Effects of Nicotine and or α-BTX on the Production of Mmps in Human Periodontal Ligament Cells Cultured in Vitro
|School||Fourth Military Medical University|
|Keywords||Matrix metalloproteinase Nicotine α - bungarotoxin Human periodontal ligament cells|
Periodontitis is a serious hazard to human infectious diseases of the oral health for initiating factor, combined with genetic, environmental, and host immune factors common pathogenic plaque. Smoking is one of the major risk factors for periodontitis, compared to non-smokers, smokers prevalence of periodontitis, tooth loss rate and symptoms and rapid progression and poor prognosis, the poor treatment reactivity. Nicotine as an important toxic components of tobacco, periodontal tissues, cells generate direct and indirect toxic effects, but the exact mechanism is unclear. Existing research results show that nicotine may be through nicotinic acetylcholine receptors (nicotinic acetylcholine receptor, nAChR) pathway, periodontal tissue damage. With the discovery of the non-neuronal cholinergic system (Non-neuronal Acetylcholine System, NNAS), a growing number of acetylcholine receptors (acetylcholine receptor, AChR) subtypes exhibit with the nervous system physiology and pathology of the broader role such as anti-inflammatory role of pro-inflammatory, pro-apoptotic and promote tumor metastasis, as a the AChR function of the study provides a new perspective. AChR including nicotinic and muscarinic receptors, nicotine nAChR agonist, through combined with the nAChR on the cell membrane, opening ion channels, increased calcium influx causing physiological and pathological responses of the cells; and through the activation of the receptor downstream pathways, such as MAPK, ERK, ultimately activate NF-κB, leading to cell nucleus changes, causing changes in gene expression. The study shows that, in the nAChR, nicotinic acetylcholine receptor α7 subtypes (α7 nicotinic acetylcholine receptor, α7nAChR) mainly mediated by the effect of nicotine on the role of tissue cells. Present in the epithelial cells of non-neural tissues, fibroblasts are find α7nAChR expression of retinal cells, and smoking-related diseases, such as to play an important role in the development of inflammation, cancer and other diseases. Task Force preliminary studies confirmed human tooth germ cells (human dental germ cells), human periodontal ligament cells (human periodontal ligament cells, hPDLCs) in the presence of α7nAChR expression, and its expression in rat periodontal tissue positioning; found a complete non-neuronal cholinergic system in human periodontal ligament cells including acetylcholine (Acetylcholine, ACh), choline acetyltransferase enzyme (choline acetyltransferase, ChAT), acetylcholinesterase (Acetylcholinesterase, AChE), nicotinic type and muscarinic nAChR. Also found, α7nAChR agonist nicotine can to raise rat periodontal tissue hPDLCs, α7nAChR expression and application α7nAChR specific antagonist α-bungarotoxin toxin (α-bungarotoxin, α-BTX) could antagonize Upregulation. The study also found that the the hPDLCs expression of pro-inflammatory cytokines interleukin-1β (interleukin, IL-1β) were consistent α7nAChR expression and nicotine-induced changes in IL-1β also be α-BTX antagonistic. Matrix metalloproteinases (Matrix metalloproteinases, MMPs) are a group of enzymes, the degradation of extracellular matrix collagen, collagen fibers is the main ingredient to maintain teeth, periodontal ligament and alveolar bone connected. Clinical trials, the periodontitis patients a variety of expression of MMPs were increased, effective treatment to decreased expression; vitro experiments have come to similar conclusions. Studies Show the nicotine through α7nAChR stimulate human retinal cells (retinal cell) expression of MMPs, thereby regulating the angiogenic effect. But nicotine can influence hPDLCs expression of MMPs, the mechanism is not clear. In this study, cultured in vitro experimental models of human periodontal ligament cells, using RT-PCR and ELISA experiments means detection and possible mechanisms of nicotine hPDLCs MMPs expression at the mRNA and protein levels to explore pathological mechanisms of smoking-related periodontitis provide a theoretical basis. Experiment periodontal ligament cells in primary culture, subculture and identification of a major method before due to orthodontic need to unplug the