Anti-Inflammatory Functions of Alverine via Targeting Src in the NF-[kappa]B Pathway

Alverine, a smooth muscle relaxant, is used to relieve cramps or spasms of the stomach and intestine. Although the effects of alverine on spontaneous and induced contractile activity are well known, its anti-inflammatory activity has not been fully evaluated. In this study, we investigated the anti-inflammatory effects of alverine in vitro and in vivo. The production of nitric oxide (NO) in RAW264.7 cells activated by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly (I:C)) was reduced by alverine. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-[alpha] (TNF- [alpha]) was also dose-dependently inhibited by treatment with alverine. In reporter gene assays, alverine clearly decreased luciferase activity, mediated by the transcription factor nuclear factor [kappa]B (NF- [kappa]B) in TIR-domain-containing adapter-inducing interferon-[beta] (TRIF)- or MyD88-overexpressing HEK293 cells. Additionally, phosphorylation of NF-[kappa]B subunits and upstream signaling molecules, including p65, p50, AKT, I[kappa]B[alpha], and Src was downregulated by 200 [micro]M of alverine in LPS-treated RAW264.7 cells. Using immunoblotting and cellular thermal shift assays (CETSAs), Src was identified as the target of alverine in its anti-inflammatory response. In addition, HCl/EtOH-stimulated gastric ulcers in mice were ameliorated by alverine at doses of 100 and 200 mg/kg. In conclusion, alverine reduced inflammatory responses by targeting Src in the NF-[kappa]B pathway, and these findings provide new insights into the development of anti-inflammatory drugs.