Effect Of Ethanol On Autophagy In PC12Cells And The Role Of P62in Ethanol-induced Autophagy

ObjectiveThe study utilized pheochromocytoma PC12cells to examine the effect of ethanol on autophagy andthe role of P62in ethanol-induced autophagy.MethodsThe inhibition effect of ethanol on proliferation of PC12cells was examined by MTT assay. Indirectimmunofluorescence was used to detect common location of LC3, a biomarker of autophagy, and P62. TheLC3fluorescence intensity in cellular cytosol was measured using a high content screening imaging system.The ultrastructural morphology of autophagic vacuoles and autophagy-lysosome were observed under thetransmission electron microscope. Protein expression of P62was detected with Western blotting analysis.ResultsEthanol at the concentration of100mmol/L,200mmol/L,400mmol/L and800mmol/L decreasedcell proliferation of PC12cells to91.97%(P＜0.05),72.63%(P＜0.01),58.23%(P＜0.01),50.82%(P＜0.01) respectively and these groups were compared to the control in serum-free medium. The studyindicated a dose-dependent inhibitory effect of ethanol on cell proliferation．Indirect immunofluorescencestaining and the high content screening imaging system showed that ethanol increased LC3fluorescenceintensity and the co-localization of LC3with P62in PC12cellular cytosol. The transmission electronmicroscope showed the ultrastructural morphology of autophagic vacuoles and autophagy-lysosome inPC12cells incubated for2h in the presence of200mmol/L ethanol. Compared with the control inserum-free medium, the protein expression of P62was increased significantly in different ethanolconcentration treatment groups (P＜0.01). The crest-time of the protein espression of P62appeared at2hafter the PC12cells were treated with200mmol/L ethanol.ConclusionEthanol could induce autophagy of PC12cells. P62may be involved in the autophagy againstethanol-induced cytotoxicity.