脱氧雪腐镰刀菌烯醇对PC12细胞自噬的影响

Effect of deoxynivalenol on autophagy of PC12 cells

【目的】研究脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)对PC12细胞自噬的影响。【方法】以不同质量浓度(0(CK),125,250,500,1 000和2 000ng/mL)的DON处理PC12细胞24h后,采用倒置显微镜、透射电镜、免疫荧光、荧光定量PCR、Western-blot等技术,研究DON对PC12细胞形态和超微结构、LC3聚点率及自噬相关基因classⅢPI3 K、Beclin-1、P62 mRNA和蛋白LC3Ⅱ、P62、classⅢPI3K表达的影响。【结果】不同质量浓度的DON均可诱导PC12细胞自噬,各试验组细胞自噬水平均高于对照组(CK),并随着DON质量浓度的增大而先增加后下降,在DON质量浓度为1 000ng/mL时达到峰值。与对照组相比,各试验组classⅢPI3 K mRNA和Beclin-1mRNA表达水平均极显著增高(P<0.01),P62mRNA表达水平随着DON质量浓度的增加总体呈下降趋势,差异达显著(P<0.05)或极显著(P<0.01)水平。Western-blot结果显示,LC3Ⅱ蛋白表达量在DON质量浓度为1 000ng/mL时最高,与对照组相比,250~2 000ng/mL DON处理组的LC3Ⅱ蛋白表达量显著(P<0.05)或极显著(P<0.01)增加;500~2 000ng/mL DON处理组classⅢPI3K蛋白表达量较对照组显著(P<0.05)或极显著(P<0.01)增加,其中以1 000ng/mL DON处理组表达量最高;与对照组相比,不同质量浓度DON处理组P62蛋白表达量均极显著降低(P<0.01),其中以1 000ng/mL DON处理组最低。【结论】DON能够诱导PC12细胞发生自噬,自噬相关基因classⅢPI3 K、Beclin-1、P62及蛋白LC3Ⅱ、P62、classⅢPI3K等参与了DON诱导PC12细胞自噬的调控。

【Objective】The aim of this research was to study the effect of deoxynivalenol(DON)on autophagy of PC12 cells.【Method】PC12 cells were exposed to DON at different concentrations(0(CK),125,250,500,1 000 and 2 000 ng/mL)for 24 h.The changes of cell morphology,cell ultrastructure,presence of LC3 puncta,autophagy-related genes classⅢPI3K,Beclin-1,P62 mRNA expressions and autophagy-related proteins LC3II,P62 and classⅢPI3K expressions were determined by inverted microscopy,transmission electron microscopy,immunofluorescence,qRT-PCR and Western-blot.【Result】Different concentrations of DON could induce cell autophagy.The cell autophagy appearance in experimental groups was higher than the control group,and the level decreased before initial increase with the increase of DON concentration with the peak at 1 000 ng/mL.Compared with control group,classⅢPI3K and Beclin-1 mRNA were significantly increased(P<0.01),while P62 mRNA was decreased significantly(P<0.05)or extremely significantly(P<0.01).Western-blot results showed that LC3II in experimental groups peaked at 1 000 ng/mL.Compared with control group,it was increased significantly(P<0.05)at 250-2 000 ng/mL.ClassⅢPI3K in experimental groups was increased significantly(P<0.05)at 500-2000 ng/mL,with peak at 1 000 ng/mL.P62 was significantly decreased(P<0.01)with the minimum at 1 000 ng/mL.【Conclusion】DON could induce PC12 cell autophagy,and classⅢPI3K,Beclin-1,P62 and LC3II participated in the regulation of cell autophagy.