The Signaling Pathway Of α7nAChR-ERK1/2-c-Fos In Smoking-related Periodontitis

Smoking is the main influence factors of periodontitis,.smoker have a higher prevalence of periodontal disease and a worse prognosis after systematic treatment. Nicotine in tobacco is proved to play an important role in the process of the destruction of periodontal tissues while the specific mechanism remains unclear. Previous research found that there existed alpha7nicotinic acetylcholine receptor in human and rat periodontal tissues and nicotine could increase its expression while this effect could be antagonisted by alpha bungarotoxin.c-Fos is a key gene in the process of osteoclast differentiation and a large number of experiments confirmed that c-Fos played an extremely important role in the osteoclast process in a variety of cells. ERK1/2pathway is an information transmission of various extracellular stimuli signal which effect on proliferation and differentiation of different cells. Experiments have confirmed that the nicotine could enhances the expression of inflammatory cytokines by combined with alpha7nAChR in gingival epithelial cells through the activated ERK signaling pathways. These results provide new thoughts for our study in the role of nicotine in the development of periodontitis:Nicotine will have effect on human periodontal ligament cells by activating alpha7nAChR and further up regulate ERK1/2signaling pathways downstream and ERK1/2signaling pathways continue activating the expression of the gene of c-Fos. So this study focus on the relationship between alpha7nAChR, ERK1/2signaling pathway and c-Fos gene expression in smoking related periodontitis and further explore role of nicotine in the development of periodontitis and its destructive effect on periodontal tissues.Part1Cell culturing and identification of hPDLCsMethods:Healthy premolars without any caries were extracted from patients (aged12-16years) for orthodontic reasons. Periodontal ligament tissues were scratched from the mid-third of the root in sterile environment. When cells surrounding the periodontal tissues reached confluencet, they c were collected and subcultured. For all the experiments, passage-5cells were used.Results:After about7days fibroblast like cells could be observed in the shape of long shuttle shape. The fibroblast like cells had a well-distributed density, regular and full grown kytoplasm with round clear nucleus in the center of it.Conclusion:We successfully set up the cell model for further experiment by culturing hPDLCs.Part2Activity and mRNA expression of c-Fos in hPDLCs after nicotine alone or in combination with α-Btx stimulation1.Methods:Fifth-passage hPDLCs were used for this experiment. Reverse transcription PCR were carried out in detecting the c-Fos mRNA expression after nicotine alone or in combination with α-Btx in different concentration and time point. Then we used real-time quantitative PCR and Western blot further confirmto test c-Fos mRNA and protein expression. 2.ResultsReverse transcriptional PCR showed that:The mRNA expression of c-Fos reached the highest point in hPDLCs after1hour of nicotine stimulation(10-4M)(P<0.05) However we adopted10-5M concentration of nicotine for follow-up study in consideration of Nicotine’s toxicity. There was a marked increase of mRNA and protein expression of c-Fos after lhr of incubation under nicotine treatment compared with the control group. This phenomenon could partially be suppressed by the selective a7nAChR antagonist a-Btx (10-8M).(P<0.01)3.ConclusionBased on the concentration-dependent and time-dependent experiment,10-5M nicotine and1hour time point were chosen for further experiment. Results indicated that nicotine might regulate the activity of c-Fos through a7nAChR.Part3The effect of ERK1/2signaling pathway on the expression of c-Fos in smoking related periodontitis1.MethodsFifth-passage hPDLCs were used for further experiment. Western blot was carried out in detecting p-ERK1/2protein expression after nicotine alone or in combination with a-Btx. Then take the same cell model, RT-PCR and Western blot was carried out in detecting c-Fos gene and protein expression changes after nicotine alone or in combination with U0126, the antagonist of ERK1/2signaling pathway.2.ResultsWestern blot results showed that:There was a marked increased protein expression of p-ERK1/2under nicotine treatment compared with the control group. While pretreating with a-BTX could partially inhibit this effect.(P<0.05) Real-time quantitative PCR and Western blot results showed that:ERK1/2antagonist U0126could obviously inhibit the c-Fos activity which was increased by nicotine.(P<0.05)3.ConclusionERK1/2signaling pathway may take part in regulating the gene and protein expression of c-Fos which was affected by nicotine.