FOXO4 ameliorates alcohol-induced chronic liver injury via inhibiting NF-κB and modulating gut microbiota in C57BL/6J mice

Background: Intestinal mucosa barrier function and gut-liver axis are impaired by ethanol in chronic alcoholic liver disease (ALD). However, the possible mechanism is not clear. This study aimed to investigate the effects of Forkhead Box O4 (FOXO4) on alcohol-induced chronic liver injury and its molecular mechanism(s). Methods: Male C57BL/6J mice were injected with or without FOXO4-WT, FOXO4-TB or NF-kappa B vectors, and fed with Lieber-DeCarli liquid diets containing 36% ethanol for eight weeks to induce chronic ALD. Thereafter, blood, liver, colon and fecal samples were collected. Biochemical parameters, endotoxin and inflammatory cytokines in the blood and antioxidant enzymes in the liver were tested by commercial kits. Histopathological changes in the liver were evaluated by HE staining. In addition, the mRNA and protein expression of FOXO4, NF-kappa B, ZO-1 and Occluding in the colon were measured by quantitative real-time PCR and Western blot, respectively. Furthermore, gut microbiota composition in the fecal samples was investigated with 16S rDNA sequencing. Results: FOXO4 significantly ameliorated liver histopathological damage. Moreover, FOXO4 reduced the serum endotoxin, biochemical parameters (ALT, AST, ALP and TG), antioxidant enzymes (ROS and MDA), inflamma- tory cytokines (IL-6, IL-1 beta and TNF-alpha), but restored the levels of GSH, SOD and IL-10. Furthermore, FOXO4 significantly inhibited the expression of NF-kappa B, p-NF-kappa B p65, p-IKK alpha and p-IKK beta, and up-regulated the expression of ZO-1 and Occludin. Additionally, FOXO4 modulated the gut microbiota composition and certain bacteria including Odoribacter, Parasutterella and Psychrobacter. Conclusion: These findings suggest that FOXO4 protects against alcohol-induced chronic liver injury via inhibiting NF-kappa B and modulating gut microbiota in C57BL/6J mice.