Methotrexate (MTX) has been used effectively to treat several neoplastic and autoimmune disorders. As a structural analog to folic acid, MTX inhibits folate metabolism by interfering with dihydrofolate reductase, thus prevents the formation of purines and pyrimidines which are precursors to nucleic acids [1]. Unfortunately, because of its non-selective impact, MTX's therapeutic potential occasionally coexists with serious multi-organ toxicity [2].

Intestinal toxicity is one of the most notable side effects of MTX chemotherapy. Uncertainty surrounds the mechanism of MTX-induced toxicity. According to previous studies, the suppressor effect of MTX on the immune system, promotes reactive oxygen species (ROS) production, lipid peroxidation, and oxidative damage. Additionally, because of the excessive ROS generation by MTX treatment, it could result in neutrophil infiltration with the subsequent release of inflammatory cytokines, leading to tissue damage [3], [4].

Interestingly, the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) participates a crucial defensive role against oxidative stress and inflammation via enhanced transcription of certain cyto-defensive genes [5], [6]. In addition to Nrf2, the peroxisome proliferator-activated receptor gamma (PPAR-γ) has been reported to protect against oxidative stress and inflammatory response in different models of intestinal injury, and can inhibit the nuclear factor-kappa B (NF-κB); the strategic regulator of inflammation; through both direct and indirect pathways [7], [8]. As well, sirtuin 1 (SIRT1) effectively regulates oxidative stress and cellular inflammatory response, and has the potential to be a modulator for stressful cellular circumstances [9], [10].

Since MTX strongly induces the production of ROS, various signals are impacted [11]. ROS induced upregulation of interleukin-6 (IL-6) is partly mediated through the transcription factor STAT3 [12]. STAT3 is activated via phosphorylation by tyrosine kinases in response to various stimulants including IL-6 cytokine, resulting in the propagation of intracellular inflammatory signaling in the intestine [13].

Nifuroxazide (NIF) is an oral antibiotic approved for several gastrointestinal infections as an effective antidiarrheal agent with high safety profile [14]. Interestingly, NIF has been demonstrated to suppress IL-6/STAT3 signaling and inflammatory perturbations in ulcerative colitis model [15]. However, there wasn't enough attention given to the therapeutic benefits of NIF in MTX-induced intestinal damage and the underlying molecular signaling pathways involved. As a result, the current study was designed to explore the potential efficacy of NIF in alleviating intestinal toxicity associated with MTX chemotherapy, pointing to the impact on Nrf2, PPAR-γ, SIRT1, NF-κB, and JAK1/STAT3 signals