Functional And Mechanism Study Of Inflammatory Microenvironment Regulating The ?7 Nicotinic Acetylcholine Receptor Of Periodontal Ligament Stem Cells In The Development Of Smoking Related Periodontitis

Chronic periodontitis,which is the most commonly seen factor for adult teeth loss,has a strong impact for the life quality of patients.Adult chronic periodontitis has a high prevalence worldwide while smoking is a high risk factor for its development.The progress of periodontitis for smokers is faster with more serious periodontal attachment loss.In addition,the toxic substances in cigarettes would reduce the effect of periodontal therapy,leading to unfavorable prognosis.Thus,clarifying the detailed pathological procedure for periodontitis which is related to smoking and making targeted prevention strategy are of critical clinical and scientific significance in improving the overall level of people's oral health.Previous studies mainly focused on the effect of nicotine toward periodontal tissue derived cells through the ?7 nicotinic acetylcholine receptor(?7 nAChR).However,periodontitis is a bacterial infectious disease.Smoking,which is a high risk factor,could only deteriorate the destruction of periodontal tissues with the help of local inflammation.As a result,researches focused on the mechanism of this disease would not fully explain the pathological development if the inflammatory condition is not taken into consideration.Besides that,large amounts of clinical evidences showed that though some smokers suffered from more severe periodontitis exhibiting more severe alveolar bone destruction,compared with non smoker periodontitis patients,their inflammatory conditions were not as bad or even better to some extent.Taken together,whether the inflammatory microenvironment of periodontitis patients would play a certain role in enhancing the bone destructive effect with the co-action of nicotine is not clear yet.Based on previous researches,this study systemically discussed the possible up regulation role of inflammatory factors on the expression of ?7 nAChR in periodontal ligament stem cells(PDLSC)through regulating the phosphorylation of GSK-3?;focusing on the bone destruction effect and mechanisms in smoking related periodontitis patients.Authors aimed to provide experimental evidences for the disease research and provide novel thoughts for the disease prevention.Experiment One: The Isolation,Culturing and Identification for PDLSCsThis experiment aimed to isolate and culture PDLSCs derived from two different kinds of periodontal tissues.The biological characteristics of the two kinds of PDLSCs were compared,wishing to provide favorable cells models for this study.Results suggested that PDLSCs could be isolated from both normal and inflammatory periodontal ligament tissues.Cells used in this study have the capability of forming single-cell-colonies and expressing mesenchymal stem cell markers positively.Under certain induction conditions,the obtained PDLSCs also possessed the multi-lineage differentiation potentials of osteogenic and adipogenic.All these results confirmed that PDLSCs obtained in this study had similar biological characteristics of mesenchymal stem cells and could be used in further experiments.Experiment Two: The Effect of Inflammatory Microenvironment on The Expression of ?7 nAChR in Human Periodontal Ligament Tissues and PDLSCsThe aim of this experiment was to observe the expression of ?7 nAChR in periodontal ligament tissues and PDLSCs from different sources under the inflammatory microenvironment in vitro and in vivo;wishing to provide preliminary possible thoughts for the effects of inflammatory microenvironment on the destructive function of nicotine.Results suggested that the expression of ?7 nAChR was up regulated in periodontal ligament tissues derived from inflammatory microenvironment.The mRNA and protein expression of ?7 nAChR in PDLSCs derived from inflammatory tissues was also increased;indicating that the inflammatory microenvironment could up regulate the expression of ?7 nAChR in human periodontal ligament tissues and tissue derived cells.This effect may play an important regulatory role in the pathological procedures of periodontitis derived from smoking.Experiment Three: The Effect of Inflammatory Microenvironment Up Regulating The Expression of ?7 nAChR in PDLSC Paticipated Alveolar Bone RemodelingThe aim of this experiment was to observe the role of PDLSCs,of which the up regulated expression of ?7 nAChR was silenced by shRNA,in participating alveolar bone remodeling under inflammatory microenvironment;hoping to clarify the biological function of ?7 nAChR in PDLSCs under inflammatory microenvironment and to provide theoretical and experimental theories for further elucidating the pathogenesis of smoking related periodontitis.Results suggested that under inflammatory microenvironment,the osteogenic differentiation of human PDLSCs was decreased,while the capacity of inducing pre-osteoclasts to differentiate into osteocalsts was significantly increased.However,after silencing the expression of ?7 nAChR,the changes of osteogenic differentiation ability and inducing osteoclasts differentiation ability of PDLSCs were not reversed;indicating that the up regulated ?7 nAChR expression in inflammatory microenvironment did not participate in the biological function changes of PDLSCs.Experiment Four: The Effect of Inflammatory Microenvironment Up Regulating The Expression of ?7 nAChR in PDLSC Paticipated Alveolar Bone Remodeling With NicotineThe aim of this experiment was to observe the role of PDLSCs,of which the up regulated expression of ?7 nAChR was silenced by shRNA,in participating alveolar bone remodeling under inflammatory microenvironment with nicotine;hoping to clarify the biological function of ?7 nAChR in PDLSCs under inflammatory microenvironment and to provide theoretical and experimental theories for further elucidating the pathogenesis of smoking related periodontitis.Results suggested that nicotine and inflammatory microenvironment had synergistic effect on PDLSC;decreasing its osteogenic differentiation ability while increasing its ability to induce pre-osteoclasts to differentiate into osteoclasts.The ?7 nAChR played an important role in this synergistic effect.After silencing the receptor,the biological behaviour changes of PDLSCs were partially reversed.These results suggested that inflammatory microenvironment played a synergistic effect with nicotine toward PDLSCs through ?7 nAChR;leading to further destructive resorption of local alveolar bone tissues in patients with smoking related periodontitis.Experiment Five: Effect of GSK-3? on The Inflammatory Microenvironment Up Regulating The Expression of ?7 nAChR in PDLSCsThis experiment aimed to clarify the possible pathway of inflammatory microenvironment up regulating the expression of ?7 nAChR in PDLSCs through in vivo and in vitro tests;hoping to provide theoretical basis for further revealing the pathogenesis of smoking related periodontitis.Results suggested that the inflammatory microenvironment could up regulate the phosphorylation level of GSK-3? in human PDLSCs;thus decreasing the activity of GSK-3?.After increasing the GSK-3? active expression,the up regulated expression of ?7 nAChR in PDLSCs under inflammatory microenvironment was partly inhibited.These results suggested that the possible mechanism of inflammatory microenvironment up regulating the expression of ?7 nAChR in PDLSCs lie in that related inflammatory factors might up regulate the phosphorylation level of GSK-3?.This finding provides a novel theoretical basis for revealing the synergistic effect of nicotine and inflammatory microenvironment in smoking related periodontitis.