AAS ›› 2013, Vol. 44 ›› Issue (6 ): 772-777.doi: 10.3969/j.issn.0529-1356.2013.06.008

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Effects of ethanol on autophagy and the role of P62 in ethanol-induced autophagy in pheochromocytoma cells

DONG Yue-juan1 SHI Zhen-yu1 LIU Shi-meng2 ZHANG Wei-juan2 HUANGFU Chao-shen2 DENG Jin-bo1 LIU Bin 1*   

  1. 1. Institute of Neurobiology,College of Nursing of Henan University, He’nan Kaifeng 475004, China; 2. Department of Biochemistry and Molecule Biology, Medical College of Henan University, He’nan Kaifeng 475004, China
  • Received:2013-02-26 Revised:2013-07-09 Online:2013-12-06 Published:2013-12-06
  • Contact: LIU Bin E-mail:lbgood5912z@sina.com

Abstract:

Objective To utilize pheochromocytoma (PC12) cells to examine the effect of ethanol on autophagy and the role of P62 in ethanol-induced autophagy. Methods The inhibition effect of ethanol on proliferation of PC12 cells was examined by MTT assay. Indirect immunofluorescence was used to detect location of LC3, a biomarker of autophagy, and P62. The LC3 fluorescence intensity in cellular cytosol was measured using a high content screening imaging system. The ultrastructural morphology of autophagic vacuoles and autophagy-lysosome was observed under the transmission electron microscope. Protein expression of P62 was detected with Western blotting analysis. Results Compared to the control in serumfree medium after 24hours, ethanol at the concentrations of 100mmol/L, 200mmol/L, 400mmol/L and 800mmol/L decreased cell proliferation of PC12 cells to 91.97% (P<0.05),72.63%(P<001),58.23%(P<0.01) and 50.82%(P<0.01) respectively, indicating a dose-dependent inhibitory effect of ethanol on cell proliferation.Indirect immunofluorescence staining and the high content screening imaging system showed that ethanol increased LC3 fluorescence intensity and the co-localization of LC3 with P62 in PC12 cellular cytosol. The transmission electron microscope showed the ultrastructural morphology of autophagic vacuoles and autophagy-lysosome in PC12 cells incubated for 2hours in the presence of 200mmol/L ethanol. Compared with the control in serum-free medium, the protein expression of P62 increased significantly in different ethanol concentration treatment groups (P<0.01). The crest-time appeared at 2hours after the PC12 cells were treated with 200mmol/L ethanol. Conclusion Ethanol may induce autophagy of PC12 cells. P62 may be involved in the autophagy against ethanol-induced cytotoxicity.

Key words: Ethanol, Autophagy, LC3, P62, PC12 cell, Indirect immunofluorescence, Transmission electron microscopy

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