Methotrexate (MTX) is from the antimetabolite group of anti-cancer drugs that is extensively utilized in the treatment of lymphoblastic leukemia, lymphoma, and autoimmune disorders, including rheumatoid arthritis and psoriasis (Dursun et al., 2025). The mechanism by which MTX operates involves the inhibition of dihydrofolate reductase and other associated enzymes that play a role in the synthesis of purines and thymidine, ultimately leading to the suppression of DNA, RNA, and protein synthesis. The interruption of these biosynthetic pathways hinders essential biochemical processes, such as the production of vital ATP molecules and cell division (AlJohani, 2021). The use of this medication may lead to various adverse effects, which include cardiological complications such as arrhythmia, gastrointestinal issues like nausea and vomiting, and psychological conditions such as depression. Furthermore, the drug may cause immunosuppression (Yang et al., 2011).

Methotrexate (MTX)-induced neurotoxicity is a recognized adverse effect, although its incidence varies depending on patient population, MTX dose, route of administration, and the underlying condition being treated, with estimates ranging from 3 % to 15 % (Apiraksattayakul et al., 2024). Neurotoxicity can occur following high-dose MTX or prolonged low-dose oral therapy and may manifest as seizures, aphasia, encephalopathy, or stroke-like symptoms. The timing of neurotoxic events is highly variable, occurring hours, days, or even years after the last MTX dose, with diverse clinical presentations leading to distinct outcomes. Multiple factors, including interindividual differences, contribute to the variability in susceptibility among patients receiving similar MTX regimens(Besora et al., 2025). Mechanistically, excessive production of reactive oxygen species (ROS) has been implicated in MTX-induced neurotoxicity, highlighting the role of oxidative stress, leading to cell cycle arrest, neuronal death, and impaired hippocampal function (Aslankoc et al., 2022). This oxidative stress not only reduces neurogenesis and memory performance but also triggers inflammatory cascades through elevated cytokine release (Kandemir et al., 2017). The brain is particularly vulnerable due to its limited antioxidant defenses, high metabolic demands, and abundance of polyunsaturated fatty acids, while neurons’ restricted glutathione synthesis further amplifies MTX neurotoxicity (Shagirtha et al., 2017). Consequently, oxidative stress is considered a central mechanism underlying MTX-induced neurotoxicity, highlighting the importance of antioxidant-based strategies for neuroprotection.

Given the pivotal role of ROS accumulation and oxidative stress in the development of MTX neurotoxicity, the induction of antioxidant and cytoprotective enzymes is essential for maintaining neuronal integrity. Indeed, under physiological conditions, cells are equipped with a robust network of enzymatic and non-enzymatic antioxidants that counteract redox disturbances. Heme oxygenase-1 (HO-1) plays a vital role in mitigating oxidative and inflammatory damage (Loboda et al., 2016). Numerous studies have demonstrated that activation of the protein kinase B (Akt) signaling cascade facilitates the induction of heme oxygenase-1 (HO-1), thereby exerting potent anti-apoptotic and anti-inflammatory effects in response to oxidative stress or tissue injury (Shi et al., 2019). Also, Sirtuin1 (SIRT1), which is mainly localized in the nucleus, regulates nuclear stability and gene transcription. It can translocate between cellular compartments, contributing to its diverse roles in cellular homeostasis. SIRT1 mediates the beneficial effects and exhibits anti-inflammatory, antioxidant, and antiaging properties. Moreover, it modulates energy metabolism and mitochondrial biogenesis, and recent studies implicate its protective role in various pulmonary diseases (Jiang et al., 2024). The increase in SIRT-1 gene expression, mediated in part by AKT signaling, may stimulate the activation of the NRF2 pathway, thereby enhancing the synthesis of key antioxidant and cytoprotective molecules such as HO-1 and GPX4 (Oguzoglu et al., 2024). Furthermore, NF-κB acts as a central regulator of pro-inflammatory gene expression, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, and thus plays a pivotal role in orchestrating inflammatory responses (Tak and Firestein, 2001).

Numerous researchers have explored the reduction and minimization of toxicity associated with chemotherapeutic agents by utilizing various natural products that possess significant antioxidant properties, aiming to mitigate the effects of oxidative stress in biological processes (Maksimovic et al., 2020). Syringic acid (SA) (4-hydroxy-3,5-dimethoxybenzoic acid), a naturally occurring benzoic acid derivative, is an abundant phenolic compound widely distributed in plants and fruits, including dates, olives, pumpkin, grapes, spices, and honey (Ha et al., 2018). It has been reported to exert a broad spectrum of pharmacological activities, including potent antioxidant, anti-inflammatory, antiapoptotic, and immunomodulatory effects. (Bolat et al., 2025b). Emerging evidence highlights its neuroprotective role in various models of brain injury. For instance, SA has been shown to alleviate oxidative stress and neuronal degeneration in ischemic brain tissues (Güven et al., 2015), while also protecting against MPTP-induced neuroinflammation (Rekha et al., 2014). Importantly, regular consumption of SA has been associated with beneficial effects on cognitive and behavioral functions, underscoring its therapeutic potential in neurodegenerative and neurotoxic conditions (Ogut et al., 2022).

Despite the clinical relevance of MTX-induced neurotoxicity, the precise molecular mechanisms underlying its cognitive and locomotor impairments remain poorly understood. MTX has been shown to induce oxidative stress, inflammation, and apoptosis, resulting in behavioral deficits in rodent models. However, strategies to prevent or mitigate these effects are limited, and natural compounds with antioxidant and anti-inflammatory properties, such as syringic acid, have not been fully investigated in this context. Therefore, the present study aimed to explore the potential neuroprotective effects of syringic acid against MTX-induced neurotoxicity by assessing cognitive and locomotor behavior, oxidative stress and antioxidant markers, inflammatory cytokines, apoptotic signaling, and activation of the PKC/CDC42 and AKT/SIRT1/HO-1 pathways, providing a comprehensive evaluation of its therapeutic potential.