| Periodontitis is irreversible and infectious disease of chronic inflammation in oral tissues with a high incidengce in oral system regardless of geographical,ethnic and age.Smokers resulted in the higher prevalence,more heavy and poorer prognosis of periodontal treatment than non-smokers.Numerous studies showed that smoking has a close relationship with periodontal pocket formation,attachment loss,alveolar bone resorption and missing teeth.Nicotine which is the main toxic substances in the tobacco plays an important role in the development of periodontitis.Early researches found that the protein and m RNA of alpha 7 nicotinic acetylcholine receptor subtypes(?7 nAChR)expressed both in the periodontal tissue of rats and human periodontal ligament cells(hPDLCs),and the activation of the receptor would active multiple inflammatory related(ERK/PI3K/Wnt/NF-?B)and bone metabolism related signaling pathways,regulating inflammatory cytokines(IL-1? ? IL-8 ? IL-6)and osteogenic/osteoclastic related factors(RUNX,RANKL/OPG)expression.These preliminary researches suggested that nicotine may aggravate inflammatory periodontal tissue damage in smoking correlation periodontitis patients by ?7 nAChR.Autophagy was involved inhuman inflammatory disease development,and ACh R which was the key target in cells affected by nicotine could be regulated and controlled by a variety of cells' autophagy process in pathophysiological process.Existing studies confirmed that nicotine was involved in nervous system disease development through ?7 nAChR regulating autophagy,but the concrete mechanism of nicotine regulating autophagy under the ?7 n ACh participating in periodontal disease is lack of research.This study intends to explore the relationship between the nicotine,?7 nAChR,autophagy and periodontitis,providing a new theory for the prevention and treatment of smoking related periodontitis.?Objectives? Author aimed to identify the effects of nicotine in inducing the autophagy of hPDLCs,and further investigae whether ?7 nAChR would take part in this process and whether the hPDLCs autophagy would control inflammatory factors erpressed in vitro.Exploring nicotine may regulate hPDLCs autophagy level by ?7 nAChR,and then involved in periodontal tissue inflammatory damage effect by regulating the erpression of inflammatory factors.In order to further explore the mechanisms of nicotine related the periodontitis.We hope to find some new mechanisms for the prevention and treatment of smoking related periodontitis.?Materials and Methods? 1.Enzyme tissue block method was used to culture hPDLCs primary cells.Periodontal tissues were collected from premolars for orthodontic treatment reasons for culturing of hPDLCs.When cells aggregated to 80%,we producted routine cell passage.Cells in the 4th passage were used to identify the cell phenotype.The Passage 4 to 6 cells are used for follow-up experiments.2.hPDLCs were treated with 10-5 M nicotine in different times,that was 0,3,6,12,and 24 hours and different concentrations,that is 0,10-7,10-6,10-5,and 10-4 mol/L for 12 h.Western blot and immunofluorescence(IF)were performed to test the most optimal time and concentration of nicotine on the autophagy level of the hPDLCs,Transmission electron microscope(TEM)was carried out to detect the form of autophagosomes in hPDLCs under this optimal condition.3.hPDLCs were treated with 10-5 mol/L nicotine and/or 10-8 mol/L ?-BTX.Western blot,IF and TEM were used to detect the autophagy related protein expression LC3?and LC3 and the formation of autophagosomes.4.hPDLCs were treated with 10-5 mol/L nicotine and/or 10-3 mol/L 3-methlyadenine(3-MA).Western blot was taken to analysis the expression of autophagy related protein LC3?and Beclin-1.Real-time PCR and Elisa were used to detect m RNA and protein expression of IL-1? and IL-8.?Results? 1.After culturing for five days,fibroblast like cells were appeared around the tissue blocks.When cells aggregated to 80%,we producted routine cell passage.Flow cytometry analysis indicated that the mesenchymal cell phenotype CD146 and CD105 were highly expressed while the endothelial cell surface marker CD31 and hematopoietic cell and molecular marking CD14 were negative expressed.These results illustrated that the cultured cells were ectomesenchymal source.2.Western blot results indicated that in the 12 h nicotine stimulating the protein expression of LC3? was up regulated.Besides that,the up regulation of the protein expression of LC3? was concentration dependent and nicotine at a concentration of 10-5 mol/L was the most optimal condition.IF and Western blot results are consistent.TEM observations indicated that nicotine could activate the autophagy level of hPDLCs by increasing the number of autophagosomes.3.Western blot,TEM and IF technique observed that the autophagy changes in hPDLCs after nicotine and/or ?-BTX stimulating for 12 h.The result indicated that ?-BTX can antagonism the formatiom of nicotine inducing hPDLCs autophagy,which showed that nicotine could regulate and controle the hPDLCs autophagy by ?7 nAChR.4.Analysis of Western blot results found 3-MA could significantly suppress the hPDLCs autophagy induced by nicotine;the experimental results of RT-q PCR and Elisa showed the remarkable increase of inflammatory factor IL-1? and IL-8 when hPDLCs is stimulated by nicotine,while 3-MA could significantly inhibit the expression changes.All these results further confirmed that nicotine would regulate the expression of inflammatory cytokines through regulating autophagy in hPDLCs.?conclusion? 1.Enzyme tissue block method could successfully culture hPDLCs in vitro,the cultuered cells were derved from mesenchymal source;2.Nicotine could activate the autophagy level of hPDLCs,the optimal nicotine working condition was 10-5 mol/L for 12 h;3.Nicotine could activate the autophagy level of hPDLCs via ?7 nAChR;4.Nicotine could regulate the expression of inflammatory cytokines through regulating hPDLCs autophagy. |
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