星形胶质细胞对天冬氨酸特异性半胱氨酸蛋白酶介导β淀粉样蛋白早期突触毒性作用的影响

Effect of Astrocytes on Caspase-mediated Early Aβ Synaptotoxicity

目的探讨星形胶质细胞(astrocyte,AS)对天冬氨酸特异性半胱氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)介导β淀粉样蛋白(β-amyloid,Aβ)早期突触毒性作用的影响,以期为进一步研究与血管性痴呆(vascular dementia,Va D)的发病机制奠定基础。方法以原代培养大鼠海马纯神经元体系(NE-S)及混合培养体系(MIX-S,主要包含神经元及AS)为研究对象,各体系分为6组:对照组、caspase-8抑制剂组、caspase-9抑制剂组、Aβ处理组、caspase-8抑制剂预处理加Aβ组和caspase-9抑制剂预处理加Aβ组。免疫荧光检测各组近胞体10μm段树突中突触后密度蛋白(postsynaptic density-95,PSD95)表达量的变化。结果 1在NE-S与MIX-S中,与对照组相比,caspase-8抑制剂组、caspase-9抑制剂组PSD95的表达量均无明显差异,Aβ处理组PSD95的表达量均显著降低(P均<0.001)。2在NE-S中,与Aβ处理组相比,caspase-9抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,caspase-8抑制剂预处理加Aβ组则无显著改变;在MIX-S中的结果则相反,即caspase-8抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,而caspase-9抑制剂预处理加Aβ组则无显著改变。3MIX-S与NE-S两种培养系统间相比较,对照组间及Aβ处理组间PSD95的表达量均无显著差异,而caspase-8抑制剂预处理加Aβ组间及caspase-9抑制剂预处理加Aβ组间PSD95的表达量差异有显著性。结论在Aβ早期突触毒性作用中,AS参与caspase-8介导的死亡受体通路激活过程,且参与抑制神经元的线粒体通路。

Objective To investigate the effect of astrocytes on caspase-mediated early β-amyloid（Aβ） neurotoxicity so as to lay a foundation for further studying of the pathogenesis of vascular dementia（Va D）.Methods We established the 4-day-old Wistar rat hippocampal primary mixed culture system（MIX-S）, neurons and astrocytes are mainly included, and the purifi ed primary neuronal culture system（NE-S）. Then each system was divided into six groups： control group, caspase-8 inhibitor group, caspase-9 inhibitor group, Aβ group, caspase-8 inhibitor pretreatment with Aβ group and caspase-9 inhibitor pretreatment with Aβ group. Immunofluorescence technique was applied to investigate the changes of the expression of postsynaptic density-95（PSD95） at 10 μm dendrites near to cell body.Results 1 Both in NE-S and MIX-S, compared with control group, the expression of PSD95 had no significant difference in the caspase-8 inhibitor group and the caspase-9 inhibitor group,while that in the Aβ group reduced significantly. 2 In NE-S, compared with the Aβ group, the expression of PSD95 rebounded significantly to the level of the control group in the caspase-9 inhibitor pretreatment with Aβ group, while no significant change was found in the caspase-8 inhibitor pretreatment with Aβ group; interestingly, the results of this part in MIX-S was reversed. 3 Compared with NE-S, the expression of PSD95 had no signifi cant change in the control group and Aβ group of MIX-S; while the difference of that was significant in the caspase-8 inhibitor pretreatment with Aβ group and caspase-9 inhibitor pretreatment with Aβ group.Conclusion Astrocyte plays a role in activation of caspase-8 mediated death receptor pathway, and also participates in inhibition of neuronal mitochondrial pathway in early Aβ synaptotoxicity.