基于NRs对海马神经血管单元线粒体自噬的调控作用研究血管性痴呆海马神经元转归及中药干预

Vascular dementia (VD) is the only type of dementia that can be prevented and treated. Outcomes of neurons in hippocampus are considered critical for the rehabilitation of cognitive function, but the exact mechanism is still remained unclear. Mitophagy can maintain the function of neurovascular unit(NVU) and affect the outcomes of neurons. With the support of several National Natural Science Foundation of China and provincial items, we found that: I. The damage of hippocampal neurovascular unit induced by NRs was closely related to the impairment of learning and memory; II. VD is always accompanied by the abnormal expression of NRs, the alteration of morphology in Hippocampal NVU, and the changes of structure and quantity in mitochondrion. Importantly, NRs is closely associated with the mitophagy induced by PINK1/parkin. The project aims to use TEM, FCM, Luminex technology, inhibitor, activator, gene knockout, and gene silence, revealing the mechanism of neuronal outcomes mediated by the mitophagy induced by PINK1/parkin and through the regulation of NRs, which affect the morphology and function of hippocampal NVU in vivo and in vitro. The theory of “invigorating the kidney and enriching essence, promoting blood circulation” is applied to disproof the pathology (Kidney essence deficiency and blood stasis blocking brain) of VD, which providing new ideas and targets for the prevention and treatment of VD.

血管性痴呆（VD）是目前唯一可防治的痴呆类型。海马神经元转归是VD有效防治的关键，然其核心机制如何？目前尚不清楚。线粒体自噬能维持NVU整体功能并对神经元转归产生影响。课题组前期在多项各级课题的支持下发现：VD的发生伴随海马线粒体自噬被抑制及NRs表达异常；NRs与海马NVU整体功能、神经突触可塑性密切相关。而NRs可通过PINK1/parkin信号通路调节线粒体自噬。本项目拟采用透射电镜、高内涵、膜片钳等技术，结合激动剂、阻断剂、基因沉默及过表达等方式，从在体、离体两方面揭示“VD状态下，NRs通过PINK1/parkin信号通路调节线粒体自噬，使海马NVU形态与功能发生改变，进而对其神经元转归产生影响”的分子机制；并用“滋肾填精、活血通络”立法方药干预，反证“肾精阴虚、瘀阻脑络”中医病机。该研究将为VD认知功能恢复提供新的理论依据与防治策略。

本项目拟采用透射电镜、高内涵等技术，结合激动剂、阻断剂、基因沉默等方式，明确了滋肾活血方可通过PINK1/Parkin 信号通路调节海马神经元CA1区线粒体自噬，降低氧化应激及炎症反应，促进神经元再生、抑制神经凋亡、增加神经可塑性的内在分子机制，即滋肾活血方干预后可上调VD大鼠海马区 Parkin、Pink1mRNA表达水平及Parkin、PHB2、Drp1、LC3II蛋白表达水平，下调Mfn1、Mfn2蛋白的表达水平，促进海马CA1区线粒体自噬，进一步影响神经元新生。滋肾活血方可通过抑制MDA、SOD、ROS、SOD、LDH表达，降低氧化应激，提高GAP-43、 SYN、AQP4表达以增加神经可塑性。滋肾活血方通过抑制NLRP3炎性小体通路，降低NLRP3、ASC、caspase-1 表达，减少促炎性因子IL-18、IL-1β的分泌。阐明了VD大鼠海马中R2B对Pink1/Parkin介导的线粒体自噬信号通路的调节作用，并证明滋肾活血方可通过上调NR2B介导Pink1/Parkin信号通路，改善VD大鼠海马神经元氧化应激损伤的分子机制。采用自噬流标记，证明了OGD/R诱导原代海马NVU后线粒体自噬增加并在复氧12小时达到最高点。采用自噬抑制剂和激动剂，证明促进自噬可以对海马神经血管单元起保护作用。采用ShRNA干扰技术证明滋肾活血方对海马NVU的保护作用与PINK1/Parkin通路介导的线粒体自噬有关。采用NR2A和NR2B抑制剂，证明滋肾活血方主要通过NR2B发挥神经保护作用，抑制NR2B可下调PINK1/Parkin通路介导的线粒体自噬。本项目目前共发表论文6篇，其中CSCD论文1篇；另录用CSCD论文 1篇，3篇SCI论文正在投稿中。