Overdose of acetaminophen (APAP), a commonly used antipyretic analgesic, can lead to severe liver injury and failure. Current treatments are only effective in the early stages of APAP-induced acute liver injury (ALI). Therefore, a detailed examination of the mechanisms involved in liver repair following APAP-induced ALI could provide valuable insights for clinical interventions.

Overall, these findings underscore the pivotal role of FAK activation in liver repair post-APAP overdose by promoting liver cell proliferation and macrophage recruitment.

4D-label-free proteomics analysis was used to identify dysregulated proteins in the liver of APAP-treated mice. RNA-Seq, hematoxylin-eosin staining, immunohistochemical staining, immunofluorescence staining, quantitative PCR, western blotting, transwell were used to explore the underlying mechanisms.

Utilizing high throughput 4D-label-free proteomics analysis, we observed a notable increase in proteins related to the "focal adhesion" pathway in the livers of APAP-treated mice. Inhibiting focal adhesion kinase (FAK) activation with a specific inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride (also called Y15), resulted in reduced macrophage numbers, delayed necrotic cell clearance, and inhibited liver cell proliferation in the necrotic regions of APAP-treated mice. RNA-Seq analysis demonstrated that Y15 downregulated genes associated with "cell cycle" and "phagosome" pathways in the livers of APAP-treated mice. Furthermore, blocking extracellular matrix (ECM)-integrin activation with a competitive peptide inhibitor, Gly-Arg-Gly-Asp-Ser (GRGDS), suppressed FAK activation and liver cell proliferation without affecting macrophage recruitment to necrotic areas. Mechanistically, ECM-induced FAK activation upregulated growth-promoting cell cycle genes, leading to hepatocyte proliferation, while CCL2 enhanced FAK activation and subsequent macrophage recruitment via F-actin rearrangement. Conclusions: Overall, these findings underscore the pivotal role of FAK activation in liver repair post-APAP overdose by promoting liver cell proliferation and macrophage recruitment.