The Protective Effects Of SIRT1/Nrf2 Signaling Pathway In Acetaminophen-Induced Liver Injury

Objective To establish a model of drug-induced liver injury induced by APAP in vivo and in vitro.To explore the role of SIRT1 in APAP-induced liver injury and to explore the related mechanisms.Methods(1)In the in vitro cultured LO2 cells and Hep G2 cells,the time and concentration of APAP were changed.Western Blot and Real-Time PCR were used to detect the effect of APAP stimulation on SIRT1 expression;(2)The expression of SIRT1 in LO2 cells was changed by interfering with SIRT1 plasmid,and the effect of APAP stimulation on the expression levels of SIRT1 and Cleaved caspase 3 was detected by Western Blot;Cat,Sod2 and Gpx-1 were detected by Real-Time PCR.The relevant antioxidant gene expression level;MTT assay for LO2 cell survival rate;determination of MDA and GSH content in LO2 cells;(3)6-8 weeks old C57BL/6J male mice were randomly divided into five groups: APAP 0 h group,APAP 3 h group,APAP 6 h group,APAP 12 h group,APAP 24 h group,using Western Blot,Real-Time PCR and IHC was used to detect the expression level of SIRT1 in mouse liver tissue at different time points by APAP treatment;the change of serum AST level was determined;(4)Single-tail vein injection of lentivirus-mediated SIRT1-1 and SIRT1-2 in 6-8 week old C57BL/6J male mice,specifically reducing the expression of SIRT1 in mouse liver and measuring serum in mice changes in AST levels;determination of MDA and GSH content in mouse liver tissue;detection of nuclear Nrf2 expression by Western Blot;detection of liver Nrf2 and its downstream antioxidant target genes Sod2,Gpx-1,GCLC and G6 pdh m RNA by Real-Time PCR;pathological tissue section and HE staining of mouse liver tissue were observed to observe the degree of SIRT1 expression in the liver of mice;TUNEL staining was performed on mouse liver tissue to study the decrease of SIRT1 expression in mouse liver tissue;(5)6-8 weeks old C57BL/6J male mice were randomly divided into four groups: Vehicle group,APAP group,SRT1720 group and APAP + SRT1720 group.The changes of serum AST levels were determined.MAD and GSH were detected in liver tissue.The expression of nuclear Nrf2 was detected by Western Blot.The expression of Nrf2 and its downstream anti-oxidation target genes Sod2,Gpx-1,GCLC and G6 pdh m RNA were detected by Real-Time PCR.Histopathological sections and HE staining were used to observe the degree of SIRT1 expression up-regulation in the liver of mice.TUNEL staining was up-regulated in the expression of apoptotic cells in mouse liver tissue.Results(1)In LO2 cells and Hep G2 cells,changes in the concentration and duration of APAP affect the expression of SIRT1,The expression of SIRT1 protein increased gradually with the increase of APAP concentration and the duration of action;(2)Lentivirus-mediated SIRT1 knockdown increased the expression of Cleaved caspase 3 protein in LO2 cells,and significantly aggravated APAP-induced cell resistance in LO2 cells;(3)Lentiviral-mediated SIRT1 gene knockdown aggravated APAP-induced liver injury in mice,decreased Nrf2 protein expression,decreased m Nrf2,m Sod2,m Gpx-1,m GCLC,m G6 pdh expression;(4)SIRT1 activator SRT1720 has strong antioxidant and protective effects on acetaminophen-induced acute liver injury.Conclusion SIRT1 has a good protective effect on acetaminophen-induced liver injury,and its mechanism may play a role in regulating Nrf2 signaling pathway and exerting anti-oxidative defense ability,thereby alleviating acetaminophen-induced liver injury.