Qiwei Jinggan Ling ameliorates acetaminophen-induced liver injury via targeting the IKKβ/NF‑κB pathway to suppress hepatic sterile inflammation

Serum ALT and AST, as well as hepatic GSH, MDA, and MPO levels, were measured using biochemical kits. Hepatic histopathology was observed by H&E staining. Serum contents of HMGB1, CXCL1, TNF-α, IL-1β, and IL-6 were determined by ELISA. mRNA levels of key pathway genes and inflammatory factors were detected by RT-qPCR.

Immunofluorescence was used to detect p-IKKβ and IL-1β expression in AML12 cells. Protein expression of pathway components and downstream inflammatory factors was assessed by Western blot. Molecular docking and surface plasmon resonance (SPR) were performed to analyze the binding of prototype absorbed components to IKKβ.Results Compared with the model group, all QJL dose groups ameliorated hepatocellular disarray and necrosis, and the medium- and high-dose groups significantly reduced APAP-induced increases in liver weight (P<0.05, P<0.01).

In the QJL high-dose group, serum ALT and AST levels as well as HMGB1, CXCL1, TNF-α, IL-1β, and IL-6 contents were markedly decreased (P<0.01), while hepatic GSH was significantly elevated and MDA and MPO were significantly reduced (P<0.01). Mechanistically, APAP significantly upregulated the ratios of p-IKKβ/IKKβ, p-IκB-α/IκB-α, and p-NF-κB/NF-κB in liver tissue, and promoted the protein expression of downstream TNF-α, IL-1β, and IL-6 (P<0.01); these changes were markedly reversed by QJL intervention (P<0.05, P<0.01).

In vitro experiments further confirmed that QJL-containing serum significantly reduced APAP-induced mRNA and protein expression of inflammatory factors in AML12 cells and suppressed phosphorylation of the IKKβ/NF-κB pathway (P<0.05, P<0.01). TNF-α rescue significantly reversed the inhibitory effects of QJL on IKKβ/NF-κB activation and inflammatory factor expression (P<0.05, P<0.01).

Molecular docking and SPR analyses showed that the prototype absorbed components of QJL, Glycyrrhetinate, Taxifolin, and Salicylic acid, could directly and effectively bind to IKKβ protein.Conclusions QJL directly binds to IKKβ and inhibits over‑phosphorylation of the IKKβ/NF‑κB pathway, thereby blocking hepatic sterile inflammation and alleviating APAP‑induced acute DILI.