[The role of the AMPK-mTOR pathway in paraquat-induce autophagy in PC12 cells]

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.