Effects Of Smoking On Autophagy And Apoptosis In Oral Mucosal Epithelial Cells

Chapter1 Effects of Cigarette Smoke on the Activity of Oral Mucosa Epithelial Cells and the level of autophagy markers[Objective]Oral mucosa epithelial cells can resist the invasion of a variety of harmful substances,is the "sentry" of oral mucosa.However,when the invasiveness of harmful substances is greater than the scavenging ability of the host,the oral mucosal epithelial cells are destroyed,thereby reducing the local defense ability of the oral mucosa.Smoking is a common external stimulus to oral mucosa,which can damage the integrity of oral mucosa epithelial cells in a variety of ways.The purpose of this part of the experiment was to screen the optimal concentration and duration of cigarette smoke extract(CSE),autophagy inhibitor CQ and autophagy inducer RAPA by observing the activity of oral mucosal epithelial cell line Leuk-1 and the expression of autophagosome marker LC3-II protein,so as to conduct follow-up experiments.[Methods]Leuk-1 cells were cultured in vitro,the CCK 8 method were used to detect the cell proliferation performance in different concentrations of CSE(0?8 %?16%?32%?64%),CQ(0?M ? 10?M ? 25?M ? 50?M ? 75?M ? 100?M)and different concentrations of RAPA(0?M?0.01?M?0.1?M?1?M?10?M)with different time(3h?6h?12h?18h?24h?36h?2d?3d?5d).Western blot was used to further detect whether the autophagosome marker LC3-II produced by the action of CSE at different concentrations and at different times on Leuk-1 cells had concentration and time dependence,so as to further determine the optimal action time and action concentration of CSE.Western blot was used to detect the concentration dependence of LC3-II produced by CQ and RAPA at different concentrations on Leuk-1 cells,so as to further determine the optimal concentration of CQ and RAPA.[Results]The results of CCK-8 experiment showed that 4% CSE and 8%CSE treated Leuk-1 cells showed no significant changes in cell viability over time.In this experiment,Leuk-1 cells were treated with different concentrations of CQ,and the cell viability did not change significantly with different treatment times.Leuk-1 cells were treated with RAPA at concentrations of 0.01?M?0.1?M and 1?M.The cell viability did not change significantly with time,but the cell growth was significantly inhibited after RAPA concentration reached 10?M for 24h(P<0.05).Western blot results showed that with the increase of CSE concentration,the expression of LC3-II in Leuk-1 cells was significantly up-regulated after 24 h treatment(P<0.05).The expression of LC3-II in Leuk-1 cells was significantly up-regulated(P<0.05)after CSE treatment of 4% at different times,and reached its peak at 24 h.With the increase of CQ concentration,the expression of LC3-II in Leuk-1 cells was significantly up-regulated(P<0.05),and showed the highest trend at the concentration of 50?M,while the expression of LC3-II in Leuk-1 cells was not increased at the concentration of 100?M.At the same time,with the increase of RAPA concentration,the expression of LC3-II in Leuk-1 cells was significantly up-regulated(P<0.05)and reached its peak at the concentration of 1?M.[Conclusions]Cigarette smoke extract(CSE)have both concentration and time dependence on LC3-II expression in oral mucosal epithelial cells.Moreover,autophagy inhibitor CQ and autophagy inducer RAPA had a concentration dependence on the expression of LC3-II in oral mucosal epithelial cells.According to the previous research results of our research team and the results of this part of the experiment,CSE concentration was determined to be 4%,CQ concentration was 50?M,RAPA concentration was 1?M,and the stimulation time was 24 h to study the effects of smoking on autophagy and apoptosis of oral mucosal epithelial cells.Chapter2 Cigarette Smoke Induces Autophagy in Oral Mucosa Epithelial Cells[Objective]Autophagy is a complex dynamic process,formation and degradation mainly includes two stages,when autophagy increases or degradation is restrained,autophagy body will increase in the number.The only result of LC3-II increase in Leuk-1 cannot explain the autophagy.We should give the joint observation of LC3-II,Beclin-1 and p62 expression and animal experiments to further verification.Beclin-1 is an important molecule in the formation of autophagosomes.When Beclin-1 protein production increases,the formation of autophagosomes increases and autophagy activity increases.Otherwise,autophagy is inhibited.Sequestosome 1(SQSTM1/p62,p62)is a substrate for autophagy degradation.When the autophagy degradation pathway is unblocked,the level of p62 decreases.When the degradation pathway was inhibited,p62 aggregation increased.CQ inhibits autophagy by inhibiting the fusion of autophagosomes and lysosomes.In this part,the effects of cigarette smoke on the expression of LC3-II,Beclin-1 and p62 in oral mucosal epithelial cells were observed in combination to investigate the effects of smoking on autophagy of oral mucosal epithelial cells.[Methods]This part of experiment is divided into cell experiment and animal experiment.(1)Cell experiment: pretreated cells with CQ,then treated cells with 4%CSE,and cultured for 24 h.Total protein was extracted and the expression levels of LC3-II,Beclin-1 and p62 were detected by Western blot.In order to provide further evidence of autophagy,we conducted immunofluorescence(IHC)experiments to observe the expression of LC3 and p62.The formation of autophagosomes was observed by transmission electron microscopy(TEM).