Study On The Effects And Mechanisms Of Ginsenoside Rg1 On Non-alcoholic Fatty Liver Disease In HepG2 Cells

Backgroud: Non-alcoholic fatty liver disease(NAFLD)is a clinical pathological syndrome,which is characterized by hepatic steatosis and fat accumulation without excessive alcoholic history.NAFLD can be divided into different degrees of liver diseases,from simple lipid deposition without inflammation to severe inflammatory reaction of fibrosis and even cirrhosis,including simple fatty liver,steatohepatitis,and cirrhosis.There currently doesn't exist any clinically recognized drug for the treatment of NAFLD,so it is urgent to develop new and effective drugs.Ginsenoside Rg1 is an active ingredient extracted from the roots of ginseng and has been widely reported to protect various liver diseases in recent years.Previous studies have confirmed that ginsenoside Rg1 can improve lipid peroxidation by increasing oxidase activity and promoting fatty acid ?-oxidation in high fat diet-induced NAFLD mice,and alleviate liver inflammation by inhibiting activation of inflammasomes;however,the role of Rg1 in the lipid deposition — the initial point of NAFLD and the possible direct target of subsequent inflammatory responses are not yet clear.Therefore,this study used palmitic acid-induced HepG2 cells as a lipid deposition and inflammation model to evaluate the anti-lipid deposition and anti-inflammatory effects of Rg1 in vitro and to elucidate its possible molecular mechanisms.Methods:1.CCK-8 : HepG2 cells were cultured in different concentrations of palmitate acid(0,0.125,0.25,0.5mM)and ginsenoside Rg1(0,10,20,40,80,100?g/mL)to explore the optimal concentration.2.Cell model and grouping: HepG2 cells were treated with 0.25 mM palmitic acid(PA)for 24 hours to construct a non-alcoholic fatty liver cell model,which was cultured with 40 ?g/mL or 80 ?g/mL Rg1 for 6hours.Control group,model group,low dose Rg1 group,high dose Rg1 group were set.HepG2 cells were treated with 1 mM free fatty acid for 24 hours,pretreated with or without 10?M AMPK inhibitor(Compound C,CC)for 1 h,and treated with 40?g/mL Rg1 for 6 hours,and the model +inhibitor group and Rg1 + inhibitor group were set.3.Intracellular lipid deposition assay: The content of intracellular triglyceride(TG)was detected by micro-method;After Oil Red O staining,the lipid deposition was observed under light microscope,and the absorbance of each group was determined by adding isopropanol to dissolve the dye.4.Detection of inflammatory markers in cellular supernatant:Automatic biochemical analyzer was used to detect alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in each group;enzyme-linked immunosorbent assay(ELISA)was used to determine the expression of proinflammatory cytokines,including IL-1?,IL-6,TNF-?.5.Detection of lipid metabolism pathway: Western blot was used to detect the changes of AMPK,p-AMPK and its downstream de novo synthesis key proteins ACC?,p-ACC?,SREBP-1c and FAS in each group.RT-qPCR was used to detect mRNA changes of SREBP-1c,FAS.6.Detection of inflammatory pathway: Western blot was employed toidentify protein changes of NF-?B P65,p-P65 and its downstream IL-6,IL-1?,TNF-?;RT-qPCR was used to detect mRNA changes of IL-6,IL-1?and TNF-?;immunofluorescence staining was used to observe NF-?B P65 nuclear translocation.7.Statistical analysis was performed with Graphpad Prism 6.0software,and all data were expressed as mean ± standard deviation(?x+s).One-way analysis of variance was used for comparison among groups,and the test standard was ?=0.05.Results:1.Ginsenoside Rg1 has obvious toxicity to HepG2 cells at 0.5mM PA and 100?g/mL,so we chose 0.25 mM PA and 40,80?g /mL G-Rg1 in the subsequent experiments.2.Ginsenoside Rg1 can cut down triglycerides and lipid deposition in HepG2 cells: Compared with the control group,the intracellular triglycerides of the model group increased significantly.After Rg1 intervention,the level of triglyceride decreased(P <0.05),and there was no significant difference between high and low doses(P >0.05).Oil Red O staining result showed that compared with the control group,the lipid droplet aggregation in the model group increased significantly,and the absorbance increased.Compared with the model group,the lipid droplets in the low-dose Rg1 group and the high-dose Rg1 group were decreased to some degree,and the absorbance was also decreased(P <0.05).3.Ginsenoside Rg1 can reduce expressions of inflammatory markers in cell culture supernatant: Compared with the control group,the ALT and AST in the cellular supernatant of the model group increased significantly,and low dose or high dose Rg1 could reduce the levels of ALT and AST in cellular supernatant(P <0.05),and there was no significant difference between low and high doses(P >0.05).The results of ELISA showed thatthe expressions of IL-1?,IL-6 and TNF-? in the model group were significantly higher than those of the control group;low dose and high dose of Rg1 could reduce IL-1?(11)IL-6 and TNF-? to different degrees,and the differences were statistically significant(P <0.05).There was no significant difference between low dose and high dose,except for IL-1?(P >0.05).4.Ginsenoside Rg1 can increase AMPK and ACC? phosphorylation and decrease the mRNA and protein expressions of SREBP-1c and FAS.Q-PCR results showed that the expressions of SREBP-1c and FAS in the model group were significantly higher than those in the control group.Compared with the low-dose and high-dose Rg1 group,the mRNA expressions of SREBP-1c and FAS were decreased to different degrees(P<0.05).There was no significant difference between the two groups(P >0.05).Western blot analysis of lipid metabolism related proteins in the AMPK pathway showed that,compared with the control group,the AMPK and ACC? phosphorylation was significantly reduced in the model group,and the expression of SREBP-1c and FAS was significantly increased.Compared with the model group,the phosphorylation of AMPK and ACC?in Rg1 groups increased significantly,and the expressions of SREBP-1c and FAS decreased significantly(P <0.05).Except for FAS,there was no significant difference in protein expressions between low and high Rg1 groups.And there was no significant difference in the expressions of totalAMPK and total-ACC? among different groups(P >0.05).5.Ginsenoside Rg1 can reduce the phosphorylation and nuclear translocation of NF-?B P65,and the mRNA and protein expressions of downstream IL-1?,IL-6 and TNF-?.Compared with the control group,the protein expressions of p-P65,IL-1?,IL-6 and TNF-? in the model group were significantly increased.After low-dose or high-dose Rg1 intervention,the expressions of p-P65,IL-1?,IL-6 and TNF-? decreased significantly,and the differences were statistically significant(P <0.05).There was no remarkable difference in total-NF-?B among different groups(P >0.05).Compared with the high-dose Rg1 group,p-NF-?B and IL-1? were decreased more significantly in the low-dose Rg1(P <0.05).Immunofluorescence showed that the NF-?B P65 protein in the control group was mainly expressed in the cytoplasm,and most of the P65 in the model group was activated into the nucleus.After treatment with low or high dose Rg1,NF-?B P65 was demonstrated to translocate to cytoplasm from the nuclear.Conclusion:Ginsenoside Rg1 can reduce triglycerides and alleviate deposition of lipid droplets in HepG2 cells;and can also reduce the releases of pro-inflammatory factors in the supernatant of HepG2 cells,including IL-1?,IL-6 and TNF-?.Ginsenoside Rg1 can improve lipid deposition and ameliorate inflammation in NAFLD cell model through AMPK/NF-?B pathway,which provides a new thought for treatment of NAFLD.