The Role Of α7Nachr In Nicotine Promoting The Poptotic Effects Of HERS/ERM

Periodontitis is one of two major oral diseases, mainly for gingival inflammation,periodontal pocket formation, alveolar bone loss, cementum damage, tooth loss, which isthe leading cause of adult tooth loss. Numerous studies confirm that smoking is a riskfactor for periodontal disease, especially for severe periodontitis. Smokers have higherprevalence of periodontitis than non-smokers and a worse prognosis, but the exactmechanism caused by smoking is still not clear.Hertwig’s epithelial root sheath (HERS) rupture during root development, thendifferentiate into cementum cells to form cementum. The remaining HERS leaves fromroot surface to forme HERS/ERM cells. HERS/ERM can transition into cementum cell forcementum repair by epithelial-mesenchymal. Studies confirm that a great change onpatients’ cementum surface morphology, micro-composition and hardness, suggestingcementum repair process directly affect the outcome of periodontitis. Nicotine, as a majortoxic components of tobacco, which has played an important role in promoting gingivalinflammation and accelerating alveolar bone loss. However, studies on the effects of nicotine on cementum repair were rarely reported, and it’s worthy of deeep study toprovides a theoretical basis for the cementum repair in patients with periodontitis.The study by constructing an experimental periodontitis rats model and culturedHERS/ERM primary cells, using HE staining to obseerve effects of nicotine on ratperiodontal tissue inflammation and HERS/ERM. By TUNEL staining, reversetranscription PCR and quantitative real-time PCR experiments to observe the effects ofnicotine on the HERS/ERM apoptosis to investigate whether nicotine by promotingHERS/ERM apoptosis thereby affecting the development of the root of repair. The maincontents and results are as follows:Part1The construction of nicotine experimental periodontitis ratmodel and the effect of nicotine on HERS/ERM numberMaterials and Methods:The16,6-week-old SD rats were anesthetized by intraperitoneal injection ofpentobarbital and fixed, receving silk ligatures around right maxillary second molar,successfully established animal model of experimental periodontitis in rats. Then theanimals were randomly assigned into2groups, of daily intraperitoneal injection: groupA(control), saline solution; group B, nicotine. Sacrificing animals at day28, isolatedbilateral maxillary molars dental periodontal tissue, making paraffin sections, using HEstaining to observe the effects of nicotine on periodontal inflammation and HERS/ERMcells.Results:HE staining observed varying degrees of inflammatory cell infiltration in both groupsligated side, heavier in nicotine group. The non-ligated side showed no inflammatoryphenomena. HERS/ERM cells were existed in the vicinity of the root bifurcation,significantly lower than the control group compared to the nicotine group.Conclusion:Molar neck silk ligation can successfully established rat model of experimentalperiodontitis, and nicotine through reducing the number of HERS/ERM to affect the cementum repair process.Part2Cell culturing and identification of rat molar HERS/ERMMaterials and Methods:Extracting molars epithelial root sheath after sacrificing PN7d SD rats. I collagenaseand dispase Ⅱ protease digestion, then culturing primary cell lines. Selective removal offibroblasts by trypsin digestion to obtain purified epithelial cells. Immunocytochemistry toidentify cells.Results:After about2~3days, shuttle fibroblasts and polygonal epithelial cell were mixedaround tissue. Polygonal epithelial cells could be observed after selective trypsin digestion,growth like cobblestone arrangement which were positive for cytokeratine. Spindlefibroblast were almost removal.Conclusions:Using enzymes to digest tissue was successfully isolated and cultured rat molarHERS/ERM, which laid the foundation for further research related to the impact ofnicotine on it.Part3Expression of α7nAChR in rat molar HERS/ERM and effectof nicotine on itMaterials and Methods:Seeded successfully cultured epithelial root sheath in six-well plates, detecting itsexpression of α7nAChR. Detecting α7nAChR expression under the α7nAChR agonistnicotine (10-5M) and/or antagonist α-BTX (10-8M) using reverse transcription PCR andquantitative real-time PCR.Results:Immunocytochemistry result displays HERS/ERM express α7nAChR. Reversetranscription PCR and quantitative real-time PCR showed that nicotine can increase α7nAChR mRNA expressin of HERS/ERM, but with α-BTX pretreatment, upregulated α7 nAChR mRNAexpression can be antagonised.Conclusions:Rat molar HERS/ERM express α7nAChR. The upregulated α7nAChR mRNAexpression by nicotine can be antagonised with α-BTX pretreatment which suggests α7nAChR may mediated the nicotine’s effects on HERS/ERM.Part4Studies of nicotine on apoptosis in rat molarsHERS/ERM cellsMaterials and Methods:Observing apoptotic effect of nicotine on the HERS/ERM by TUNEL staining.Reverse transcription PCR and quantitative real-time PCR experiments detected mRNAexpression of bcl-2and bax with α7nAChR agonist nicotine(10-5M) and/or antagonistα-BTX (10-8M).Results:TUNEL staining showed that nicotine can make HERS/ERM cell dehydration,cytoplasmic concentration, karyopyknosis and fracture. Reverse transcription PCR resultsshowed that: compared with the control group,10-5M nicotine significantly up-regulatingbax mRNA expression in rat molars HERS/ERM while down-regulating bcl-2mRNAexpression. Quantitative real-time PCR experiments further validate the results, and thedifference between bcl-2and bax was statistically significant (P <0.05).Conclusion:Nicotine can promote apoptosis of HERS/ERM, and may exert its pro-apoptotic effects bydown-regulating the expression of bcl-2while up-regulating the expression of bax.