Lactobacillus reuteri attenuates methotrexate-induced liver injury via modulation of oxidative stress and inflammation through HO-1/GPX4 and NF-κB/NLRP3 pathways

Abstract

Methotrexate (MTX) is a chemotherapeutic agent widely used in cancer and autoimmune diseases; however, its clinic use is considerably limited owing to its severe hepatotoxicity. Although gut microbiota is implicated in various liver diseases, its specific role and mechanism in MTX-induced liver injury remain unclear. We established a MTX-induced liver injury model in mice and assessed hepatic damage through biochemical markers and histopathological analysis. Gut microbiota depletion was conducted using an antibiotic cocktail (ABX) and microbial composition was analysed via 16S rRNA sequencing. We observed that MTX administration induced hepatic pathological and oxidative stress injury by increased hepatic MDA levels, elevated serum AST and ALT activities, and reduced SOD and GSH activities. Depletion of the gut microbiota using ABX resulted in comparable liver injury in MTX-treated and saline-treated mice, suggesting that gut microbiota contributed to MTX-induced liver injury. Further, 16S rRNA sequencing demonstrated that MTX reduced gut microbial diversity and disturbed gut microbial composition. Among the altered bacteria, Lactobacillus reuteri was negatively associated with liver injury index. Furthermore, L. reuteri supplementation attenuated MTX-induced hepatic pathological and oxidative stress injury by inhibiting hepatic MDA content, AST and ALT activities, and promoting the activities of SOD through enhancing the HO-1/GPX4 signalling pathway. In addition, L. reuteri alleviated hepatic inflammation responses by reducing pro-inflammatory cytokines through inhibiting the NF-κB/NLRP3 signalling pathway. In conclusion, our results demonstrated that MTX-induced liver injury is dependent on the gut microbiota and L. reuteri supplementation can help prevent MTX-induced liver injury.