Tehran University of Medical Sciences Acta Biochimica Iranica 0001-5261 2026 4 2026 06 21 98 105 Narges Najafi Kajabad Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Mehdi Fazeli Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Mohammad Foad Noorbakhsh Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Milad Faraji Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran 2026 05 17 2026 05 26 Objectives: Methotrexate (MTX) is an effective chemotherapeutic and immunosuppressive agent; however, its clinical use is limited due to nephrotoxicity associated with oxidative stress. This study aims to evaluate the protective effects of silymarin, citral, and thymoquinone against MTX-induced renal injury in rats. Methods: Forty-eight adult male Sprague–Dawley rats were randomly assigned into six groups (n=8): healthy control, cosolvent control (DMSO), MTX-only, MTX+citral (30 mg/kg), MTX+silymarin (50 mg/kg), and MTX+thymoquinone (10 mg/kg). Treatments were administered intraperitoneally for 10 consecutive days. Methotrexate (20 mg/kg, i.p.) was administered to all groups except the healthy control. Serum urea and creatinine levels were measured, while renal tissues were evaluated for total antioxidant capacity (TAC), malondialdehyde (MDA), and histopathological alterations. Results: MTX administration significantly increased serum urea, creatinine, and renal MDA levels while decreasing TAC compared with the healthy control group. Silymarin significantly reduced serum urea and creatinine levels and restored TAC compared with the MTX group (p<0.05). Thymoquinone significantly improved TAC and reduced MDA levels (p<0.05), although its effects on serum renal biomarkers were not statistically significant. Citral significantly decreased MDA levels (p<0.01) but showed limited effects on TAC and renal function markers. Histopathological examination demonstrated vascular and glomerular congestion with hemorrhage in the MTX group, whereas treatment groups exhibited varying degrees of structural preservation, with silymarin showing the greatest improvement. Conclusions: Oxidative stress plays a major role in MTX-induced nephrotoxicity. Among the tested compounds, silymarin demonstrated the most consistent nephroprotective effects, while thymoquinone and citral provided partial antioxidant and histological protection. These findings support the potential use of natural antioxidants as adjunctive strategies against MTX-induced renal injury. https://abi.tums.ac.ir/index.php/abi/article/view/191 https://abi.tums.ac.ir/index.php/abi/article/download/191/141