Regulation Of SIRT3/FOXO1 Signaling Pathway On Oxidative Stress In Nonalcoholic Fatty Liver Disease Model Cells

Objective: Nonalcoholic fatty liver disease(NAFLD)model cells were established by inducing oleic acid(OA)in HepG2 cells in vitro to investigate the mechanism of SIRT3 regulating oxidative stress in NAFLD cells and intervention of Salvianolic acid B(Salvianolic acid,Sal B).Methods: HepG2 cells were split into control group,model group(OA)and intervention group(OA+Sal B).The control cells were cultured in complete medium for 24 hours;the model cells were treated with medium containing 1 mmol/L OA for 24 hours;the cells in the intervention group were treated with medium containing 1 mmol/L OA for 24 hours,and then cultured with Sal B for 3 hours.After the intervention,the accumulation of intracellular fat was observed by oil red O staining,and the relevant lipid biochemical indicators of the supernatant were detected to confirm the successful modeling.Intracellular ROS levels were measured using a fluorescent probe kit,and MDA content was determined by a Malondialdehyde(MDA)kit.The mRNA expression levels of SIRT3 and SOD2 were detected by RT-qPCR,and the protein expression levels were detected by Western Blot.Immunoprecipitation(IP)was used to detect the acetylation level of Forkhead transcription factor O1(Forkhead box O1,FOXO1).SIRT3 overexpression plasmid was used to up-regulate SIRT3 and SIRT3-siRNA to down-regulate SIRT3,followed by Sal B intervention.The most suitable transfection ratio was screened by fluorescence microscopy,and the relative expression of SIRT3 and acetylated FOXO1 was tested by Western Blot and IP.Results: Sal B could effectively reduce the contents of ALT,AST,TG and TC in OA-induced NAFLD model cells,compared with the model group.There were significant differences between the groups(F values were 1240.075,471.989,97.53,39.824,respectively,P values were <0.05).The MDA content of the three groups was significantly different(F=336.67,P<0.01).The MDA content of the model group was more than that of the control group,and the intervention group was significantly lower than the model group.Sal B can effectively alleviate the accumulation of ROS in the NAFLD model,and the ROS content in the three groups is significantly different(P<0.01).The mRNA expression levels of SIRT3 and SOD2 in the three groups were significantly different(F values were 884.2,2372.2,P<0.05).The protein expression levels of SIRT3 and SOD2 in the three groups weresignificantly different(F=296.9,279.4,P<0.05).Compared with the control group,the mRNA and protein levels of SIRT3 and SOD2 in the model group were significantly reduced,and the mRNA and protein levels of SIRT3 and SOD2 in the model group were higher than those in the intervention group.The protein expression levels of acetylated FOXO1 in the three groups were significantly different(F=199.8,P<0.01),the model histone was significantly higher than the control group,and the intervention group was significantly lower than the model group.Transfection of SIRT3 overexpression plasmid SIRT3 expression was effectively increased,and the expression of intracellular acetylated FOXO1 was decreased.The expression of SIRT3 was further increased after Sal B intervention,and the expression of acetylated FOXO1 was reduced.After siRNA transfection,the expression of SIRT3 was effectively decreased,and the expression of acetylated FOXO1 was increased.Sal B intervention After only slightly enhancing the expression of SIRT3,the inhibition of acetylated FOXO1 was not obvious.Conclusion: The expression of SIRT3 and SOD2 in NAFLD model cells is lower than that in normal hepatocytes.The expression of acetylated FOXO1 is increased,cell damage is aggravated,and intracellular lipid and peroxide accumulation are increased.Sal B can attenuate lipid accumulation and oxidative stress in NAFLD model cells by regulating SIRT3/FOXO1 pathway,and has a certain therapeutic effect on NAFLD.