褪黑素对缺血性卒中白质损伤的保护作用及机制研究

White matter injury（WMI）is an important pathological change that dictates the long-term neurological function deficits in stroke patients. The protection strategies on WMI have recently attracted tremendous attention. Previous studies reported that the melatonin can protect against WMI; howerver, the mechanism remains undefined. In our preliminary studies, we have obtained the exciting results showing that 1) the melatonin strengthened integrity of the WMI and ameliorated long-term motor deficits; 2) the cell culture showed that the melatonin inhibited the PTEN activity in oligodendrocyte (OL), and directed the microglia polarization toward the neuroprotective M2 phenotype. We postulate that the melatonin protects against WMI via 1) promoting Akt activity via inhibiting PTEN activity; 2) anti-inflammatory action via directing the microglia polarization toward the neuroprotective M2 phenotype. In the current proposal, we will further explore the underlying mechanisms in both in vitro and in vivo models of ischemia. The overall hypothesis is that the melatonin alleviates WMI after cerebral ischemia through dual mechanisms: 1) the melatonin directly protects OL by inhibiting PTEN activity and enhancing the activation of Akt survival pathway; 2) the melatonin provides indirect protection to OL through directing the microglia polarization toward the neuroprotective M2 phenotype via activating IL-4/STAT6 pathway. A combination of state-of-the-art approaches including diffusion tensor imaging and adeno-associted virus (AAV)-mediated gene transfection will be used to test this hypothesis. The success of this project will provide a novel therapeutic strategy that targets the WMI after ischemia and leads to improved long-term neurological function in stroke survivors.

白质损伤是导致卒中病人长期神经功能障碍的重要原因，针对白质损伤的保护成为卒中研究的热点。研究显示，褪黑素可保护卒中导致的白质损伤，但机制不清。我们前期工作发现，褪黑素可增加卒中小鼠白质完整性和改善其长期运动功能；细胞实验显示在褪黑素作用下少突胶质细胞（OL）中PTEN活化显著被抑制，小胶质细胞亚型向神经保护性M2型转化。由此推测，褪黑素可能通过两个方面保护白质：通过抑制PTEN激活促进Akt活性，增加OL存活；通过诱导小胶质细胞极化，发挥抗炎作用，保护OL。本研究基于动物和细胞模型，采用弥散张量成像、嵌套式原代细胞共培养、病毒转染等技术，证明褪黑素通过双重机制保护白质：①通过抑制OL中PTEN活性并激活Akt生存信号通路，直接保护OL；②通过激活IL-4/STAT6通路，诱导小胶质细胞亚型由毒性M1型向保护性M2型转化，发挥抗炎作用，间接保护OL。本研究将为卒中长期神经功能恢复提供新思路。

白质损伤是导致卒中病人长期神经功能障碍的重要原因，针对白质损伤的保护成为卒中研究的热点。前期研究显示，褪黑素可保护卒中导致的白质损伤，但机制尚不清楚。本项目基于动物和细胞模型，采用RNA干扰、抑制剂、嵌套式细胞共培养等方法，进一步明确褪黑素能够对脑卒中导致的白质损伤提供保护并能够促进长期神经功能恢复；机制上，一方面褪黑素通过抑制PTEN活性及激活Akt信号通路而发挥直接的神经保护作用；另一方面褪黑素通过激活IL-10/STAT3信号通路，调控小胶质细胞由促炎性表型向抗炎性表型极化，抑制促炎反应，从而促进脑组织结构和功能修复。此外，项目实施过程中也取得以下新的研究发现：1）发现褪黑素能够促进脊髓损伤后神经功能恢复，诱导小胶质细胞向抗炎性表型极化，其中促炎性表型的小胶质细胞标志物mRNA水平与神经功能恢复效果呈负相关性，而抗炎性表型的小胶质细胞标志物mRNA水平与其呈正相关性，说明对小胶质细胞表型极化调控可能是褪黑素减轻脊髓损伤的重要机制之一。2）阐释了抑制TLR4/NF-κB信号通路可调控小胶质细胞表型由促炎性型向抗炎性转化，减少促炎性炎症反应，是坎地沙坦间接抵抗缺血性脑损伤重要的分子机制；3）揭示姜黄素治疗能够调控小胶质细胞表型由促炎性向抗炎性转化，抑制脑内促炎反应，增加抗炎反应，从而促进脑卒中后长期感觉运动功能的恢复。本项目的研究结果将为脑卒中神经功能恢复治疗提供新思路。