Hesperidin attenuates methotrexate-induced cardiotoxicity: Modulation of Nrf2 antioxidant defense and NF-κB/iNOS proinflammatory signaling

Methotrexate (MTX) is a commonly used chemotherapeutic and immunosuppressive agent, although serious adverse effects, including cardiotoxicity, sometimes compromise its clinical use and effectiveness. This work was designed to investigate the mechanisms underlying MTX-induced cardiac injury and to evaluate the potential cardioprotective efficacy of hesperidin (HES), a bioflavonoid derived from citrus fruits, known for its potent antioxidant and anti-inflammatory properties. Thirty-two adult male Sprague-Dawley rats were randomly divided into four groups (n = 8/group) and treated for six weeks: Control, HES (200 mg/kg/day, via orogastric tube), MTX (5 mg/kg/week, via intraperitoneal injection), and HES+MTX co-treatment. Serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels were measured. The cardiac tissue homogenates were analyzed for lipid peroxidation (malondialdehyde [MDA]), nitric oxide (NO), reduced glutathione (GSH), and superoxide dismutase (SOD) activity. Histopathological changes were assessed by hematoxylin and eosin (H&E) staining, while the immunoreactivity levels of nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear factor-κB (NF-κB), and inducible nitric oxide synthase (iNOS) were measured by quantitative immunohistochemical analysis. Methotrexate treatment induced myocardial damage, manifested by a considerable increase in serum CK-MB and LDH activity, along with histological changes. MTX triggered oxidative and nitrosative stress, evidenced by a significant increase in tissue MDA and NO levels, accompanied by a notable decrease in endogenous GSH levels and SOD activity. This redox imbalance was associated with reduced immunoexpression of Nrf2 and stimulation of the pro-inflammatory NF-κB/iNOS signaling axis. Conversely, co-treatment with HES significantly preserved myocardial architecture and restored redox equilibrium, demonstrated by enhanced Nrf2 immunoreactivity and suppressed NF-κB and iNOS expression. These results indicate that hesperidin effectively attenuates MTX-induced cardiotoxicity. The cardioprotective effects of HES appear to involve its dual capacity to enhance Nrf2-dependent endogenous antioxidant defenses and attenuate the NF-κB/iNOS-driven inflammatory response. Therefore, HES may hold potential as a supplementary agent to minimize the cardiotoxic complications associated with MTX regimens, warranting further investigation.