Resveratrol enhances methotrexate-mediated suppression of inflammatory cytokines via NF-κB and MAPK pathway inhibition in THP-1 cells

Methotrexate (MTX) is widely used in the management of inflammatory diseases; however, its clinical use is limited by dose-dependent toxicities. Resveratrol, a natural polyphenol, exhibits potent anti-inflammatory properties by modulating multiple intracellular signaling pathways. We hypothesized that co-treatment with resveratrol could enhance MTX’s anti-inflammatory efficacy while mitigating its cytotoxic effects. THP-1 monocytic cells were stimulated with lipopolysaccharide (LPS) and treated with MTX (5 μg/ml) alone or in combination with resveratrol (50 or 100 μg/ml). Cell viability and cycle progression were assessed by flow cytometry. Western blotting evaluated phosphorylation of NF-κB p65 and ERK1/2 (p44/42 MAPK) pathway proteins. Cytokine and chemokine profiles were measured via qPCR and enzyme-linked immunosorbent assay (ELISA). Resveratrol provided dose-dependent protection against MTX-induced cytotoxicity. At 100 μg/ml, it significantly reduced necrotic cell death and enhanced cell cycle progression. Combined treatment with MTX and resveratrol resulted in marked suppression of inflammatory signaling, including average 55% reduction in phosphorylated NF-κB p65 and 40% in p-ERK1/2 ( p  < 0.0001) at both concentrations tested. Cytokine analysis revealed over 75% reduction in TNF-α, IL-6, and IFN-γ compared to LPS controls, alongside a 3-fold increase in IL-10 at transcriptional level. Chemokine suppression included CCL2, CCL3, CCL4, CCL5, and CXCL10.

Resveratrol enhances MTX’s anti-inflammatory effects via dual inhibition of NF-κB and MAPK pathways, while reducing MTX-induced toxicity. This combination yields broad cytokine and chemokine suppression and promotes anti-inflammatory responses. These findings support the potential of MTX-resveratrol co-therapy as a safer, more effective strategy for managing chronic inflammatory diseases.