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

Int Immunopharmacol. 2015 Dec;29(2):504-512. doi: 10.1016/j.intimp.2015.10.005. Epub 2015 Oct 18.

Abstract

Objective: GTS-21, a selective α7 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-κB 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)-1β, IL-6, or tumor necrosis factor (TNF)-α 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)-κB was analyzed by confocal fluorescence microscopy. In addition, α7 nicotinic acetylcholine receptors (α7 nAChR) were detected on RAW264.7, and the α7 nAChR-specific antagonist was adopted to verify whether the effect of GTS-21 was mediated by α7 nAChR.

Results: The CCK-8 assay showed that GTS-21 did not significantly affect cell proliferation. The production of IL-1β, IL-6, and TNF-α decreased after being treated with GTS-21 in LPS-stimulated RAW 264.7 cells. GTS-21 also suppressed LPS-induced phosphorylation of NF-κBp65, IKKα/β, IκBα, and Akt, as well as NF-κB p65 nuclear translocation. Moreover, α7 nAChR was expressed on the surfaces of RAW264.7 cells, and the α7 nAChR-specific antagonist almost completely prohibited the inhibitory effect of GTS-21 on NF-κB activation.

Conclusion: These findings indicate that GTS-21 suppresses LPS-induced inflammation by inhibiting the Akt/NF-κB signal pathway through α7 nAChR. GTS-21 has a potential application in inflammatory disease therapy.

Keywords: Akt; GTS-21; Inflammation; NF-κB; RAW264.7 cells; α7 nicotinic acetylcholine receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzylidene Compounds / pharmacology*
  • Bungarotoxins / pharmacology
  • Coumarins / pharmacology
  • Cytokines / biosynthesis*
  • Inflammation / metabolism*
  • Lipopolysaccharides / pharmacology
  • Mice
  • NF-kappa B / biosynthesis*
  • NF-kappa B / genetics
  • Nicotinic Antagonists / pharmacology*
  • Oncogene Protein v-akt / biosynthesis*
  • Oncogene Protein v-akt / genetics
  • Pyridines / pharmacology*
  • RAW 264.7 Cells
  • Signal Transduction / drug effects
  • alpha7 Nicotinic Acetylcholine Receptor / drug effects*

Substances

  • Benzylidene Compounds
  • Bungarotoxins
  • Coumarins
  • Cytokines
  • Lipopolysaccharides
  • NF-kappa B
  • Nicotinic Antagonists
  • Pyridines
  • alpha7 Nicotinic Acetylcholine Receptor
  • coumarin-alpha-bungarotoxin conjugate
  • 3-(2,4-dimethoxybenzylidene)anabaseine
  • Oncogene Protein v-akt