The Mechanism Of Silybin Against Lipid Accumulation And Lipid Peroxidation By Activating NRF2 Signaling Pathway And Down-regulating CD36 Expression

Background:Non-alcoholic fatty liver disease(NAFLD)is one of the main causes of liver death and liver transplantation in recent years,and its pathogenesis is related to excessive accumulation of free fatty acids(FFA)in hepatocytes and lipid peroxidative damage.Excessive influx of FFA into the liver leads to steatosis in hepatocytes and induces oxidative stress,which ultimately leads to liver damage.The vast majority of FFA in the circulation is transported into the liver by fatty acid transporters.Among them,the fatty acid transporter CD36 is one of the important transporters for the uptake of FFA by hepatocytes,and its expression is regulated by nuclear receptors such as LXRα.The activation of LXRα is closely related to the NRF2 signaling pathway responsible for regulating redox balance.Silybin has good pharmacological effects in protecting the liver and gallbladder,reducing lipid and glucose,antioxidant,anti-inflammatory,anti-cancer,anti-fibrosis,etc.It is widely used in the treatment of hepatobiliary-related diseases in clinical.Studies have shown that Silybin is often accompanied by up-regulation of nuclear factor NRF2 expression in the treatment of NAFLD.So,can Silybin fight against lipid accumulation and lipid peroxidation by activating the NRF2 signaling pathway and downregulating the expression of the fatty acid transporter CD36? This deserves further study and attention.Objective:The FFA-induced lipid peroxidation model was constructed by using HepG2 cells,and the effect of Silybin on NRF2 signaling pathway and CD36 expression were further studied,as well as its anti-lipid accumulation and anti-lipid peroxidation mechanisms.It is hoped that this study will provide theoretical and experimental basis for the clinical treatment of Silybin in diseases related to lipid metabolism disorders.Methods:1.HepG2 cells were treated with FFA(sodium palmitate:sodium oleate = 1:2)for 24 h to build the lipid peroxidation model.2.Based on the above model,the oil red O staining experiment and the determination of TG,TC,SOD,MDA and other indicators were used to study the anti-lipid accumulation and anti-lipid peroxidation effects of Silybin.RT-qPCR,Western-blot and other methods to study the effect of Silybin on NRF2 signaling pathway and CD36 expression.3.Based on the above studies,HepG2 cells model that silences NRF2 was constructed usingRNA interference technology.Then the anti-lipid accumulation and anti-lipid peroxidation effects of Silybin after NRF2 silencing and its effects on NRF2 signaling pathway and CD36 expression were investigated.Results:1.The content of intracellular lipid droplets,TG,TC and MDA were significantly increased,the content of SOD was significantly decreased after FFA intervention for 24 h in HepG2 cells,which indicated that the lipid peroxidation model was successfully constructed.2.The mRNA expression of NRF2 had no significant difference in the intervention of FFA for 24 h in HepG2 cells,but the mRNA expression of LXRα and CD36 were significantly up-regulated.There was no significant difference in the expression of total NRF2 protein,but the mRNA expression of LXRα and CD36 were significantly up-regulated.And the expression of NRF2 nuclear protein was significantly decreased,the expression of NRF2 cytoplasmic protein was significantly increased,the protein expression of FXR and SHP was significantly decreased,and the total protein and nuclear protein of LXRα were significantly increased.The protein expressions of the adipogenic genes SREBP1,FAS and lipid metabolism-related genes PPARα,CPT-1 were significantly increased.The protein expression of the fatty acid transporter CD36 was also significantly increased.3.The lipid droplet content,TG content,TC content and MDA content in HepG2 cells were significantly decreased after 24 h of simultaneous intervention with Silybin,while the SOD content was significantly increased.However,there was no significant difference in NRF2 and LXRα mRNA expression,and CD36 mRNA expression was significantly decreased.After the intervention of Silybin,the nuclear activation of NRF2 was increased,the expressions of FXR and SHP proteins were up-regulated,the nuclear activation of LXRα was decreased,and the expressions of SREBP1,FAS,CD36 and other proteins were down-regulated.The protein expression of lipid metabolism-related gene LXRα and its downstream target gene CPT-1 were also down-regulated after Silybin intervention for 24 h.4.In HepG2 cells which was silenced NRF2,there was no significant difference in TG and SOD content between the Silybin intervention group and the model group,as well as the expression of FXR and SHP protein,LXRα nucleoprotein and CD36 protein.Compared with the Silybin intervention group without NRF2 silencing,the TG-lowering and SOD-raising effects of Silybin were significantly weakened in the NRF2-silenced Silybin intervention group.And the effects of Silybin on up-regulating FXR and SHP protein expression,down-regulating CD36 protein expression,and reducing LXRα nuclear activation were also significantly attenuated.Conclusions:1.Silybin has a good resistance to lipid accumulation and lipid peroxidation caused by FFA in HepG2 cells.2.The anti-lipid accumulation and anti-lipid peroxidation effects of Silybin in HepG2 cells are mainly related to activating the NRF2 signaling pathway,up-regulating the expression of FXR and SHP proteins,and then inhibiting the activation of LXRα into the nucleus and down-regulating the expression of the fatty acid uptake gene CD36.