GTS-21 attenuates lipopolysaccharide-induced inflammatory cytokine production in vitro by modulating the Akt and NF-κB signaling pathway through the α7 nicotinic acetylcholine receptor

Objective: GTS-21, a selective alpha 7 nicotinic acetylcholine receptor agonist, has recently been established as a promising treatment for inflammation. However, the detailed molecular mechanism of GTS-21 in suppressing pro-inflammatory cytokine production is only partially explored. The study aimed to analyze cytokine expression suppressed by GTS-21 with lipopolysaccharide (LPS)-induced inflammation in vitro and to gain insights into the role of Akt/NF-kappa B signaling pathway in this process. Materials and methods: Cell Counting Kit-8 (CCK-8) assay was performed to detect drug cytotoxicity. RAW 264.7 cells were stimulated with LPS and treated with GTS-21. Interleukin,(IL)-1 beta, IL-6, or tumor necrosis factor (TNF)-alpha production was detected using enzyme-linked immunosorbent assay. Western blot was used to assess the expression patterns of signal transduction protein. Nuclear translocation of nuclear factor (NF)-kappa B was analyzed by confocal fluorescence microscopy. In addition, alpha 7 nicotinic acetylcholine receptors (alpha 7 nAChR) were detected on RAW264.7, and the alpha 7 nAChR-specific antagonist was adopted to verify whether the effect of GTS-21 was mediated by alpha 7 nAChR. Results: The CCK-8 assay showed that GTS-21 did not significantly affect cell proliferation. The production of IL-1 beta, IL-6, and TNF-alpha decreased after being treated with GTS-21 in LPS-stimulated RAW 264.7 cells. GTS-21 also suppressed LPS-induced phosphotylation of NF-kappa Bp65, IKK alpha/beta, I kappa B alpha, and Akt, as well as NF-kappa B p65 nuclear translocation. Moreover, alpha 7 nAChR was expressed on the surfaces of RAW264.7 cells, and the alpha 7 nAChR-specific antagonist almost completely prohibited the inhibitory effect of GTS-21 on NF-kappa B activation. Conclusion: These findings indicate that GTS-21 suppresses LPS-induced inflammation by inhibiting the Akt/NF-kappa B signal pathway through alpha 7 nAChR. GTS-21 has a potential application in inflammatory disease therapy. (C) 2015 Published by Elsevier B.V.