MPTP对小鼠多巴胺能神经元细胞MN9D凋亡的影响

Effects of MPTP on apoptosis of mouse dopaminergic neuron MN9D cells

目的:探讨MPTP对小鼠多巴胺能神经元细胞MN9D凋亡的影响及其可能机制。方法:MN9D细胞分别以0、1μmol/L MPTP处理(对照组和MPTP组),或分别以pcDNA4、pcDNA4-BACE2转染(对照组和BACE2组),采用Western blot法检测BACE2、Cleaved Caspase-3和内源性DSG2蛋白的表达。MN9D细胞分为DSG2组、DSG2+BACE2组和DSG2+BACE2+BACE抑制剂组,分别转染pENTER-DSG2+pcDNA4、pENTER-DSG2+pcDNA4-BACE2以及转染pENTER-DSG2+pcDNA4-BACE2后用1μmol/L的BACE抑制剂处理,采用Western blot法检测DSG2全长以及DSG2各片段的表达。结果:与对照组相比,MPTP组中BACE2及Cleaved Caspase-3表达均上升(P<0.05);与对照组相比,BACE2组Cleaved Caspase-3表达增加,内源性DSG2表达减少(P<0.05)。与DSG2组相比,DSG2+BACE2组中全长DSG2表达下降,DSG2羧基末端片段以及胞外DSG2氨基末端片段均增多(P<0.05),而DSG2+BACE2+BACE抑制剂组中全长DSG2及各DSG2片段的表达与DSG2组相似(P>0.05)。结论:MPTP可通过上调BACE2促进MN9D细胞凋亡,其机制可能与BACE2对DSG2的剪切有关;BACE2和DSG2可能参与了帕金森病的发病。

Aim:To investigate the effects of MPTP on apoptosis of mouse dopaminergic neuron MN9D cells and its possible mechanism.Methods:MN9D cells were treated with 0 or 1μmol/L MPTP(control group and MPTP group),or transfected with pcDNA4 or pcDNA4-BACE2(control group and BACE2 group),respectively.Western blot was used to detect the expressions of BACE2,Cleaved Caspase-3 and endogenous DSG2 proteins.MN9D cells were allocated into DSG2 group,DSG2+BACE2 group and DSG2+BACE2+BACE inhibitor group,and cells were transfected with pENTER-DSG2+pcDNA4,pENTER-DSG2+pcDNA4-BACE2,and pENTER-DSG2+pcDNA4-BACE2 and treated with 1μmol/L BACE inhibitor,respectively.Western blot was used to detect the expressions of full-length DSG2 and DSG2 fragments.Results:Compared with control group,the expressions of BACE2 and Cleaved Caspase-3 in MPTP group were increased(P<0.05).Compared with control group,the expression of Cleaved Caspase-3 in BACE2 group overexpressing BACE2 was increased,and that of endogenous DSG2 reduced(P<0.05).Compared with DSG2 group,the expression of full-length DSG2 decreased,and the carboxyl terminal fragment and the amino terminal fragment of extracellular DSG2 in DSG2+BACE2 group increased(P<0.05).The expressions of full-length DSG2 and DSG2 fragments in DSG2+BACE2+BACE inhibitor group were similar to those in DSG2 group(P>0.05).Conclusion:MPTP could promote apoptosis by up-regulating BACE2,which might be laid to BACE2′s cutting DSG2;BACE2 and DSG2 may be involved in the pathogenesis of Parkinson′s disease.