脑内小胶质细胞P2X7受体介导HIV相关神经变性病变及柚皮苷作用研究

The development of human immunodeficiency virus (HIV) related dementia (HIV associated dementia, HAD) is associated with the severity of immunosuppression. The brain macrophages and microglia cells (macrophages and microglia, M/M) play an important role in HAD through secreting the substance of immune and inflammation. The excessive activation of microglial cells is a major reason for the development of nerve inflammation in the neurodegenerative diseases. ATP as a signal molecule of adjusting microglia function can activate P2X7 receptor on microglia. P2X7 receptors play an important role in the regulation of microglia function, and involved in inflammation and immune response and also closely related with neurodegenerative diseases. Therefore, P2X7 receptor may become the new targets for the prevention and treatment of HAD. Naringin is a kind of natural flavonoids, which has anti-inflammatory properties, suggesting it may inhibit P2X7 receptor, thus play antagonistic action on HAD. In this project, we will mainly combine the experiment of systemic level in rat model of cognitive impairment on HAD with the experiment of cellular level in cell damage model induced by HIV-1 capsule glycoprotein gp120, to observe the pathophysiological roles of P2X7 receptors on rat hippocampal microglia in the neuropathic degenerative diseases caused by HIV-1 infection. The study will make sure the effects of naringin on HAD mediated by P2X7 receptor of hippocampal microglia and its possible mechanism, so that we may look for a new way for prevention and treatment of HAD.

人类免疫缺陷病毒相关痴呆(HAD)的病情与免疫抑制的程度有关。通过免疫、炎症物质的分泌，脑内巨噬细胞和小胶质细胞在HAD中起重要的作用。在神经退行性疾病中，小胶质细胞的过度激活是产生神经炎症的最重要原因。ATP作为小胶质细胞功能的信号分子可激活P2X7受体。P2X7受体参与调节小胶质细胞功能活动，加之P2X7受体涉及炎症与免疫反应，并与神经退行性疾病密切相关。因此，P2X7受体可能成为HAD防治的新靶点。柚皮苷是一类天然黄酮类，具有抗炎作用的特性，提示柚皮苷可能抑制P2X7受体，进而产生抗HAD作用。本项目将HAD的认知障碍大鼠模型的整体水平研究与HIV-1包膜糖蛋白gp120细胞损伤模型的细胞水平研究结合，观察大鼠海马小胶质细胞P2X7受体在HIV-1感染所致神经退行性病变中的病理生理作用，确定柚皮苷对大鼠海马小胶质细胞P2X7受体介导HAD的作用及其可能机理，为HAD的防治探寻新方式。

该项目旨在通过建立艾滋病-痴呆复合征（ADC）的认知障碍大鼠模型和HIV-1包膜糖蛋白gp120细胞损伤模型，观察全身或局部应用柚皮苷对大鼠海马小胶质细胞P2X7受体介导gp120所致ADC的作用及可能机理，探寻ADC防治的新方式。.在整体水平上观察了全身应用柚皮苷对大鼠海马小胶质细胞P2X7受体介导gp120所致大鼠学习记忆障碍的影响。重复测量方差分析显示，不同浓度gp120组大鼠逃避潜伏期和寻找目标错误次数具有时间效应性，随着水迷宫实验天数的增加，各组大鼠的逃避潜伏期逐步缩短以及寻找目标平台的错误次数逐渐减少；同时时间与gp120因素之间存在交互效应；单因素方差分析显示，各组大鼠逃避潜伏期和出现错误次数均存在组间差别，不同浓度gp120组大鼠逃避潜伏期和寻找目标象限的错误次数与对照组比均显著增多，说明侧脑室灌注gp120能引起大鼠认知障碍，拟艾滋痴呆症动物模型造模成功。与对照组比较，不同剂量gp120组大鼠海马P2X7受体蛋白和mRNA的表达均有所上升，表明P2X7受体参与gp120诱导大鼠认知障碍。各剂量柚皮苷治疗组大鼠的逃避潜伏期和寻找目标平台的错误次数与gp120模型组相比均有所缩短，说明柚皮苷对gp120所致大鼠认知障碍有改善作用，同时各剂量柚皮苷治疗组P2X7受体蛋白和mRNA的表达与模型组相比均有所下降，说明柚皮苷可抑制P2X7受体介导的gp120所导致大鼠认知障碍。结果显示，gp120组大鼠海马P65蛋白的表达比对照组高，表明P65蛋白参与柚皮苷改善P2X7受体介导gp120所致大鼠认知功能障碍的病理过程。.细胞水平上发现，gp120对BV2小胶质细胞和原代培养小胶质细胞均具有损伤作用，柚皮苷对其损伤具有保护作用；分析显示，与Ctrl组相比，gp120可诱导细胞生成TNFα、IL-1β等炎性因子及NO、ROS等过氧化物，且柚皮苷对以上指标均具有抑制作用；同时，柚皮苷也能抑制gp120诱导的小胶质细胞P2X7 mRNA和蛋白表达上调效应，且细胞内P65 NFκB的表达与之类似。.以上研究结果表明，海马小胶质细胞P2X7受体参与gp120所致大鼠认知障碍和小胶质细胞损伤，柚皮苷对其具有一定的改善作用，为ADC发病机理和防治提供理论依据和新途径。