were both decreased(P<0.05).CPT1 and ACOX1 expression increased in the G-Rg1 group at different concentrations,with the most significant increase at 50μM(P<0.05).RT-q PCR results showed that the m RNA level was consistent with the protein level.CPT1 and ACOX1 m RNA expressions in the model group were decreased(P<0.05).CPT1 and ACOX1 m RNA expressions were increased in G-Rg1 group at a concentration of 50μM(P<0.05).7.Effect of G-Rg1 on expression of genes related to lipid export in Hep G2 cells induced by FFA: The results of western blot showed that compared with the control group,MTTP and Apo B100 protein expressions in the model group were decreased(P<0.01).Both MTTP and Apo B100 protein expressions were increased after treatment with G-Rg1 of concentration at 50μM(P<0.05).The results of RT-q PCR showed that,Both MTTP and Apo B100 were decreased(P<0.05).MTTP m RNA was increased in the G-Rg1 group at different concentrations(P<0.05),and Apo B100 m RNA was increased at 50μM concentrations(P<0.01).8.Effect of G-Rg1 on transcription factors PPARα and PPAR γ:Western blot and RT-q PCR results showed that,the protein and m RNA expression of PPARα in the model group all decreased(P<0.05).The protein and m RNA expression of PPARα were increased in the G-Rg1 group at different concentrations(P<0.01).The expression of PPAR γprotein and m RNA was completely opposite to that of PPARα.PPAR γprotein and m RNA expression in the model group were increased(P<0.01).PPAR γ protein and m RNA expression were decreased in the G-Rg1 group at different concentrations(P<0.01).Conclusion1.G-Rg1 can reduce the contents of TG and TC in the lipid accumulation model of Hep G2 cells induced by FFA,reduce the lipid accumulation in cells,and ameliorate the cellular lipogenesis.2.G-Rg1 can reduce the MDA content of Hep G2 cells treated with FFA,improve the activity of SOD,and have the functions of anti-oxidation and improving lipid peroxidation.3.G-Rg1 can inhibit lipid uptake and synthesis by inhibiting the expression of PPAR γ,thus reducing lipid inflow and lipid accumulation in liver cells.4.G-Rg1 can promote lipid oxidation and lipid export by enhancing the expression of PPARα,thus enhancing lipid outflow and ameliorating lipid degeneration of liver cells. |
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