Essential Role And Signalling Pathway Of α7 Nicotinic Acetylcholine Receptor In Nicotine-associated Periodontitis

Background: Periodontitis, which causing periodontium destruction, pocket formation, alveolar bone resorption, and eventually teeth exfoliation, is one of the most common chronic diseases in adults. Numerous data demonstrated that tobacco smoking is one of the major risk factors for periodontitis. In addition, nicotine, the major component in tobacco smoke, has been proven to play an important role in smoking- associated periodontitis. However, the mechanisms behind the effects of nicotine on periodontal destruction are still not well understood.Our previous studies demonstrated the functional expression of lphaα7 subunit of nicotinic acetylcholine receptors (α7 nAChR) in rat periodontal tissues. What's more, nicotine acted directly and specifically throughα7 nAChR to affect periodontitis activity. However, initial data regarding how theα7 nAChR modulates nicotine's effects on periodontitis, such as regulating cells behavior and pro-inflammatory cytokines expression of PDL cells in vitro, or affecting periodontal tissue destruction in vivo, are still lacking.Objective: The aim of this study was to investigate how theα7 nAChR regulates nicotine's effects on PDL cells behavior, inflammatory related cytokines expression and periodontal tissue destruction, and to explore the signalling pathway downstream ofα7 nAChR after its activation.Materials and methods: In vitro, human PDL cells were cultured with or without different concentration of nicotine (Concentration ranging from 0.1μM to 10μM), alpha-bungarotoxin ( -Btx, inhibitor ofα7 nAChR, 10nM), U0126 (inhibitor of ERK1/2, 10μM), SB203580 (inhibitor of P38, 10μM), SP600125 (inhibitor of JNK, 10μM) and LY-294,002 (inhibitor of PI3K, 10μM). Cell proliferation, adhesion and migration were measured by WST-1 assay, attachment assay and wound healing assay, respectively. The expression ofα7 nAChR, IL-1?, IL-6, TNF- , RANKL and OPG in PDL cells were explored by Real Time-PCR, Immunofluorescence and Western blot assay.In vivo, twenty-four male Sprague-Dawley rats were anesthetized and scanned by an in vivo micro-CT system (day 0, baseline). They were then tied by silk ligatures around cervixes of the second maxillary molar while the contra lateral one was left untreated. The rats were randomly divided into four groups, and received intraperitoneal injections of saline, nicotine (1.62 mg/kg) and/or -Btx (10μg/kg), respectively. All the animals were then scanned at dayα7, 14, 21, and 28 after ligatures placement. The alveolar bone loss below the roof of the furcation, and above the root apex of the second maxillary mola was three-dimensionally selected and analyzed. All rats were sacrificed at day 28. The maxillas were harvested and paraffin serial sections were cut. Hematoxylin and eosin examination, and immunohistochemical staining ofα7 nAChR, IL-1β, IL-6, TNF-α, RANKL, OPG and TRAP were performed.Results: In vitro, The expression ofα7 nAChR was significantly up-regulated (about 4.α7-fold) by the administration of nicotine at the concentration of 10μM, and this effect could be partially suppressed by pretreatment with -Btx. Nicotine significantly induced the migration, but inhibited the proliferation and adhesion of cultured PDL cells in a dose dependent manner. Moreover, whileα7 nAChR expression by PDL cells was down-regulated by pretreatment of -Btx, abrogated functions of cell behaviors were observed.In vivo, compared with the control group, nicotine administration up-regulated the number of TRAP positive cells in periodontal tissues, of which the periodontal destruction was correlated. However, the changes were insignificant when pretreated with -Btx. Nicotine alone caused moderate bone microstructure deteriorations begin with decreasing Tb.Th and increasing Tb.Sp, and latter the BVF and Tb.N loss. Nicotine combined with ligature induced robust bone loss that occurred earlier and last over time. The expression ofα7 nAChR in periodontal tissue was up-regulated by nicotine administration, whereas -Btx treatment partially suppressed this effect.Both in vitro and in vivo, induced IL-1β, IL-6, TNF-α, RANKL and reduced OPG expression were also related with nicotine administration and partially suppressed byα-Btx pretreatment, which were correlated withα7 nAChR expression. In addition, ERK1/2 and PI3K inhibition significantly diminished nicotine induced IL-1β, IL-6 and TNF- expression in vitro.Conclusion: The present studies demonstrate that nicotine inhibits human PDL cells attachment, proliferation and enhances PDL cells migration through activation ofα7 nAChR. In addition, Nicotine itself causes moderate alveolar bone microstructure deteriorations, and nicotine combined with ligature induces robust bone loss that occurred earlier and last over time. Furthermore, the nicotine induced IL-1β, IL-6, TNF- and RANKL expression, and reduced OPG expression were regulated by periodontalα7 nAChR, partially through ERK1/2 and PI3K pathway. All those effects are critical for the inflammatory reaction, periodontal destruction and alveolar bone loss in the process of periodontitis.Our work indicates that nicotine may affect periodontal tissue destruction and inflammatory cytokines expression through regulation ofα7 nAChR in smoking-associated periodontitis, which may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.