限时进食通过Sirt1/Nrf2通路抑制铁死亡改善代谢相关脂肪性肝炎OA

Objective To investigate the effects of time-restricted feeding(TRF)on improving metabolic-associated steatohepatitis(MASH)and the underlying molecular mechanisms involved.Methods(1)A MASH model was established in C57BL/6J mice using a high-fat,high-cholesterol diet.Twenty-four mice were randomly assigned to normal control(NC),normal time-restricted feeding(NT),model(M),and model time-restricted feeding(MT)groups(n=6 per group).Mice were anesthetized and weighed after 14 weeks,and serum samples were collected.Serum levels of total cholesterol(TC),triglycerides(TG),aspartate aminotransferase(AST),alanine aminotransferase(ALT),malondialdehyde(MDA),and ferrous ions(Fe2+)were measured.Livers were harvested to calculate the liver index.Oil Red O,hematoxylin-eosin(HE),and Masson's trichrome staining were used to evaluate hepatic steatosis degree,inflammatory infiltration,and fibrosis.Protein expression levels of silent information regulator 1(Sirt1),nuclear factor E2-related factor 2(Nrf2),acyl-coA synthetase long-chain family member 4(ACSL4),transferrin receptor 1(TfR1),solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),and tumor necrosis factor alpha(TNF-α)were detected by Western blot.(2)Additionally,an in vitro MASH model was established in human HepG2 cells using oleic acid and cholesterol stimulation,and a fasting model was established with serum deprivation.Cells were divided into Control,serum-deprived(FBS-),M,and M+FBS-groups.The ferrostatin-1(Fer-1)ferroptosis inhibitor was employed to investigate the relationship between ferroptosis and MASH/TRF.Sirt1 activity was inhibited using EX-527 to investigate the relationship between Sirt1 and Nrf2-mediated ferroptosis.Lipid accumulation in hepatocytes was observed with Oil Red O staining.HepG2 TC,TG,ALT,and AST levels were measured using kits.Western blot analysis was used to assess Sirt1,Nrf2,TfR1,ACSL4,SLC7A11,GPX4,and TNF-α protein expression levels in HepG2 cells.Results(1)Compared with MASH mice,TRF significantly reduced body weight and TC,TG,ALT,AST,MDA,and Fe2+serum levels(P<0.01).Liver Fe2+levels and TNF-α expression were also decreased(P<0.01),while hepatic steatosis and fibrosis were improved.Western blot analysis revealed that TRF intervention significantly increased Sirt1,Nrf2,SLC7A11,and GPX4 protein levels(P<0.01)while decreasing those of TfR1 and ACSL4 in the livers of MASH mice(P<0.01).(2)Compared with the M group,serum deprivation intervention reduced TC,TG,ALT,AST,MDA,and TNF-α levels in oleic acid-cholesterol-induced HepG2 cells(P<0.01),effectively reducing the number of lipid droplets.Western blot analysis indicated that serum deprivation elevated Sirt1,Nrf2,SLC7A11,and GPX4 protein levels(P<0.01)while decreasing those of TfR1 and ACSL4(P<0.01).SLC7A11 and GPX4 protein levels increased following Fer-1 intervention(P<0.01),while those of TfR1 and ACSL4 decreased(P<0.01).Following EX-527 intervention,Sirt1,Nrf2,SLC7A11,and GPX4 protein levels decreased(P<0.05 or P<0.01),while TfR1 and ACSL4 levels significantly increased(P<0.01),attenuating the ameliorative effects of serum deprivation on fat accumulation and injury in the M group(P<0.05 or P<0.01).Conclusions TRF may improve metabolic-associated fatty liver disease by inhibiting ferroptosis,with its protective mechanism potentially involving Sirt1/Nrf2 pathway mediation.