香烟烟雾提取物对MIN6细胞JNK及p38MAPK表达影响

Effect of cigarette smoke extract on JNK,p38MAPK expressions in MIN6 cells

目的探讨体外香烟烟雾提取物(CSE)对胰岛MIN6细胞形态及活力的影响,及对胞内丝裂原活化蛋白激酶(MAPKs)信号转导通路ERK、JNK、p38MAPK表达的影响。方法体外培养MIN6细胞至生长对数期后,设不同浓度CSE(20、50、100和200μg/m L)实验组,同时设对照组,48 h后,镜下观察MIN6细胞形态学改变,并应用CCK-8法检测CSE对MIN6细胞的增殖抑制率;同时应用蛋白印迹法(WB)检测细胞内ERK、JNK、p38M APK蛋白及磷酸化程度。结果与对照组比较,100和200μg/m L剂量下的细胞形态出现明显异常,胞核固缩,细胞呈梭形样改变;与对照组比较,MIN6细胞活力出现下降,20、50、100、200μg/m L各组细胞活力分别为(97.32±2.67)%、(94.67±5.33)%、(87.71±12.3)%和(74.23±25.8)%;与对照组相比,ERK、JNK、p38MAPK蛋白表达变化不大,但随着CSE剂量的增加,p-JNK和p-p38MAPK磷酸化程度明显增加(与对照组相比,P<0.05),并呈一定的剂量-效应关系。结论 CSE能够导致M IN6细胞形态异常、抑制其细胞增殖活力,其机制可能与JNK和p38MAPK信号通路的激活相关。

Objective To investigate the effect of cigarette smoke extract （CSE） on expressions of c-Jun N-terminal kinase （JNK） and p38 mitogen-activated protein kinase （MAPK） in MIN6 cells. Methods MIN6 cells in logarithmic growth period were cultured in vitro and then treated with different concentrations of CSE （20,50,100,200 μg/mL） and a vehicle （control） group was also established. After 48 hours＇ treatment of CSE, the change in morphology of the cells was observed with inverted microscope and cholecystokinin （CCK-8） was used to assess CSE-mediated anti-proliferative effect on MIN6 cells. Meanwhile, Western blot analysis was applied to determine cellular protein expressions of extracellular-signal-regulated kinase （ERK） ,JNK,p38MAPK,and corresponding phosphorylation level. Results Compared with the control group,obvious abnormal morphology was found in the cells under the treatment of 100 μg/mL and 200 μg/mL CSE,including nuclear condensation and spindle like change. Compared with the control group, CSE at different dosages significantly inhibited the viability of MIN6 cells, with the cell viabilities of 97.32 ± 2.67%, 94. 67 ± 5.33% ,87.71 ± 12% （ all P 〈 0. 05 ）, and 74. 23 ± 25.80% （ P 〈 0. 01 ） for the cells treated with 20,50,100, and 200 μg/mL CSE,respectively. In addition,CSE effectively activated the JNK and p38MAPK signaling pathway. With the increment of CSE concentration, the phosphorylation level significantly increased when compared to that of the control group （ P 〈 0.05 ）. Conclusion Cigarette smoke extract could decrease the cell viability of MIN6 cells and the effect may due to the increased expressions of p-JNK and p-p38MAPK.