Uncontrolled inflammation is a critical pathological response that can damage tissues and lead to disease. This study aimed to evaluate the anti-inflammatory potential of ethyl 4-(4-bromophenyl)-2-diazo-4-(formyloxy) butanoate (6) and explored the underlying mechanisms. Zebrafish models with copper sulfate, tail transection, and LPS microinjection were used to evaluate the anti-inflammatory efficiency of 6 through neutral red staining, H&E staining, and RT-qPCR analysis. A LPS-stimulated inflammatory RAW264.7 cells model was employed, and the anti-inflammatory effects of 6 were explored by siRNA knockdown, Western blotting analysis, and immunofluorescence staining. CETSA, DARTS and SPR experiments were used to examine the interaction between 6 and ɑ7nAChR. 6 exhibited significant anti-inflammatory activities in both zebrafish and RAW264.7 cells. In zebrafish, 6 attenuated inflammatory responses as evidenced by the reduced migration of inflammatory cells, improved histological outcomes, and downregulated expressions of inflammatory genes. In RAW264.7 cells, 6 inhibited NF-κB signaling as reflected by the decreased level of phosphorylation of IκBɑ at Ser32 in total cell protein and NF-κB p65 in nuclear protein which was further confirmed by immunofluorescence. 6 also decreased the levels of inflammatory factors TNF-ɑ and IL-6 as detected by RT-qPCR. Furthermore, 6 activated ɑ7nAChR and the blockage of ɑ7nAChR counteracted the inhibitory effects of 6 on the NF-κB pathway. CETSA, DARTS, and SPR experiments provided direct evidence for the interaction between 6 and ɑ7nAChR. Our finding demonstrates that 6 with a novel skeleton exerts potent anti-inflammatory effects through the inhibition of the NF-κB pathway by targeting ɑ7nAChR.
