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 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.


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.


CONCLUSIONS
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.
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Ye
Y
Yue
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China; Institute of Orthopedics, Chinese PLA General Hospital, Fuxing 28# Road, Beijing, China.
Ruoxi
R
Liu
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Wenxiang
W
Cheng
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Yiping
Y
Hu
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Jinchao
J
Li
The Department of Orthopedics, The Second Clinical Medical College, Jinan University, Shenzhen, Guangdong, China.
Xiaohua
X
Pan
Institute of Orthopedics, Chinese PLA General Hospital, Fuxing 28# Road, Beijing, China. Electronic address: pengjdxx@126.com.
Jiang
J
Peng
Shenzhen Bioactive Materials Engineering Key Lab for Medicine, Translational Medicine R&D Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Electronic address: peng.zhang@siat.ac.cn.
Peng
P
Zhang
D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology*; D002038:Bungarotoxins / Q000494:pharmacology; D003374:Coumarins / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis*; D007249:Inflammation / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D051058:Oncogene Protein v-akt / Q000096:biosynthesis* / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology*; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000187:drug effects; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000187:drug effects*
D001597:Benzylidene Compounds; D002038:Bungarotoxins; D003374:Coumarins; D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D018733:Nicotinic Antagonists; D011725:Pyridines; D064569:alpha7 Nicotinic Acetylcholine Receptor; C033451:coumarin-alpha-bungarotoxin conjugate; C088936:3-(2,4-dimethoxybenzylidene)anabaseine; D051058:Oncogene Protein v-akt
Akt; GTS-21; Inflammation; NF-κB; RAW264.7 cells; α7 nicotinic acetylcholine receptor