Folate Attenuates Ulcerative Colitis via PI3K/AKT/NF-κB/MLCK Axis Inhibition to Restore Intestinal Barrier Integrity

Vitamin homeostasis plays a critical role in inflammatory bowel disease management, yet the protective mechanisms and clinical utility of specific vitamins remain incompletely characterized. Within this context, a two-sample Mendelian randomization analysis leveraging genetic instruments for measuring circulating vitamin levels identified folate as a protective factor against ulcerative colitis (UC). To validate these findings, a DSS-induced colitis model was established with serial serum folate measurements. Therapeutic folate supplements were subsequently administered, followed by a comprehensive evaluation of epithelial barrier modulation through in vivo and recombinant TNF-α/IFN-γ-induced in vitro models. This included assessment of junctional proteins, ultrastructural analysis by transmission electron microscopy, and functional quantification of barrier integrity using transepithelial electrical resistance with paracellular permeability assays in epithelial monolayers. Molecular mechanisms were investigated through RNA sequencing complemented by immunoblot validation of key pathway components. The results demonstrated decreased serum folate levels in DSS-induced colitis mice, whereas folate supplementation ameliorated disease severity and attenuated intestinal inflammation and histopathological damage. Crucially, folate restored epithelial barrier structural integrity and function both in vivo and in vitro. Mechanistically, folate mediated barrier restoration through suppression of the PI3K/AKT/NF-κB/MLCK/MLC2 signaling axis. Collectively, the results of this study provide mechanistic insights that support the use of folate as an active therapeutic molecule in patients with UC.