Modulation of Interleukin-6 and Transforming Growth Factor -β Signaling Pathways by Saffron and Silymarin in Methotrexate-Induced Hepatotoxicity

This study examined whether saffron (Crocus sativus) and silymarin (Silybum marianum), used alone or together, can protect the liver against methotrexate (MTX)-induced acute injury in rats, with a focus on interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) signaling. Ethanol extracts of saffron and silymarin were standardized by GC–MS, and thirty adult male rats were randomly assigned to five groups (n = 6): control, MTX alone (20 mg/kg, intraperitoneally, on day 7), saffron + MTX (100 mg/kg/day orally for 14 days + MTX), silymarin + MTX (100 mg/kg/day orally for 14 days + MTX), and saffron + silymarin + MTX. We measured serum liver enzymes (ALT, AST, ALP, LDH), inflammatory markers (CRP, IL-6, TGF-β) and oxidative stress indices (MDA, GPX1), and examined liver tissue with H&E and immunohistochemistry for IL-6 and TGF-β. MTX caused liver damage, with significant rises in liver enzymes, CRP, IL-6, TGF-β and MDA, and reductions in albumin, total protein and GPX1 (p < 0.05). Histology showed inflammatory cell infiltration, necrosis and strong IL-6 and TGF-β staining in hepatocytes. Saffron or silymarin treatment improved these changes, partially normalizing biochemical markers, restoring GPX1, lowering IL-6 and TGF-β and reducing inflammatory and necrotic lesions. The combined saffron and silymarin regimen produced the greatest protection. These findings suggest that saffron and silymarin, especially in combination, can limit MTX-induced acute hepatotoxicity by reducing oxidative stress and downregulating IL-6 and TGF-β signaling, helping to preserve liver structure and function and supporting their potential use as natural adjuncts to lessen drug-induced liver injury.