| Tobacco smoking has been recognized as the main risk factor for thedevelopment and progression of periodontal diseases.More than4000chemicals,including high concentrations of free radicals and some oxidizing materrials,arepresent in tobacco smoke and they contribute a lot to the progression ofperiodontal diseases, thus after periodontal therapy, the healing procedure wouldbe delayed. In previous studies, we have demonstrated that functional α7nAChR, which is not only a predominant subunit composition of nAChRs butalso a potent target of nicotine binding receptor, presents in the periodontaltissues of human and rats. the up-regulation of α7nAChR levels byadministration of nicotine could be partially suppressed by pretreatment with α-BTX, a α7nAChR antagonist. The presence of α7nAChR in the periodontaltissue opens another route to exploring the mechanisms that underlie the effectsof nicotine in periodontitis. However, the mechanisms under the α7nAChR signaling pathway are not well understood. NF-κB, a protein complexes,comprising a family of transcriptional factors. It has been reported that NF-κBwas highly activated in subjects who had chronic periodontitis compared tohealthy controls. So, to further discover the pathways mediating α7nAChRsignaling in oral Periodontal cells exposed to nicotine, we chose NF-κB to bethe target to investigate its role as the signaling transmitter.Part1cell culturing and identification of hPDLCsMethods: Human teeth were extracted from three healthy patients （meanage12years） for orthodontic reasons. Periodontal ligament tissues weredissociated from the mid-third of the root of premolars to avoid the intermixingof gingival or dental pulp tissues.When the cells surrounding the explantsreached confluencet, cell layers were collected and subcultured. For all theexperiments, passage-6cells were used.Results: After about37days of original generation culturing we could seefibroblast like cells in the shape of long shuttle run out of the round of the tissueclumps. In the procedure of culturing, the long shuttle cells aligned in a radiationway. The fibroblast like cells had a well-distributed density, regular and fullgrown kytoplasm and round clear nucleus.Conclusion: We successfully set up the model for the experiment.Part2Gene expression of NF-κB P65subunit and activity of NF-κBafter nicotine alone and in combination with α-BTX stimulationMethods: To discover the effect of nicotineon NF-κB gene expression in human periodontal cells, both dose-and time-dependent studies were conducted.The specific primers for the PCR were chosen based on the nucleotide sequenceof the p65subunit of NF-κB. In the time-dependent study, the cells were placedin serum-free medium and then incubated with nicotine for some periods of timeranging from0（control） to24h. In the dose-dependent studies, the cells wereincubated with nicotine at concentrations ranging from0（control） to100ng/mlfor24h. To determine the effect of α-BTX on the activity of NF-κB, PDLcells were incubated for24h and then divided into four groups. Each groupreceived one of the following treatments randomly:（1） no treatment;（2）nicotine (10-5M);（3） α-BTX (10-8M);（4） α-BTX (10-8M) followed by nicotine(10-5M) after30min.Results: The electrophoretogram of RT-PCR detected gene expression ofNF-κB in PDL cells, but nicotine didn’t lead to significant alterations of NF-κBboth in time-and dose-dependent manners compared with the control group.Isolated protein of NF-κB from the cells’ nucleus after nicotine treatmentshowed that the protein expression was enhanced. There was a marked increaseprotein expression in nucleus after24hr of incubation under nicotine treatmentcompared with the control group. This phenomenon could partially besuppressed by the selective α7nAChR antagonist α-BTX (10-8M).（P<0.01）Conclusion: The effect of intranuclear of NF-κB p65subunit could beenhanced after nicotine stimulating on PDL cells. And this effect could besuppressed by α-BTX, the antagonist of α7nAChR. which indicates that the transcription of NF-kB is mediated by α7nAChR in PDL cells. However, thedifference of mRNA expression of NF-κB between groups was not statisticallysignificant after stimulated by nicotine. There was also no difference of geneexpression between groups for P65in the nucleus.Part3Effects of nicotine, alone and in combination with α-BTX or PDTC,on intracellular IL-1βã€IL-8mRNA levels and Activity of NF-κB in PDLcellsMethods: To determine the effect of PDTC||on the activity of NF-κB,PDLcells were incubated for24h and then divided into five groups, with each groupreceiving one of the following treatments randomly:（1） no treatment;（2）nicotine(10-5M);（3） PDTC (10-5M) followed by nicotine (10-5M) after30min;（4） PDTC(3×10-5M) followed by nicotine (10-5M) after30min;（5） PDTC (5×10-5M)followed by nicotine (10-5M) after30min. To determine the effect of PDTC onIL-1βã€IL-8gene expression, PDL cells were incubated for24h and thendivided into five groups, with each group receiving one of the followingtreatments randomly:（1） no treatment;（2） nicotine (10-5M);（3） α-BTX (10-8M);（4）α-BTX （10-8M） followed by nicotine （10-5M） after30min;（5） PDTC (5×10-5M);（6） PDTC （5×10-5M） followed by nicotine (10-5M) after30min.Results: A marked increase of NF-κB from the nucleus in protein levelwas observed after24hr’s incubation with nicotine compared with the controlgroup. This effect could partially be blocked after the cells pre-incubation withPDTC (5×10-5M) for30min.（P<0.01）. The electrophoretogram of RT-PCR showed that IL-1β and IL-8could be partially blocked when pre-incubation ofPDL cells with α-BTX (10-8M) or PDTC (5×10-5M) for30minConclusion: The expression of IL-1β and IL-8secreted by PDL cells couldbe enhanced after treatment of nicotine. This effect could be partially blockedby pre-treatment with α-BTX and PDTC. These results indicate that nicotinemay affect PDL cells functions through the cholinergic pathway mediated by theα7nAChR and the transcriptional factor NF-κB. |
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