Abstract: 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 concentrations 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, 12 h), 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=0.016 7). 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=0.016 7). 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=0.016 7). 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=0.016 7), 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.

Key words: Acetaminophen, Toll-like receptor 4, Programmed necrosis, Ferroptosis, Inflammation