Effects of programmed necrosis and ferroptosis regulated by toll-like receptor 4 on acetaminophen-induced liver injury
Objective To explore whether toll-like receptor 4(TLR4)further affects the process of acetaminophen(APAP)induced liver injury and its mechanism by regulating programmed necrosis and ferroptosis.Methods Human normal hepatocytes L-02 were cultured in vitro and cell viability was detected by the CCK-8 method,and the concentra-tions of APAP and TAK-242 were evaluated.The experiment was divided into control group,APAP groups(1,4,12 h)and APAP+TAK-242 groups(1,4,12h),and the TLR4 mRNA levels were compared in each group.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in cell homogenates of different groups were detected;The levels of nuclear factor-κB(NF-κB),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected in each group;The levels of high mobility group box 1(HMGB1),receptor interacting protein kinase 1(RIP1)and receptor interacting protein kinase 3(RIP3)were detected in each group;The intracellular Fe2+content and the level of NF-κB,P53,recombinant solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4)were detected in each group.Results The concentration of 5 mmol/L APAP and 100 nmol/L TAK-242 was determined by the CCK-8 method.Compared to the control group,the TLR4 mRNA levels of the APAP groups were positively regulated at each time point(P<0.05);Compared to the APAP groups,the levels of TLR4 mRNA in the APAP+TAK-242 groups were negatively regulated at the corresponding time points(P<0.05/3=0.0167).Compared to the control group,the levels of ALT and AST in the APAP groups increased at each time point(P<0.05);Compared to the APAP groups,the levels of ALT and AST in the APAP+TAK-242 groups decreased at the corresponding time points(P<0.05/3=0.016 7).Compared to the control group,the mRNA expressions of NF-κB,IL-6 and TNF-α were up-regulated in the APAP groups at each time point(P<0.05);Compared to the APAP groups,the mRNA expressions of NF-κB,IL-6 and TNF-α were all down-regulated in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.0167).Compared to the control group,the levels of HMGB1,RIP1,and RIP3 increased in the APAP groups at all time points(P<0.05);Compared to the APAP groups,the levels of HMGB1,RIP1,and RIP3 decreased in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.0167).Compared to the control group,the content of Fe2+,NF-κB and P53 was increased(P<0.05),but the levels of SLC7A11 and GPX4 decreased in the APAP groups at all time points(P<0.05);Compared to the APAP groups,the content of Fe2+,NF-κB and P53 were decreased(P<0.05/3=0.0167),but the levels of SLC7A11 and GPX4 increased in the APAP+TAK-242 groups at the corresponding time points(P<0.05/3=0.016 7).Conclusion Inhibition of TLR4 can negatively regulate programmed necrosis by regulating the TLR4/HMGB1 signaling pathway and can negatively regulate the inflammatory response and ferroptosis by regulating TLR4/NF-κB signaling pathway to alleviate APAP-induced liver injury.
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