Therapeutic effects of Rosmarinus officinalis against methotrexate-induced submandibular salivary gland injury in male albino rats.

Methotrexate (MTX) is a chemotherapeutic drug extensively used for the treatment of malignancies and autoimmune disorders. However, its clinical use is limited by cytotoxic effects on normal tissues, mediated by oxidative stress-induced depletion of antioxidant defenses and activation of apoptotic pathways. Rosmarinus officinalis (rosemary) contains bioactive compounds with established antioxidant and anti-apoptotic properties, indicating potential therapeutic effects against MTX-induced tissue injury. This study aimed to investigate the potential therapeutic effect of Rosmarinus officinalis in attenuating the cytotoxic effects of MTX on the submandibular salivary gland (SMSG) of male albino rats. This experimental animal study included twenty-eight adult male albino rats (aged 4-6 months), randomly allocated into four equal groups (n = 7): the control group received saline; the rosemary group received Rosmarinus officinalis methanolic extract (RME) (200 mg/kg, i.p.) once daily for 10 days; the MTX group received a single dose of MTX (40 mg/kg, i.p.) on day 1 only; and the MTX + rosemary group received MTX (40 mg/kg i.p.) on day 1, followed by RME (200 mg/kg i.p.) for 10 days. On day 11, the submandibular salivary glands were dissected and rinsed in 0.9% saline, and the right glands were divided into two parts: one preserved fresh for biochemical assessment of reduced glutathione (GSH), and the other fixed for histological and immunohistochemical analysis of cleaved caspase-3 and nuclear factor erythroid 2-related factor 2 (Nrf2). The control and rosemary groups exhibited normal glandular architecture, with the rosemary group showing the highest GSH concentration and Nrf2 expression. In contrast, the MTX group showed pronounced degenerative and atrophic changes in the acinar and ductal structures, coinciding with strong caspase-3 immunoreactivity, significantly reduced Nrf2 expression, and depleted GSH concentration (P ≤ 0.05). In the MTX + rosemary group, the glandular architecture showed partial improvement compared with the MTX group, associated with reduced caspase-3 immunoreactivity, recovered Nrf2 expression, and increased GSH concentration relative to the MTX group (P ≤ 0.05). Rosmarinus officinalis treatment significantly attenuated MTX-induced SMSG injury, as evidenced by enhanced Nrf2 immunoexpression, increased GSH concentration, and reduced cleaved caspase-3 expression. These findings suggest that rosemary exerts antioxidant and anti-apoptotic effects, contributing to the preservation of glandular integrity against MTX toxicity. Taken together, Rosmarinus officinalis may represent a promising adjunctive therapeutic strategy for attenuating MTX-induced glandular damage, warranting further investigation.