Research About Nicotine's Toxicity And Its Mechanism On Periodontal Ligament Fibroblasts (PDLFs)

BackgroundNicotine, main cytotoxicity component of cigareete,can cause many diseases. Signaling pathways on apoptosis are well-matured mechanism of cell regulation of nicotine.Although the cytotoxicity of periodontal tissue from nicotine are known, there are no study on apoptosis mechanism of periodontal disease caused by nicotine.ObjectiveTo confirme nicotine's cytotoxicity on human PDLFs, and to research its cytotoxicity mechanism by clarifying the signal transduction pathway. The research was to lay a basis for the prevention and therapy of periodontal disease.MethodsCell culture:human periodontal ligament fibroblasts. Proliferations on periodontal ligament fibroblasts were detected with MTT. The cultured cell stimulated by three nicotine concentrations selected from MTT results were assessed by flow cytometry (FCM) in order to observing the changes about cell cycle and cell apoptosis rate of 48h. Reversing transcription-polymerase (RT-PCR) was performed to examine the gene expressions of PI3K, Bax, Akt and Bcl-2 after PDLFs cell exposing in 3 groups concentrations for 24h. Subsequently, protein activation of caspase 3 was examined by western blot staining assay, and discussed the mechanism according to the experiment results.Results1. Research about nicotine's cytotoxicity on PDLFs PDLFs were cultured in the presence of nicotine at various concentrations(10000μg/ml, 1000μg/ml, 100μg/ml, 10μg/ml, 1μg/ml,0.1μg/ml and 0.01μg/ml) on 24h,48h and 72h. The growths of cell were examined by MTT methods. With the increase of the concentration of nicotine, the value of A490 decreased gradually; and time is the same. With concentration increased, the cell DNA contents of G0G1 phase are increased compared with normal control group, while the phase of G2 and S gradually declined (P<0.05). The analysis of annexinV-FITC/PI double-labeling combined with flow cytometry shows that:there occur higher apoptosis rates as cell presented at varied concentrations. According to the order of apoptosis:1000μg/ml>10μg/ml>0.1μg/ml. The above show that proliferation inhibition, cell cycle arreset and apoptosis promotion of nicotine activated on PDLFs cell.2. Mitochondrial signaling pathway of PDLFs induced by nicotineCa2+ fluorescence intensity of PDLFs increased significantly, and with the nicotine dose increased, intracellular Ca2+ fluorescence intensity gradually elevated. It was significantly increased that mRNA expression levels of Bax and Caspase3 of different groups (1μg/ml, 10μg/ml and 100μg/ml) compared with the control group at 24h. Furthermore, with increasing doses of nicotine, Bax and Caspase3 gene expression levels gradually elevated. However, Bcl-2 expression decreased. Moreover, the protein expression levers of Caspase3 gradully increased. The conclusion was that nicotine can cause PDLFs extracellular Ca2+ influx, intracellular [Ca2+] increase. Bcl-2 gene expression was significantly reduced, Bax increases. Eventually, it lead to the release of Cyt C, the occurrence of PDLFs cell apoptosis.3. Protection effection of PI3K/Akt signaling pathway in the PDLFs apoptosis induced by nicotineIt was significantly increased that mRNA expression levels of PI3K and Caspase3 of different groups (1μg/ml,10μg/ml and 100μg/ml) compared with the control group at 24h. Furthermore, with increasing doses of nicotine, PI3K and Caspase3 gene expression levels gradually elevated. However, Akt decreased. Moreover, Thr308 and Ser473 phosphorylation sites of Akt in each experimental group were significantly higher (P<0.01). The conclusions are that PI3K/Akt involved in nicotine-induced apoptosis, and played a role in inhibiting apoptosis. but the contributing factor of apoptosis induced by nicotine is stronger than PI3K/Akt signaling pathway, and eventually activation of Caspase3 expression. Caspase3 expression signals the occurrence of apoptosis.ConclusionsNicotine may inhibit cell proliferations at the same time of DNA synthesis of PDLFs. Nicotine may inhibit cell proliferations at the same time of DNA synthesis of PDLFs. And the apoptosis rates are escalated with nicotine's concentration increased. It is the apoptosis mechanism of nicotine stimulated on PDLFs that including promoting apoptosis effection, Mitochondria signaling pathway, and protection effection, PI-3K/AKT pathway. Eventually Caspase3 perform a series of apoptosis events.