酒精诱导嗜铬细胞瘤细胞自噬及其与P62作用的关系

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

目的采用不同浓度酒精作用于大鼠嗜铬细胞瘤（PC12）细胞，观察细胞自噬发生及其与P62变化的关系。方法用四甲基偶氮唑盐比色法（MTT）观察酒精对PC12细胞生存率的影响；间接免疫荧光法检测细胞自噬标志性蛋白LC3和P62的变化；高内涵活细胞成像系统检测细胞LC3荧光强度；透射电镜检测细胞自噬的超微结构；Western blotting方法检测P62蛋白量的表达。结果50-800mmol／L浓度酒精对PC12细胞的增殖有显著的抑制作用，呈浓度依赖性。酒精致PC12细胞自噬标志性蛋白LC3在细胞核周围密度增高，并与P62形成点状聚集共定位，其中在200mmol／L浓度酒精作用2h，PC12细胞LC3自噬荧光强度最高；透射电镜也观察到酒精作用的PC12细胞质中自噬体和自噬溶酶体。Western blotting结果显示，不同浓度酒精处理PC12细胞2h，P62蛋白表达量显著增加（P〈0．01）；用200mmol／L浓度酒精处理PC12细胞，P62蛋白表达在2h达到最高值。结论酒精诱导PC12细胞的自噬作用，P62蛋白参与自噬调控过程。

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 〈 0.01 ） ,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 eytosol. 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.