Effects Of Nicotine And Or α-BTX On The Production Of Mmps In Human Periodontal Ligament Cells Cultured In Vitro

Periodontitis is an infective disease which has harmful effects on people's oral health. It is started with plaque, combining the effects of inheritance, environment and immunity of host. Smoking is an important risk factor for periodontitis, as a result, smokers suffer a higher morbidity and frequency of tooth loss, more serious symptoms, faster progression of sickness, poorer prognosis and responding worse to treatments. Non-smokers and those who have quit smoking have better conditions than smokers. Nicotine, the most important component in tobacco, has both direct and indirect effects on periodontal tissues as well as periodontal cells. However, the mechanism remains unknown. There are more and more evidences indicating the crucial role of nicotinic acetylcholine receptor signaling pathway in periodontal destruction.With the discovery of non-neural cholinergic system, there are increasing evidences showing that the non-neural cholinergic system exhibit quite different physical and pathological functions compared to those in the central nervous system, such as anti-inflammatory or proinflammatory effects, apoptosis, accelerating tumor transformation, which provides a promising thought for studying the mechanism of diseases. Acetylcholine receptors include nicotinic acetylcholine receptor and muscarinic acetylcholine receptor. Nicotine is an agonist of nicotinic acetylcholine receptor (nAChR) situating on the surface of cell membrane, which could open the ion channel, increase inflow of calcium ion, and lead to the physical and pathological functioning. It then activates downstream pathways of acetylcholine receptor, such as MAPK, ERK1/2, and NF-κB to change the gene expression and modification. There are evidences suggesting that among various subtypes of nicotinic acetylcholine receptors,α7 subunit acts the vital role in the interaction of nicotine and relative cells. Recently,α7 subunit has been detected in various tissues such as epithelial cells, fibroblasts. Especially in smoking-related diseases it really plays an important role.Previous experiments in our groups have discovered the presence ofα7nAChR in human tooth germ cells, human periodontal ligament cells (hPDLCs) and rat's periodontal tissues. We also found out the whole cholinergic system in hPDLCs, including acetylcholine (ACh), choline acetyltransterase (ChAT), and acetylcholinesterase (AChE), muscarinic and nicotinic AChR. In addition, nicotine up-regulated the expression ofα7nAChR in hPDLCs and rat's periodontal tissues, which could be restrained by specificα7nAChR antagonist,α- bungarotoxin (α-BTX). Meanwhile, the expression of proinflammatory cytokine IL-1βacts in accordance with the expression ofα7nAChR, which could also be inhibited byα-BTX.Matrix metalloproteinases (MMPs) are a series of proteases, which can degrade extracellular matrix (ECM), including collagen. Collagen is a major component of peoriodontium lying between alveolar bone and cementum, acting as a vital role in maintaining the balance of periodontal homeostasis. Clinical experiments suggest that patients suffering periodontitis have higher MMPs levels, which can be decreased by effective treatment. Experiments in vitro also have similar results. Althoughα7nAChR can mediate the effects of nicotine on MMPs levels in angiogenesis in retina cells,it's largely unknown whether nicotine has the ability to regulate the expression of MMPs in hPDLCs.PartⅠ: Culture and identification of human periodontal ligament cells in vitro1 MethodWe used caries-free premolars of 12-16-year-old teenagers extracted for orthodontic purpose. Primary culture was done with aseptically shaving off the ligament tissues of the middle part of teeth root. After about 7 days, cells crept from the tissue block, and then went on with sub culturing. Identify the origin of these cells by staining with anti- vimentin and anti-keratin.2 ResultsHuman periodontal ligament cells were successfully cultured in vitro, of vimentin positive and keratin negative through staining with anti-vimentin and anti-keratin antibodies,3 ConclusionsWe have cultured human periodontal ligament cells successfully in vitro, and we ascertain that the cells were from mesenchymal tissues.PartⅡ: Measurement of nicotine on the expression of MMP-1 and 3 in human periodontal ligament cells using RT-PCR and ELISA1 MethodAfter counting the total number of cells, we used 5th passage hPDLCs for next experiments and divided them into two groups. When all the cells attached the bottom of the culturing board and had reached the confluency of about 80%, then for one group, we added nicotine (10-4M) into culture-medium, while nothing but culture-medium for the second group. After 24h of culturing, we took the spent culture supernatant for ELISA and cells for RT-PCR.2 ResultsRT-PCR results showed that the three bands are at the correct position and the expression level of MMP-1 and 3 in nicotine treated group are much higher than the control. ELISA experiment yielded the similar result with RT-PCR.3 ConclusionsResults above shows that nicotine up-regulated the expression of MMP-1,3 in hPDLCs compared to control group both at the mRNA and protein levels, indicating that nicotine can change the amount of MMP-1, 3 to influence the physical and pathological conditions of periodontal tissues.PartⅢ: Measurement of nicotine and/orα-BTX on the expression of MMP-1 and 3 in human periodontal ligament cells using RT-PCR and ELISA1 MethodhPDLCs at passage 5 were used for the experiment and divided into four groups:①nicotine group (10-4M);②α-BTX group(1.25×10-9M);③α-BTX and nicotine group (the same concentration as group1 and 2);④control group. Then we performed ELISA and RT-PCR as we did in Experiment PartⅡ.2 ResultsIn RT-PCR experiment, all the bands presented are at the right position. The expression of MMP-1 and 3 in group 1 are much higher than the control group. It seemed thatα-BTX alone didn't affect the expression of MMP-1 or MMP-3. However, group3, which containing the same amount of nicotine andα-BTX, showed the similar amount of gene expression with the control. The result of ELISA was in accordance with that in RT-PCR.3 ConclusionsIn normal conditions, hPDLCs expressed very little amount of MMP-1, and the quantity of MMP-3 is even less. With the stimulus of nicotine, both expression of MMP-1 and 3 increased, and this could be inhibited by specificα7nAChR antagonist,α-BTX at mRNA and protein levels. It suggests that nicotine may change the expression of MMP-1 and 3 in hPDLCs, and this effect may be mediated byα7nAChR.