4-DMB, a diclofenac derivative, alleviates DSS-induced ulcerative colitis in mice by binding TNFR1 to inhibit inflammation.

Novel diclofenac (Dic) hybrids with methyl 2-bromobenzoate (2-DMB) and methyl 4-bromobenzoate (4-DMB) modifications via ester linkages have been synthesized and proven to have anti-inflammatory potential in vitro, suggesting that further development of these compounds may lead to a potential anti-inflammatory drug. In this study, the anti-inflammatory activity of these compounds was evaluated both in vitro and in vivo, and the underlying mechanisms were explored using a RAW 264.7 cell model of LPS-induced inflammation. Compared with 2-DMB, 4-DMB showed more potent anti-inflammatory activity in LPS-treated RAW 264.7 cells, with no cytotoxicity. Moreover, 4-DMB exhibited greater protective efficacy than 5-aminosalicylic acid (5-ASA) against DSS-induced colitis in mice. Notably, the results from cellular studies indicated increased protein and mRNA levels of TNF-α following treatment with 4-DMB, suggesting an effect of 4-DMB on TNFR. Furthermore, cellular thermal shift assays (CETSA), isothermal titration calorimetry (ITC), and molecular docking studies collectively demonstrated that 4-DMB competes with TNF-α for binding to TNFR1. In addition, network pharmacology analysis combined with Western blot validation indicated that the anti-colitis effect of 4-DMB is closely associated with the inhibition of the AKT/ERK/NF-κB signaling pathway. Critically, siRNA-mediated knockdown of TNFR1 significantly suppressed the AKT/ERK/NF-κB signaling and inflammatory responses, demonstrating the essential role of TNFR1 in mediating the AKT/ERK/NF-κB signaling. Taken together, these findings indicate that 4-DMB mitigates colitis by directly binding to TNFR1 and inhibiting the AKT/ERK/NF-κB signaling pathway. This research underscores the potential clinical relevance of 4-DMB for the treatment of inflammatory disorders, particularly ulcerative colitis.