JNK/AP-1信号通路在百草枯诱导肺泡上皮细胞间充质转变中的作用
Regulation of JNK/AP-1 signaling pathway in paraquat-induced mesenchymal transition of epithelial cells
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摘要:
目的 探讨c-jun氨基末端激酶（JNK）/转录因子激活蛋白-1（AP-1）信号通路在百草枯致大鼠Ⅱ型肺泡上皮细胞间发生上皮-间充质转变（EMT）中的作用。
方法 体外培养大鼠Ⅱ型肺泡上皮细胞RLE-6TN，终浓度0、25、50、100、200、300、400μmol/L百草枯处理细胞24 h，终浓度200 μmol/L百草枯分别处理细胞0、12、24、36、48、60、72 h，倒置相差显微镜观察细胞形态学的改变；四甲基偶氮唑盐（MTT）检测细胞相对存活率，确定剂量和时间-效应关系；流式细胞仪Annexin V-FITC/PI法检测细胞凋亡；细胞划痕实验检测细胞迁移能力；Transwell小室法检测细胞侵袭能力；免疫荧光、免疫印迹法（Westernblot）分别检测上皮标志蛋白E-cad、ZO-1和间质标志蛋白FSP-1、α-SMA表达水平；Westernblot检测JNK/p-JNK、c-jun/p-c-jun、c-fos/p-c-fos蛋白表达水平；双荧光素酶报告基因实验检测AP-1的转录活性，评估百草枯对JNK信号通路的激活作用。
结果 200 μmol/L百草枯诱导细胞由卵圆形或多边形的上皮细胞变成长梭形的间质细胞，细胞间连接消失，且时间越长形态学改变越明显。与对照组相比，100、200、300、400 μmol/L百草枯染毒组的细胞存活率明显降低（P<0.05），200 μmol/L百草枯诱导细胞不同时间（24、36、48、60、72 h）的细胞存活率明显降低（P<0.05）。200 μmol/L百草枯诱导细胞不同时间（12、24、36h）：细胞凋亡率随诱导时间的延长而增加，尤其36h诱导组的细胞凋亡率较对照组明显升高，差异有统计学意义（P<0.05）；细胞迁移指数、侵袭细胞数均明显升高（P<0.05）；上皮细胞标记蛋白E-cad、ZO-1表达下调，且下调的趋势随时间延长更加明显（P<0.05）；间质细胞标记蛋白FSP-1、α-SMA表达随时间的延长逐渐升高（P<0.05）；与对照组相比，100、200、300 μmol/L百草枯染毒可诱导p-JNK、p-c-jun、p-c-fos蛋白表达明显上调；200、300 μmol/L百草枯染毒组较对照组比较，AP-1转录活性明显升高，差异有统计学意义（P<0.05）。
结论 百草枯呈剂量和时间依赖方式影响上皮细胞活性。百草枯能够诱导大鼠Ⅱ型肺泡上皮细胞发生EMT，JNK/AP-1信号通路可能参与了百草枯诱导肺泡上皮细胞EMT的发生。
Abstract:Objective To explore the role of c-jun N-terminal kinase (JNK)/activated protein-1 (AP-1) signaling pathway in paraquat (PQ)-induced epithelial-mesenchymal transition (EMT) of rat type Ⅱ alveolar epithelial cells.
Methods RLE-6TN cells were incubated with designed concentrations of PQ (0, 25, 50, 100, 200, 300, and 400 μmol/L) for 24 h or 200 μmol/L PQ for 0, 12, 24, 36, 48, 60, and 72 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined by MTT assay to evaluate dose/time-effect relationship. Cell apoptosis was detected by Annexin V-FITC/PI. The migration and invasion abilities of cells were detected by wound healing and Transwell assays. The protein expressions of epithelial phenotype markers (E-cad and ZO-1) and mesenchymal phenotype markers (FSP-1 and α-SMA) were determined by immunofluorescence and Western blot respectively. The protein expressions of JNK/p-JNK, c-jun/p-c-jun, c-fos/p-c-fos were detected by Western blot. The activity of AP-1 was measured by luciferase reporter assay to evaluate the activation of JNK signaling pathway by PQ.
Results An elongated shape and a lack of tight cell-cell adhesions of mesenchymal cells were induced by 200 μmol/L PQ treatment in a time-dependent manner. The cell viability decreased in the cells treated with PQ at 100, 200, 300, and 400 μmol/L (P<0.05) or with 200 μmol/L PQ for 24, 36, 48, 60, and 72 h (P<0.05). After 200 μmol/L PQ treatment for 12, 24, and 36 h, the percentage of apoptosis cells increased with more exposure time, especially in the 36h treatment group as compared with the control group (P<0.05); cell migration index and invasive cells increased; the protein expressions of epithelial phenotype markers (E-cad and ZO-1) significantly decreased while those of mesenchymal phenotype markers (α-SMA and FSP-1) dramatically increased in a time-dependent manner after PQ exposure (P<0.05). Compared with the control group, after exposure to PQ (100, 200, and 300 μmol/L) for 24 h, the protein expressions of p-JNK, p-c-jun, and p-c-fos significantly increased (P<0.05). The PQ treatment at 200 and 300 μmol/L induced a significant increase of AP-1 activity (P<0.05).
Conclusion PQ affects cell viability of epithelial cells in a dose-and time-dependent manner. PQ can induce EMT in rat alveolar epithelial cells, in which JNK/AP-1 signaling pathway is involved.
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