(2)Animal experiment: the mouse smoking model was established.All the mice were randomly divided into two groups,group A was the negative control group exposed to air(n=6),and group B was the smoking group(n=6).The total tissue protein was extracted and the expressions of LC3-II,Beclin-1 and p62 in the buccal mucosa of mice were detected by Western blot.The expressions of LC3 and p62 in the buccal mucosa of mice were detected by immunohistochemistry.[Results]This study found that LC3-II expression increased after CQ treatment alone(P<0.05),but no significant changes were observed in p62 and Beclin-1 expression,indicating that CQ blocked the formation of autophagy lysosomes after CSE treatment alone,p62 expression was significantly down-regulated(P<0.05),while LC3-II and Beclin-1 expression increased(P<0.05),indicating the occurrence of autophagy.Compared with the group treated by CSE alone,the expression of LC3-II and p62 in the CQ and CSE combined treatment group was significantly increased(P<0.05),while the expression of Beclin-1 was not significantly increased(P<0.05).Immunofluorescence results showed that LC3 and p62 in Leuk-1 cells treated with 4%CSE and CQ increased significantly(P<0.05).Transmission electron microscopy observation showed that autophagosomes in the 4%CSE group increased significantly compared to the control group.Western blot results in animal experiments showed that the expression of LC3-II and Beclin-1 in the smoking group was significantly increased(P<0.05),while the expression of p62 was significantly decreased(P<0.05).The results of immunohistochemistry of the buccal mucosa epithelial cells in mice showed that the expression of LC3 in oral mucosa epithelial cells of the smoking group was increased(P<0.05),while the expression of p62 was significantly decreased(P<0.05).It can be seen that the results of cell experiment and animal experiment are consistent.[Conclusions]Cigarette smoke can induce autophagy in oral mucosa epithelial cells.Chapter3 The Role of Autophagy Inhibitors and Inducers in the Regulation of Apoptosis Induced by Cigarette Smoke[Objective]Apoptosis is an autonomous and orderly way of cell death which is strictly controlled by a variety of genes and plays an important role in maintaining homeostasis of the internal environment.The disorder of apoptosis is closely related to the occurrence and development of many diseases.The Caspase-3 protein family is one of the most critical proteins in the process of cell apoptosis,and the level of Caspase-3 protein(a key protease in the Caspase-protease family)can indirectly reflect the level of cell apoptosis.In this study,changes in expression levels of Cleaved caspase3(Ccasp3),a molecule related to apoptosis of buccal epithelial cells and oral mucosal epithelial cells after cigarette treatment were detected to investigate the effects of smoking on apoptosis of oral mucosal epithelial cells.More and more scholars pay close attention to the regulation function of autophagy for cell apoptosis.To investigate the role of autophagy in the apoptosis of oral mucosal epithelial cells induced by CSE,oral mucosal epithelial cells were treated with CSE combined with autophagy inhibitor CQ and autophagy inducer RAPA in this study.The expression levels of apoptosis-related proteins were detected by Western blot.Annexinv-FITC/PI double staining flow cytometry was used to detect the apoptosis rate to observe the role of autophagy in the regulation of CSE-induced apoptosis.[Methods]The expression levels of apoptosis-related protein Caspase-3 were observed by Western blot in both cell and animal experiments to investigate the effect of smoking on apoptosis of oral mucosal epithelial cells.To investigate the role of autophagy in CSE-induced apoptosis of oral mucosal epithelial cells,the following methods were used for detection and verification.Annexin V-FITC/PI double staining flow cytometry was used to detect and analyze the apoptosis rate of cells in different treatment groups(Control,4%CSE,50?M CQ,50?M CQ+4%CSE)to investigate the effect of autophagy inhibitor CQ on CSE-induced apoptosis.Secondly,autophagy inducer RAPA was used to pretreat leuk-1 cells to activate autophagy activity.Annexin V-FITC/PI double staining flow cytometry was used to detect and analyze the apoptosis rate of cells in different treatment groups(Control,4%CSE,1?M RAPA,1?M RAPA+4%CSE)to investigate the effect of autophagy inducers on CSE-induced apoptosis.[Results]Both cell and animal experiments showed that cigarette smoke treatment increased the expression of apoptosis-related protein Caspase-3 in oral mucosal epithelium.Western blot results showed that CQ pretreatment further increased the level of Ccasp3 in CSE-induced Leuk-1 cells,while RAPA pretreatment reduced the level of Ccasp3 in CSE-induced Leuk-1 cells(P<0.05).Flow results showed that the apoptosis rate of CQ pretreatment group was higher than that of CSE treatment group(P<0.05).In contrast,the apoptosis rate of the RAPA pretreated group was lower than that of the CSE group(P<0.05).[Conclusions]Cigarette smoke can induce the apoptosis of oral mucosa epithelial cells,autophagy inhibition can aggravate the apoptosis of oral mucosa epithelial cells,and autophagy activation can inhibit the apoptosis of oral mucosa epithelial cells induced by CSE,suggesting that autophagy plays an inhibitory role in the apoptosis of oral mucosa epithelial cells induced by CSE.