Objective: To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) . Methods: The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 μmol/L PQ for 24 hours, and the cells were induced by 300 μmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 μmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3Ⅱ, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot. Results: Compared with the control group, the cell survival rate of the 100, 200, 300, 400 μmol/L PQ group decreased significantly, and showed a dose-dependent pattern (P<0.05) . The survival rate of cells treated with 300 μmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) (P<0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 μmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased (P<0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased (P<0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3Ⅱand Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher (P<0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300μmol/L PQ-treatd groups, with statistically significant difference (P<0.05) . Conclusion: AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.
目的： 探讨AMPK-mTOR信号转导通路在百草枯（paraquat，PQ）诱导大鼠肾上腺髓质嗜铬细胞瘤细胞（PC12）自噬异常中的调控作用。 方法： 用终浓度0、25、50、100、200、300、400 μmol/L PQ处理PC12细胞24 h，300 μmol/L PQ处理细胞不同时间（6、12、24、48 h），四甲基偶氮唑蓝（MTT）检测细胞相对存活率，确定剂量/时间-效应关系；终浓度0、100、200、300 μmol/L PQ处理细胞24 h，分光光度法检测培养上清中乳酸脱氢酶（LDH）活力；二氯荧光素双醋酸盐（DCFH-DA）法检测细胞内活性氧（ROS）水平；单丹磺酰尸胺（MDC）染色法观察自噬溶酶体的表达及分布变化；免疫荧光（IF）检测微管相关蛋白1轻链3（LC3）表达水平；免疫印迹法（Western blot）检测自噬相关蛋白LC3Ⅱ、p62、Beclin1及AMPK-mTOR信号通路蛋白p-AMPK、p-mTOR表达水平。 结果： 与对照组比较，100、200、300、400 μmol/L PQ染毒组的细胞存活率明显降低，且呈剂量依赖关系，差异有统计学意义（P<0.05）。300 μmol/L PQ处理细胞不同时间（6、12、24、48 h），细胞存活率随染毒时间的延长而下降，其中12、24、48 h染毒组的差异有统计学意义（P<0.05）。与对照组比较，100、200、300 μmol/L PQ染毒组细胞上清液中LDH活力明显升高，细胞内ROS荧光强度明显增高，差异有统计学意义（P<0.05）；MDC染色结果显示，PQ染毒组的自噬溶酶体在核周分布密度降低、荧光强度减弱、自噬水平降低；免疫荧光结果显示，PQ染毒组的LC3荧光强度减弱，细胞自噬水平下降，与MDC染色结果一致。Western blot结果显示，与对照组比较，PQ染毒组的自噬相关蛋白LC3Ⅱ、Beclin1表达水平均明显降低，而p62蛋白表达水平升高，差异有统计学意义（P<0.05）。200、300 μmol/L PQ染毒组的p-AMPK蛋白表达水平降低，p-mTOR蛋白表达水平升高，差异有统计学意义（P<0.05）。 结论： PQ致PC12细胞损伤过程中，细胞自噬水平降低，AMPK-mTOR信号通路发挥调节作用。.
Keywords: AMPK-mTOR singal pathway; Autophagy; Dopaminergic neurons; Paraquat.