ROS/JNK/caspase 3信号轴在薄荷味电子烟烟液诱导牙周膜干细胞凋亡中的作用

The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells

目的:探讨薄荷味电子烟暴露对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)的细胞毒性及促凋亡机制。方法:自正畸拔除的健康前磨牙的牙周膜中分离、培养得到PDLSCs,取第3代细胞,流式细胞术分析表面干细胞标志物。将薄荷味电子烟烟液(尼古丁浓度为59 mg/mL)加入细胞培养基,使各暴露剂量组细胞培养基中的尼古丁终浓度分别为0.1μg/mL、1.0μg/mL、10μg/mL、50μg/mL,0.1 mg/mL、0.2 mg/mL和0.5 mg/mL,于不同时间点(24、48、72 h)检测各组细胞的细胞活力(CCK8法)及细胞凋亡情况(7-AAD及Annexin V双染后流式细胞术分析、TUNNEL分析)。采用荧光探针DCFH-DA检测细胞活性氧(reactive oxygen species,ROS)水平,共聚焦显微镜下观察及流式细胞分析。采用蛋白免疫印迹法(Western blot)分析ROS/JNK/caspase 3信号轴相关蛋白[c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)、c-Jun、磷酸化c-Jun(p-Jun)、Bcl-2、Bax和活化的半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase 3)]的表达水平,采用免疫荧光染色分析p-JNK的表达。分别添加ROS清除剂N-乙酰半胱氨酸(Nacetyl-l-cysteine, NAC)及MAPK/JNK特异性阻断剂SP600125预处理细胞,分析其对电子烟诱导的细胞凋亡的影响。采用Graph Pad 5.0软件包对数据进行统计学分析。结果:成功分离、培养出hPDLSCs,流式细胞术分析显示间充质干细胞表面标志分子CD29、CD90、CD105及CD146表达阳性。各电子烟暴露剂量组3个时间点(24、48、72 h)细胞活力情况相比,差异有统计学意义(P<0.001);各浓度组细胞凋亡情况相比,差异有统计学意义(P<0.001)。与0.0 mg/L浓度对照组相比,≥50μg/mL暴露剂量组呈浓度依赖性方式显著抑制细胞活力,促进细胞凋亡比例增加,并且上调细胞ROS水平。Western印迹分析提示,电子烟暴露可浓度依赖并以时间依赖激活MAPK/JNK磷酸化,NAC及SP60025预处理能反转电子烟暴露导致的上调的p-JNK及活化型caspase 3水平,降低电子烟暴露诱导的PDLSCs凋亡率。结论:ROS/JNK/caspase 3信号轴参与薄荷味电子烟暴露诱导的PDLSCs细胞凋亡。

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 reasons.Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry.Then the cells were exposed to different doses of menthol-favored E-liquid(at 59 mg/L nicotine concentration)in the culture median(the final nicotine concentrations were 0.1μg/mL,1.0μg/mL,10μg/mL,50μg/mL,0.1 mg/mL,0.2 mg/mL and 0.5 mg/mL,respectively)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)production 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 apoptosis 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)different 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≥50μg/mL,cell viability was significantly reduced,and the proportion of apoptotic cells and the cellular ROS level was significantly 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.Either 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 apoptosis of hPDLSCs.