Protective effects of ganoderic acid C2 from Ganoderma lucidum against aflatoxin B1-induced splenorenal injury: Inhibition of NOX4-mediated oxidative stress and inflammation.

Aflatoxin B1 (AFB1) is a mycotoxin known to induce splenorenal toxicity through oxidative stress and chronic inflammation, yet effective protective strategies remain limited. Ganoderic Acid C2 (GA-C2), a triterpenoid isolated from Ganoderma lucidum, possesses antioxidant and anti-inflammatory potential, but its role against AFB1-induced toxicity has not been fully clarified. In this study, seventy-two male BALB/c mice were treated with AFB1 (0.75 mg/(kg·day)) alone or in combination with GA-C2 (5, 10, 15 mg/(kg·day)) or the NADPH oxidase 4 (NOX4) inhibitor GKT137831 (30, 60 mg/(kg·day)) for 30 days. Physiological indices, histopathology, serum injury markers, cytokines, and oxidative stress parameters were evaluated. Potential molecular targets were predicted by network pharmacology and molecular docking, and validated by quantitative reverse transcription-polymerase chain reaction and western blot. Principal component analysis was used to assess clustering of inflammatory and oxidative stress responses. GA-C2 significantly protected against AFB1-induced splenorenal toxicity by restoring body and organ weights, preserving tissue morphology, and reducing renal injury biomarkers. It attenuated oxidative stress and inflammation while enhancing antioxidant defenses. Mechanistically, GA-C2 inhibited NOX4, restored protein kinase B (Akt)/glycogen synthase kinase 3 beta (GSK3β) signaling, activated nuclear factor erythroid 2-related factor 2, and suppressed nuclear factor kappa-B activation. Molecular docking confirmed strong binding between GA-C2 and NOX4, and the protective effects of GKT137831 further validated the NOX4-Akt/GSK3β axis as a therapeutic target. These findings demonstrate that GA-C2 protects against AFB1-induced splenorenal injury and highlight its translational potential as a natural antidote in food safety.