Objectives: Methotrexate (MTX) is a highly effective chemotherapeutic drug used to treat neoplastic disorders and autoimmune conditions; however, its therapeutic utility is often limited by dose-limiting hepatotoxicity. We designed this study to assess the protective efficacy of betulinic acid (BA), a naturally occurring pentacyclic triterpenoid, on MTX-induced liver injury, via the NLRP3/Caspase-1 inflammasome signaling pathway.

Materials and Methods: Male mice were randomly divided into four groups, including control, BA (25 mg/kg, i.p.), MTX (20 mg/kg, i.p.), and co-administration of BA and MTX. Upon conclusion of the experimental period, mice were euthanized. Blood and hepatic tissue specimens were collected to perform biochemical and histopathological investigations.

Results: MTX significantly increased serum levels of liver enzymes in comparison with controls. The levels of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), lipid peroxidation, and nitric oxide were increased; total thiols, GPx, superoxide dismutase (SOD), and catalase (CAT) activities were decreased; and the expression of hepatic NLRP3 and caspase-1 was increased. BA pretreatment significantly reduced liver enzyme levels, suppressed lipid peroxidation and IL-1β and, TNF-α, restored thiol content, CAT, SOD activities, and downregulated NLRP3 and Caspase-1 expression. Histological analysis confirmed that BA alleviated MTX-induced liver injury.

Conclusion: BA showed potent attenuation of MTX-mediated hepatic damage through modulation of oxidative stress and inhibiting the NLRP3/Caspase-1 inflammasome activation. Our data suggest that the administration of BA could be a viable supportive strategy to minimize the liver injury secondary to MTX treatment.