Probiotics, folic acid, and vitamin C ameliorate leflunomide-induced hepatotoxicity and gastric injury via Nrf2/TGF-β1 signaling

Leflunomide (LEF) is a disease-modifying antirheumatic drug whose prolonged use may be limited by organ toxicity. This study evaluated whether probiotics, folic acid plus vitamin C, or their combination attenuate LEF-induced hepatic and gastric injury and modulate Nrf2 and TGF-β1 expression. Forty male albino rats were randomly allocated to five groups (n = 8/group): control, LEF, LEF plus folic acid and vitamin C, LEF plus probiotics, and LEF plus folic acid, vitamin C, and probiotics. LEF was administered at 10 mg/kg body weight, while folic acid (500 µg/kg), vitamin C (500 mg/kg), and probiotics (5 × 109 CFU/mL/rat/day) were administered orally for four weeks. Serum AST, ALT, albumin, and total protein were assessed. Hepatic and gastric MDA and GSH levels, Nrf2 and TGF-β1 expression, histopathological changes, and immunohistochemical expression of cleaved caspase-3, TNF-α, and IL-1β were evaluated. LEF increased AST, ALT, MDA, inflammatory markers, and tissue injury, while reducing albumin, total protein, and GSH and altering Nrf2 and TGF-β1 expression. Adjunctive treatments attenuated these changes, with the combined regimen showing the greatest improvement. These findings suggest that combined probiotic, folic acid, and vitamin C supplementation may mitigate LEF-induced hepatic and gastric injury by enhancing antioxidant defenses and modulating fibro-inflammatory responses.