CD73在小鼠脊髓损伤后巨噬细胞向M2型极化中的作用及其机制研究

Various signal microenvironments in the injured area after spinal cord injury can polarize macrophages / microglia, in which M1-polarized macrophages release pro-inflammatory cytokines, aggravate spinal cord injury, and M2-type polarized macrophages promote axonal growth and repair spinal cord injury. In vitro studies have shown that adenosine can promote macrophage / microglia to M2-type polarization through the A2A and A2B receptors, where CD73 is the rate of adenosine that breaks down extracellular adenosine (ATP, ADP) Enzymes, Our previous studies showed that increased inflammatory response and apoptosis, and poor recovery of neurological dysfunction after spinal cord injury were found after the spinal cord injury in CD73 knockout mouse model. At the same time, the deletion of CD73 gene can reduce the trend of macrophages / microglia to M2 polarization. Therefore, we hypothesize that 1) CD73 plays a role in regulating the polarization of macrophages / microglia to M2 in spinal cord injury and 2) CD73 positively regulates adenosine-A2A receptor-p38 pathway by promoting adenosine synthesis to promote M2 type polarization of macrophages / microglia. This topic provides a new strategy and experimental basis for the clinical treatment of spinal cord injury by preventing secondary damage caused by inflammatory reaction and promoting nerve repair after spinal cord injury.

脊髓损伤后损伤区的各种信号微环境可使巨噬/小胶质细胞发生极化，其中M1型极化的巨噬细胞能释放促炎症细胞因子，加重脊髓损伤，M2型极化的巨噬细胞具有促进轴突生长作用，能修复脊髓损伤。有体外研究表明，腺苷可通过A2A和A2B受体促进巨噬/小胶质细胞向M2型极化，其中CD73是将胞外腺嘌呤核苷(ATP、ADP)分解产生腺苷的限速酶，我们的前期研究表明，CD73基因敲除小鼠脊髓损伤造模后小鼠脊髓炎症反应加剧，凋亡增加，神经功能障碍恢复较差。同时CD73基因的缺失能够减少巨噬/小胶质细胞向M2型极化的趋势。因此我们提出假设：1）CD73在脊髓损伤中具有调节巨噬/小胶质细胞向M2型极化的作用；2）CD73通过促进腺苷合成，正向调节腺苷-A2A受体-p38通路来促进巨噬/小胶质细胞的M2型极化。本课题为有效防止脊髓损伤后炎症反应造成的二次损伤，并同时促进神经的修复，临床上治疗脊髓损伤提供新的实验依据。

脊髓损伤后的病理过程可划分为原发性损伤和继发性损伤两个阶段。前者很难干预和逆转，而由前者引起的继发性损伤，其损伤范围及严重程度会远大于原发性损伤，其中炎症反应是继发性脊髓损伤过程种最重要的病理过程，损伤区多细胞、多系统、多机制参与炎症、免疫、化学反应,导致外周血源性巨噬细胞和脊髓损伤处小胶质细胞的激活、积聚、吞噬坏死组织碎片、分泌炎性细胞因子、生长及神经营养因子，促进髓鞘及轴突再生，导致神经元细胞死亡等，最终导致脊髓损伤加重。作为主要的参与者，巨噬/小胶质细胞会发生极化，其中M1型极化的巨噬细胞能释放促炎症细胞因子，加重脊髓损伤，M2型极化的巨噬细胞具有促进轴突生长作用，能修复脊髓损伤。既往体外研究表明，腺苷可通过A2A和A2B受体促进巨噬/小胶质细胞向M2型极化，其中CD73是将胞外腺嘌呤核苷(ATP、ADP)分解产生腺苷的限速酶，CD73基因敲除小鼠脊髓损伤造模后小鼠脊髓炎症反应加剧，凋亡增加，神经功能障碍恢复较差。同时CD73基因的缺失能够减少巨噬/小胶质细胞向M2型极化的趋势。此外，体外实验还发现小鼠脊髓损伤后脊髓组织中构成炎症小体的NLRP3、ASC、Caspase-1表达增高，而炎症小体通常是由Nod样受体、适配器蛋白和pro-caspase-1所构成的小分子多蛋白复合物。经典的炎症小体形成可促进细胞内caspase-1的剪切活化，是细胞焦亡起始过程调节的关键环节。基于上述文献资料及前期结果，本项目深入探究了继发性脊髓损伤中的炎症反应机制，通过一系列严谨的体内外实验证实了：.1）CD73在脊髓损伤中具有调节巨噬/小胶质细胞极化的作用；.2）CD73正向调节腺苷-A2B受体、p38通路促进小胶质细胞向M2极化；.3）小胶质细胞焦亡是脊髓损伤后炎症反应的重要组成部分；.4）CD73通过PI3K/AKT/Foxo1信号通路缓解GSDMD介导的脊髓损伤小胶质细胞焦亡；.5）TLR4在脊髓损伤后通过激活PI3K/AKT通路lncRNAF630028O10Rik促进小胶质细胞焦亡；.6）CD73通过促进Ado/cAMP/PKA/CREB通路抑制神经元凋亡。.本课题为有效防止脊髓损伤后炎症反应造成的二次损伤、促进神经的修复提供了新的实验依据。