人神经黑色素过度激活小胶质细胞：多巴胺能神经细胞进行性变性的新机制

Human Neuromelanin Induced Microglial Overactivation:a Novel Mechanism for the Progressive Dopaminergic Neurodegeneration

目的探讨小胶质细胞在中脑黑质致密带(SNpc)人神经黑色素(HNM)导致多巴胺(DA)能神经细胞变性中的作用和机制。方法(1)采用HNM处理中脑胶质细胞重组培养体系,检测DA能神经细胞摄取能力,探讨小胶质细胞对HNM损伤作用的影响。(2)采用小胶质细胞培养体系,通过免疫细胞化学染色、检测免疫炎性及神经毒性因子,从功能及形态上研究HNM对小胶质细胞的激活作用。结果(1)经5.0μg/mL HNM干预后10%、20%和30%小胶质细胞重组培养体系DA能神经细胞摄取能力分别为纯神经细胞培养体系(对照组)的58%、51%和35%,与对照组比较差异有统计学意义(P<0.05);40%、50%和60%星形胶质细胞重组培养体系中DA能神经细胞摄取能力分别为对照组的97%、92%和93%,与对照组比较差异无统计学意义(P>0.05)。(2)经5.0μg/mLHNM干预后中脑神经-胶质细胞混合培养体系中激活的小胶质细胞数为对照组的5.77倍(P<0.05),小胶质细胞体积明显增大,形状不规则,呈棒状和/或阿米巴样,胞浆深染。(3)经5.0μg/mLHNM干预后小胶质细胞产生大量的细胞内活性氧类物质、一氧化氮、肿瘤坏死因子α、白细胞介素1β和前列腺素E2,与对照组比较差异均具有统计学意义(P<0.05)。结论HNM从功能及形态上激活小胶质细胞,促进了DA能神经细胞的进行性变性,为抑制小胶质细胞过度激活成为治疗PD的新靶点提供了一定理论依据。

Objective To investigate the role and mechanism of microglia in dopaminergic neurodegeneration induced by human neuromelanin （HNM） from substantia nigra pars compacta （SNpc） by using multiple primary mesencephalic culture systems. Methods （1）In primary mesencephalic glia-reconstituted cultures, the role of microlgia on HNM-elicited dopaminergic neurodegeneration was evaluated by measuring DA uptake. （2）In microglia-enriched cultures, functional and morphological microglial overactivation by HNM was explored by staining microglia with OX-42 antibody and detecting the production of a series of pro-inflammatory and neurotoxic factors. Results （1） 5.0 μg/mL HNM-induced DA uptake were 58%, 51% and 35% of neuron-enrichend cultures （control）in 10%, 20% and 30% microglia-reconstituted cultures respectively, which were statistically significant compared with control group;5.0 μg/mL HNM-induced DA uptake were 97%, 92% and 93% of neuron-enrichend cultures （control）in 40%, 50% and 60% astroglia reconstituted cultures respectively, which weren’t statistically significant compared with control group;（2）5.0 μg/mL HNM increased the number of activated microglia, which was 5.77 folds of control group, and was statistically significant;HNM changed the morphology of microglia indicated by enlarging cell body, irregular shape such as rod and/or ameboid-like shape and intensified staining;（3）5.0 μg/mL HNM produced a large amount of intracellular reactive oxygen species, nitrite oxide, tumor necrosis factors α, interleukin1β and prostaglandin E2, which were all statistically significant compared with control group. Conclusions HNM overactivated microglia functionally and morphologically,accelerating dopaminergic neurodegeneration.Thus inhibition of microglial overactivation might be a novel target for PD therapy.