小胶质细胞TLR4介导自噬稳态和表型转化在脑白质缺血后髓鞘损伤中的作用

Myelin loss, always accompanied with activation of microglia (MG), is the main neuropathologic process of whiter matter ischemia (WMI) and cerebral small vessel disease. Immunologic process mediated by different phenotypes of MG activation is one of the crucial factors for the re-myelination. Toll-like receptor 4 (TLR4) is highly expressed on MG. Activation of TLR4 could result in neuro-inflammation. Our previous study showed that MG was significantly activated and mounting autophagosomes accumulated in MG during WMI. Modulation of MG phenotype towards M2 could ameliorate myelin damage and improve cognitive function after WMI. TLR4 knockout could inhibit MG inflammation and the over-activation of autophagy. Accordingly, we suggest that TLR4 on MG is activated during WMI, resulting in autophagy imbalance and inflammatory phenotype of MG, and finally neuro-inflammation injury. In the present project, we specifically knock out the microglial TLR4 by Cre-loxP system, try to investigate and elucidate the roll of TLR4 mediated MG autophagy after WMI and the potentially molecular mechanisms of MG polarization, which might provide a novel method for the clinical research of the cognitive impairment after WMI.

髓鞘脱失是慢性脑白质缺血(WMI)及脑小血管病的主要病理改变，往往伴随小胶质细胞(MG)的持续活化。不同表型MG介导的炎症微环境改变是影响受损髓鞘修复的重要因素。Toll样受体4(TLR4)高表达于MG上，激活并介导神经炎症级联反应。文献报道，TLR4介导的自噬反应参与调节外周巨噬细胞的功能状态重构，抑制TLR4或自噬可显著减轻巨噬细胞的炎性损伤反应。我们的前期研究发现，WMI后MG被显著激活，自噬小体大量积聚；调控MG表型向M2转化可以减轻WMI后的髓鞘损伤，改善认知功能障碍；TLR4敲除可以抑制MG活化，减轻自噬的过度激活。基于此我们假设，WMI后MG的TLR4被激活，诱导MG自噬失衡并促进MG表型向促炎状态转化，介导神经炎症损伤过程。本研究拟通过选择性基因敲除手段，阐明TLR4调控MG自噬和表型转化的分子机制，以改善髓鞘修复的微环境，为脑白质缺血后认知功能障碍的临床研究提供新的思路。

髓鞘脱失是慢性脑白质缺血（WMI）及脑小血管病的主要病理改变，往往伴随小胶质细胞(MG)的持续活化。不同表型MG介导的炎症微环境改变是影响受损髓鞘修复的重要因素。Toll样受体4（TLR4）高表达于MG上，激活并介导神经炎症级联反应。我们的研究发现，TLR4基因敲除可以诱导小胶质细胞表型向抑炎型转化，减少低灌注引起的髓鞘损伤。慢性脑白质缺血后小鼠胼胝体内小胶质细胞自噬水平明显被激活，呈现自噬应激态，利用自噬抑制剂抑制小鼠自噬水平可以改善脑白质缺血性脱髓鞘损伤。而TLR4基因敲除后自噬相关蛋白表达减少，也可使小胶质细胞自噬水平恢复到稳定的动态平衡。同时，在离体原代培养小胶质细胞经过LPS刺激后，小胶质细胞LC3和beclin-1等自噬相关蛋白上调，而TLR4基因敲除后小胶质细胞的自噬相关蛋白表达减少，与体内一致。进一步我们发现LPS诱导的促炎标志物的上调可被自噬抑制剂（wortmannin和 bafilomycin A1）阻断，而小胶质细胞中抗炎标志物的表达水平未受自噬抑制剂的影响。这些结果表明，自噬参与了小胶质细胞的促炎标志物的激活。同时TLR4敲除可以抑制LPS诱导的自噬水平的上调，给予自噬激动剂rapamycin 后可以逆转TLR4敲除对自噬激活的抑制作用，说明TLR4是自噬诱导的促炎标志物激活的上游通路。进一步我们发现TLR4依赖的自噬可能是通过STAT1/6信号通路介导小胶质细胞的表型转化。本项目的延伸研究还发现低灌注脑白质损伤中存在一类血管周边的小胶质细胞，具有自噬水平明显增高的特点，小胶质细胞自噬是其清除髓鞘碎片的关键通路。本项目的成功开展为临床治疗缺血性脑白质损伤提供了新的干预靶点及理论依据。