HMGB1/TLR3:视神经炎症损伤的新机制

The key role of inflammation in optic nerve injury was triggered by retinal microglia, however the mechanism is umcompletely discovered. In previour project we found that the release of inflammatory factor was downregulated, the "pressure" of optical regenration was lower, the regenerative ability was strenthened, which demonstrated that TRIF was is involved in optical inflammatory injury. TRIF is the downstream adaptor of TLR3 and TLR4. However, the expression of TLR3 was higher that that of TLR4, which reminds us that TLR3/TRIF axis is the main upstream signaling pathway in optic nerve injury, the ligand was undiscovered. In ischemia model, it is showed that HMGB1 was the endogenous ligand which mediate inflammation in central nervous system.The following experiment has demonstrated the hypothesis that HMGB1 activates TLR3 in microglia. In order to deeply invistigate the mechanism of the phenomenone, we plan to explore the onset of HMGB1/TLR3 in optical nerve injury, the time-dependent change of HMGB1 and TLR3, the recovery and regeneration of optic nerve and retina when HMGB1/TLR3 was limited or inhibited. We also plan to discover the downstream and effect of TLR3 signaling when activated by optical injury. The aim of this project is to show the up-stream signaling pathway which induce inflammation in optic nerve injury, and to provide new target of the therapy.

小胶质细胞介导的炎症反应是导致视神经损伤的重要因素之一，但机制不完全清楚。我们前期发现，TRIF敲除鼠视神经损伤减轻、再生增强，提示TRIF直接参与视神经炎症损伤。但视神经损伤后，何种途径导致TRIF激活仍不清楚。已有研究显示，TLR3、TLR4是TRIF上游受体，HMGBs是TLR3和TLR4的共同配体。预实验发现视神经损伤后TLR3表达显著高，而TLR4无明显变化，同时HMGBs中仅有HMGB1明显增高。为此我们提出HMGB1-TLR3/TRIF是引起视神经视伤的上游关键环节的假说。本项目拟进行：1）阐明小胶质细胞HMGB1-TLR3在视神经损伤中的作用及启始机制；2）探讨干预HMGB1-TLR3途径对视神经形态和功能恢复的效果。本研究可望揭示炎症反应导致视神经损伤的上游关键环节，不仅为深入研究视神经损伤的机制奠定基础，而且可望为视神经损伤的治疗提供新靶点，既有理论意义，又有临床价值。

1..结题摘要：视网膜神经节细胞（RGC）死亡是患者视觉丧失的根本原因。胶质细胞介导的炎症反应是导致RGC损伤的重要因素。课题组研究发现，视神经损伤后，小鼠fVEP感光电生理逐渐丧失，FG标记的RGC大量丢失，视神经轴突退变无法再生。在损伤后1-7d，CD11b、TRIF及MyD88的mRNA水平高表达。其中CD11b与TRIF和MyD88呈现相反变化趋势，即CD11b在损伤后1天急剧增高，而后下降；而TRIF和MyD88在损伤后的1-7天逐渐增高。流式细胞学分析显示80%以上的CD11b为Müller细胞所表达，应用免疫组织化学染色对CD11b进行定位，结果显示CD11b主要表达于Müller细胞的RGC侧末端。电镜结果显示Müller细胞出现囊泡样内容物，胞外基质增生。ic3b为补体系统重要分子，是CD11b的特异配体，我们敲除C3后发现CD11b仍高表达，提示CD11b在Müller细胞上受有其他配体激活。RGC死亡过程中释放HMGB1，应用IP及质谱实验研究发现CD11b结合蛋白为HMGB1，若应用NIF抑制CD11b则加剧RGC死亡， 造成Toll信号通路下游MyD88和TRIF活化并引发剧烈炎症反应，而应用LV过表达CD11b或Leukadherin-1激活使CD11b过表达7天则抑制MyD88和TRIF活化，同时降低TNF-α，IL-1β和IFN-β分泌。提示CD11b参与负向调控TLRs信号途径。体外研究证实Müller细胞在NIF处理24h后条件培养基对培养的RGC有细胞毒性作用。RGC轴突在CD11b过表达后的再生距离大于对照组（WT ONI 和LV-GFP ONI）。视神经损伤后，RGC释放的量效和时效为视神经导致的炎症损伤的关键问题。为此本项目进行了：应用分子生物/电镜/流式细胞术等，以小鼠视神经损伤模型研究RGC-Müller神经胶质单元无菌性炎症机制；阐明HMGB1激活CD11b的关键环节；揭示CD11b调控Toll-TRIF/MYD88信号途径的具体机制；干预CD11b达到减轻RGC死亡的效果。本研究可望阐明RGC-Müller神经胶质单元中CD11b负向调控Toll信号的机制，不仅为炎症负反馈提供理论依据，也为神经-胶质研究提供思路。