health of caries-free molars of young people aged 12-16, rinse it under sterile conditions, scraped root in 1/3 of the periodontal ligament organizations, the the tissue explant for hPDLCs primary culture. Tissue blocks the peripheral cells climbed out and proliferation, conventional passage, and immunohistochemical staining cells observed vimentin, keratin expression, identification of cell sources. About a week after the main results by explant culture, its edge after another successful fiber-like cells climbed out after routine passage, inverted spindle cell is known under the microscope, outline a clear, spindle-shaped, star-shaped, rare polygonal; plump cytoplasm oval-shaped nucleus clear, prominent nucleoli and more common split phase; cells covered the bottom of culture flask, arranged in a whirlpool or radial. Immunohistochemical staining showed that cultured cells anti-vimentin staining, anti-cytokeratin negative. 3 main conclusions of the success of in vitro the hPDLCs primary culture experimental model; positive staining of cultured cells anti-vimentin, anti-cytokeratin negative, confirming derived from mesenchymal. Experimental RT-PCR and ELISA detection of nicotine on human periodontal ligament cells MMP-1, 3 expression method take growth well the 5th generation hPDLCs count after two 25mL flasks were seeded in adherent 24h, were added with and without nicotine (10-4M) 5% fetal bovine serum, low sugar DMEM. 24h, culture supernatants for ELISA detection and extraction of cells RNA, reverse transcription cDNA used for RT-PCR experiments were compared between the two groups of MMP-1, 3 mRNA and protein expression. The main results of agarose gel electrophoresis showed: in 203bp, 716bp and 667bp at the β-actin and MMP-1, single zone, and nicotine groups express higher than that of the control group, a statistically significant difference (P lt ; 0.05); expression of α-BTX group and nicotine group of α-BTX similar compared with the control group, no statistical significance (P gt; 0.05). Translation of the actual concentration of the ELISA test, according to the OD value of each well, the nicotine group were significantly higher than the control group, a statistically significant difference (P lt; 0.05); α-BTX group and α-BTX the nicotine group of expression, and the control group compared, no statistical significance (P gt; 0.05). RT-PCR and ELISA results show that the control group of hPDLCs, MMP-3 mRNA and protein expression, but the expression level is weak, the 3 main conclusions. Nicotine role of MMP-1, 3 expression enhanced nicotine may promote hPDLCs secretion of MMP-1, 3, periodontal tissue impact. Experimental three-RT-PCR and ELISA detection of nicotine and / or α-BTX on human periodontal the membrane cell MMP-1, 3 expression method to take the growth of good 5th generation of hPDLCs,, count after culture flasks were seeded in four 25mL are grouped as follows: (1) nicotine group (10-4M); ② α-BTX group (1.25 x 10-9M): (3) of α-BTX nicotine group; ④ control group. 24h, cells was extracted RNA, reverse transcription cDNA, RT-PCR amplification and agarose gel electrophoresis; draw centrifugation of cell supernatants for ELISA experiments. 2 The main results of the RT-PCR experiments results application software for statistical analysis, significantly up-regulated in the nicotine group, the expression of MMP-1, 3, compared with the control group, the difference was statistically significant (P lt; 0.05); α-BTX group and α -BTX the nicotine group of expression similar to the control group, no statistical significance (P gt; 0.05). ELISA test, according to the translation of the actual concentration of each group OD value, the statistical analysis showed that nicotine group was the strongest, compared with the control group, a statistically significant difference (P lt; 0.05); α-BTX group and α-BTX nicotine group expression, compared with the control group, no statistical significance (P gt; 0.05). The three main conclusions in the mRNA and protein levels in the control group of hPDLCs, MMP-3 expression is weak, nicotine can upregulate of hPDLCs, MMP-1, 3 expression, consistent with the results of Experiment II. After joining α7nAChR specific antagonist α-BTX significant antagonistic effect of nicotine on MMP-1, 3 raised effect, suggesting nicotine regulation hPDLCs secretion of MMP-1, 3, may by α7nAChR path mediated.