Abstract: Objective To investigate the role of hepatocellular nuclear factor-κB (NF-κB) signaling pathway in increasing hepatic sensitivity to lipopolysaccharide (LPS) mediated and drug-induced indirect liver injury caused by triptolide (TP). Methods In the in vivo experiment, female C57BL / 6N mice were randomly divided into the control group (Con), TP sequential LPS group (TP+LPS), and LPS sequential TP group (LPS+TP). Six hours after coadministration of TP and LPS, blood biochemistry, liver weight coefficient and hematoxylin eosin staining (HE) were detected to assess liver injury. Polymerase chain reaction (PCR) was used to detect the levels of inflammatory factors and genes related to anti-apoptosis. The expressions of apoptosis / anti-apoptosis related proteins were detected by Western Blot (WB) and immunohistochemistry (IHC), and the enzyme activity of apoptosis related proteins was also measured. Two hours after administration of LPS, the protein expressions of NF-κB inhibitory protein α (IκBα) and p65 were detected by WB. The translocation of p65 from the cytoplasm to the nucleus was observed by IHC. Changes in the levels of anti-apoptosis related genes were determined by PCR. In the in vitro experiment, the LDH content, enzyme activity of apoptosis related proteins, and levels of apoptosis / anti-apoptosis related proteins were detected after coadministration of TP and TNF-α. After administration of TP, changes in degradation of IκBα protein induced by TNF-α were determined by WB, and translocation of p65 was observed via immunofluorescence (IF). Phorbol 12-myristate 13-acetate (PMA) was selected to activate NF-κB signaling pathway in AML12 cells. The enzyme activity of apoptosis related proteins, protein levels of apoptosis / anti-apoptosis related proteins, and the content of LDH were measured to evaluate the changes caused by PMA pre-administration. Results Compared with the Con group, mice in the (TP+LPS) group showed a significant increase in aminotransferase levels, liver weight coefficient, and inflammatory factors, with significant pathological changes in the liver. However, there were no significant changes in the (LPS+TP) group compared with the Con group. The enzyme activity of caspase-8, expressions of apoptosis-related proteins, and IHC positive regions of cleaved caspase-3 increased significantly in the liver tissue of the (TP+LPS) group, and the expression of the anti-apoptotic protein FLIP showed a significant reduction, while the above-mentioned indexes were not significantly changed in the (LPS+TP) group. Pre-administration of TP significantly inhibited LPS-induced degradation of IκBα and p65 entry into the nucleus, and downregulated the expressions of anti-apoptosis related genes. In AML12 cells in in vitro, after coadministration of TP and TNF-α at a non-toxic concentration, the release of LDH and the expressions of apoptosis related proteins were significantly increased while the expressions of anti-apoptotic proteins were significantly decreased. TNF-α induced degradation of IκBα and the entry of p65 into the nucleus were significantly inhibited by TP. After pre-activation of the NF-κB signaling pathway in AML12 cells by administration of PMA, the expressions of apoptosis-related proteins, the enzyme activity of caspase-8 and the release of LDH were significantly decreased, while the expression of anti-apoptotic proteins was significantly increased. Conclusion NF-κB signaling pathway can make a big difference to the liver injury resulting from different sequential administrations of TP and LPS. TP can impair hepatocytes' ability to withstand the stimulation of exogenous pathogens and apoptosis signaling by inhibiting NF-κB activation, triggering a significant increase in apoptosis among hepatocytes, ultimately leading to the development of indirect drug-induced liver injury.

Key words: Tripterygium wilfordii Hook.f., Triptolide, Indirect Drug-Induced Liver Injury, Lipopolysaccharide (LPS), Apoptosis, Anti-Apoptosis, Hepatocellular Nuclear Factor-κB(NF-κB), Mice