The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in hu-man periodontal ligament stem cells
PURPOSE:To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells(hPDLSCs),as well as the underlying mechanism of electronic cigarette(E-cig)-induced cell apoptosis.METHODS:PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic rea-sons.Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry.Then the cells were ex-posed to different doses of menthol-favored E-liquid(at 59 mg/L nicotine concentration)in the culture median(the final nicotine concentrations were 0.1 μg/mL,1.0 μg/mL,10 μg/mL,50 μg/mL,0.1 mg/mL,0.2 mg/mL and 0.5 mg/mL,respec-tively)for different period of times(24,48 and 72 h).The cell viability was analyzed by CCK-8 assay.Cell apoptosis was evaluated by flow cytometry(7-AAD and Annexin V staining)and TUNEL assay.Reactive oxygen species(ROS)produc-tion was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry.The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK,JNK,c-Jun,p-c-Jun,Bcl-2,Bax and cleaved-caspase 3)were analyzed by Western blot.Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK.After addition of NAC,a ROS scavenger,and MAPK/JNK specific blocker SP600125,their effects on E-cig-induced cell apop-tosis were evaluated.Statistical analysis was performed with Graph Pad 5.0 software package.RESULTS:Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers(CD73,CD90 and CD105)were positively expressed.CCK8 assay indicated cell viability was significantly(P＜0.001)differ-ent among all concentration groups at various time points(24,48 or 72 h),and the difference in apoptosis rate among all concentration groups was also statistically significant(P＜0.001).After exposure to E-liquid with nicotine concentration ≥50 μg/mL,cell viability was significantly reduced,and the proportion of apoptotic cells and the cellular ROS level was sig-nificantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL).Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner.Ei-ther NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3.CONCLUSIONS:ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apopto-sis of hPDLSCs.
Electronic cigarettePeriodontal ligament stem cellsApoptosisReactive oxygen species
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