星形胶质细胞程序性坏死维持脊髓损伤后慢性炎症的作用及机制

There still lacks effective treatment for spinal cord injury (SCI) currently, partially because the mechanism of pathology of SCI is not very clear. Unlike peripheral injury, the innate immune response after SCI is characterized by M1-type macrophages/microglia-mediated chronic inflammation, but the mechanism of its maintenance remains unknown. We have demonstrated that reactive astrocytes die through RIPK3- and MLKL-mediated necroptosis after SCI. We further found that necroptosis of astrocytes polarized macrophages to M1 phenotype in vitro, while knockout of RIPK3 reduced the expression of pro-inflammatory factors after SCI. Therefore, we hypothesize that necroptosis of astrocytes may be involved in maintaining M1-type macrophages/microglia-mediated chronic inflammation after SCI. In this project, we will testify this hypothesis from aspect of damage associated molecular pattern (DAMP) released by necroptotic astrocytes, like HMGB1 and mitochondria, through application of multiple methods, including cell culture, morphology, molecular biology, genetics and animal behavior. The project could reveal the role and mechanism of necroptosis of astrocytes in chronic inflammation after SCI and provide new insights into treatment for SCI.

脊髓损伤（SCI）目前仍缺乏有效的治疗手段，部分原因在于对SCI后病理机制的认识不够充分。与外周不同，SCI后的固有免疫反应以M1型巨噬/小胶质细胞介导的慢性持续性炎症为特征，但其维持机制尚不清楚。申请人前期研究发现：SCI后星形胶质细胞发生RIPK3/MLKL介导的慢性程序性坏死（necroptosis）；体外坏死的星形胶质细胞可诱导巨噬细胞向M1表型极化；体内敲除RIPK3可减轻SCI后的M1型炎症。据此，我们推测星形胶质细胞的necroptosis是维持SCI后M1型巨噬/小胶质细胞介导的慢性炎症的重要因素之一。本课题拟采用细胞培养、形态学、分子生物学、遗传学及动物行为学等方法，从坏死星形胶质细胞释放损伤相关分子模式（HMGB1及游离线粒体）的角度验证上述假设，阐明星形胶质细胞necroptosis维持SCI后慢性炎症的机制，以期为SCI的修复治疗提供新思路。

脊髓损伤（SCI）给个人、家庭和社会带来沉重负担，但目前仍缺乏有效的治疗措施，其有效救治已成为急需突破的领域，而明确SCI后病理机制是特异性治疗的前提。SCI后，以M1型巨噬/小胶质细胞介导的促炎免疫贯穿整个病程，其是SCI后修复困难的重要原因之一，但维持机制尚不十分明确。.本项目从星形胶质细胞necroptosis的角度，以其坏死后释放的高迁移率族蛋白1（HMGB1）为切入点，研究其在SCI后巨噬/小胶质细胞M1表型极化，及巨噬/小胶质细胞表型调控在损伤后免疫微环境调节、功能恢复中的潜在价值。我们目前的研究结果显示：SCI后，发生necroptosis的星形胶质细胞可释放HMGB1，且外源性HMGB1可诱导体外培养的小胶质细胞向M1型表型极化，利用HMGB1特异性受体抑制剂证实RAGE/NF-κB通路介导了其M1表型极化机制。更重要的是，体内SCI后给予HMGB1抑制剂或其受体抑制剂均可减少M1型巨噬/小胶质细胞数量及其介导的促炎性免疫，而改善的免疫微环境可促进损伤区周围神经元及少突胶质细胞存活，且能改善SCI后动物行为学恢复，论文发表在Journal of Neuroinflammation（2020，17（1）:295. IF:5.79）。另外，我们发现天然药物Quercetin可调控SCI后巨噬/小胶质细胞极化，减轻其向M1表型极化及其介导的促炎性免疫反应，进而抑制炎症诱导的少突胶质细胞死亡，改善SCI后脱髓鞘病变、促进动物运动功能恢复，论文发表在Journal of Neuroinflammation（2019，16（1）: 1-15. IF:5.70）。因此，本项目的研究阐明了SCI后M1型巨噬/小胶质细胞极化机制及其在功能恢复中的价值，为基于调控necroptosis及SCI后促炎免疫，促进运动功能恢复的修复策略提供了的实验依据。