别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

王彤彤; 陈治池; 叶鑫; 边维; 杜娟娟; 傅维达; 陈梦娇; 李俊楠; 孙臣友

doi:10.16098/j.issn.0529-1356.2020.04.001

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解剖学报 ›› 2020, Vol. 51 ›› Issue (4) : 473-482. DOI: 10.16098/j.issn.0529-1356.2020.04.001

神经生物学

别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制

王彤彤^1,2,3陈治池^1,3叶鑫^1,3边维^1,3 杜娟娟^3,4 傅维达⁵ 陈梦娇⁵ 李俊楠⁵孙臣友^1,3*

作者信息 +

Allopregnanolone restores dopaminergic neurons and motor performance in Parkinson’s disease mice and its molecular mechanisms

WANG Tong-tong^1,2,3CHEN Zhi-chi^1,3 YE Xin^1,3 BIAN wei^1,3 DU Juan-juan^3,4 FU Wei-da⁵ CHEN Meng-jiao⁵ LI Jun-nan⁵ SUN Chen-you^1,3*

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文章历史 +

摘要

目的探讨别孕烯醇酮（APα）对帕金森病（PD）小鼠黑质-纹状体多巴胺能系统及行为学的影响及可能的分子机制。方法 90只3月龄体重为20~25 g雄性C57BL/6小鼠1侧纹状体被注射6-羟多巴胺（6-OHDA）复制小鼠PD模型，再给予APα及γ-氨基丁酸A受体（GABAAR）拮抗剂荷包牡丹碱（Bic）。采用ELISA检测血清及大脑皮质APα含量和纹状体多巴胺水平，免疫组织化学法检测黑质多巴胺能神经元及其纹状体纤维投射数量的变化，Western blotting检测中脑胞膜GABAAR、胞质及胞核各蛋白水平的变化，并通过免疫共沉淀验证它们之间的相互作用，观察小鼠行为学的变化。结果 PD小鼠大脑皮质内源性APα水平显著降低，黑质多巴胺能阳性神经元及其纹状体纤维投射含量下降，胞膜、胞质和胞核各蛋白的水平也明显下降，小鼠的运动功能发生障碍。在给予外源性APα处理后，上述情况均有所改善。但在应用Bic后，上述调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号呈现相反的变化，免疫共沉淀结果显示，蛋白之间存在相互作用。结论外源性的APα通过增加其内源性水平调控GABAAR/钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3/脑源性神经营养因子信号通路促进PD模型小鼠黑质-纹状体多巴胺能神经系统和小鼠行为学的改善。

Abstract

Objective To investigate the effects of allopregnanolone(APα) on the dopaminergic neurons in substantia nigra, striatal dopaminergic neural fibers and behavioral performance in Parkinson’s disease (PD) model mice, as well as its possible molecular mechanisms. Methods C57BL/6 adult male mice with 20-25 g at 3-month old (n=90) were successively injected with 6-hydroxydopamine (6-OHDA) to generate a PD animal model. APα and its receptor γ-aminobutyric acid A receptor (GABAAR) antagonist—bicuculline (Bic) were successively injected. ELISA was used to detect the APα or dopamine concentration in the serum, cerebral cortex and striatum. The number of tyrosine hydroxylase (TH) in the substantia nigra (SN) and striatal dopaminergic neural projections were examined by immunohistochemical staining. The expression levels of GABAAR in membrane fractions and Ca²⁺/calmodulin-dependent protein kinase Ⅱ δ3 (CaMKⅡδ3), phosphorylated CaMKⅡδ3 (p-CaMKⅡδ3), brain derived neurotrophic factor (BDNF), and cyclin-dependent kinases 1 (CDK1) were detected by Western blotting in the cytoplasmic or nuclear fractions. The interaction of CaMK Ⅱδ3/p-CaMK Ⅱδ3 with BDNF and CDK1 was verified by immunoprecipitation. In addition, the behavioral changes of animals were recorded. Results There was a significant decrease in the endogenous APα levels of the cerebral cortex, or the dopaminergic neurons of the SN, or the striatal dopaminergic neural fibers, or various protein levels of the membrane, cytoplasmic and nuclear fractions, as well as motor function in PD mice. As compared with PD mice, the abovementioned results were ameliorated in the mice administrated with APα, which were pulled down in mice pre-treated with Bic. The immuno-precipitated result indicated that CaMKⅡδ3 or p-CaMKⅡδ3 interacted with BDNF or CDK1. Conclusion By increasing the endogenous APα levels, exogenous APα treatment can improve SN-striatum dopaminergic neurons in PD model mice by regulating GABAAR/CaMKⅡδ3/BDNF/CDK1 signaling pathway, which in turn promotes behavioral performance in 6-OHDA-lesioned mice.

导出引用

王彤彤陈治池叶鑫边维杜娟娟傅维达陈梦娇李俊楠孙臣友. 别孕烯醇酮对帕金森病模型小鼠黑质多巴胺能神经元的影响及可能的分子机制[J]. 解剖学报. 2020, 51(4): 473-482 https://doi.org/10.16098/j.issn.0529-1356.2020.04.001

WANG Tong-tong CHEN Zhi-chi YE Xin BIAN wei DU Juan-juan FU Wei-da CHEN Meng-jiao LI Jun-nan SUN Chen-you. Allopregnanolone restores dopaminergic neurons and motor performance in Parkinson’s disease mice and its molecular mechanisms [J]. Acta Anatomica Sinica. 2020, 51(4): 473-482 https://doi.org/10.16098/j.issn.0529-1356.2020.04.001

中图分类号： Q189

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