基于外泌体介导OPCs分子信号探讨补肾益髓方促进多发性硬化髓鞘再生的作用机制

Multiple sclerosis (MS) is a common central demyelinating disease. It has great significance to promote remyelination for improving neurologic injury of MS. The inhibitory environment produced due to demyelination limits the recruitment and differentiation of oligodendrocyte precursor cells (OPCs), resulting in difficulty of remyelination. The studies show that a kind of exosomes delivering biological information regulates remyelination by mediating molecular signaling of OPCs. Previous studies had proved that Bushen Yisui formula promoted remyelination of MS. Preliminary findings showed that the formula increased the expression of transmembrane protein of exosomes, and the exosomes treated by the formula had the neuroprotective effect. It is speculated that the action of the formula in promoting remyelination is related with intervening exosomes which mediate OPCs functions, but the molecular mechanism is unclear. Therefore, in this study, focusing on exosomes, the expression of key proteins such as leukemia inhibitory factor (LIF)/ LIF receptor, semaphorin (Sema) 3A / neuropilin (NP) 1, the transcription factor Gtx and microRNA (miR)-219 and other signaling molecules influencing OPCs functions in exosomes obtained from the serum and cerebrospinal fluid of mice with experimental autoimmune encephalomyelitis (EAE), secreted by OPCs with OPCs model in vitro will be measured, by high-throughput analysis techniques of liquid chip and transcriptome sequencing. The mechanisms of the formula on promoting remyelination by intervening exosomes mediating OPCs molecular signaling will be investigated. It will provide new ideas for illustrating the scientific connotation of tonifying kidney and producing marrow in treatment of MS.

多发性硬化（MS）是常见的中枢脱髓鞘疾病。促进髓鞘再生对改善MS神经损伤有重要意义。脱髓鞘产生的抑制环境影响了少突胶质前体细胞（OPCs）募集及分化，导致髓鞘再生困难。研究表明一种携带生物信息的外泌体通过介导OPCs分子信号调控髓鞘再生。前期研究证实补肾益髓方可促进MS髓鞘再生，初步发现本方增加外泌体跨膜蛋白表达，其处理的外泌体有神经保护作用，推测本方促进髓鞘再生与其干预外泌体介导OPCs功能有关，但分子机制尚不清楚。故本课题利用EAE小鼠模型，结合体外OPCs模型，以外泌体为研究重点，采用液相悬浮芯片、转录组测序等高通量分析技术，检测小鼠血清、脑脊液及OPCs分泌的外泌体中影响OPCs功能的关键蛋白LIF/LIFR、Sema3A/NP1、Gtx和miR-219等信号分子的表达，探讨补肾益髓方干预外泌体介导OPCs分子信号促进髓鞘再生的作用机制，为阐释补肾益髓治疗MS的科学内涵提供新思路。

多发性硬化（MS）是常见的中枢神经炎性脱髓鞘疾病。促进髓鞘再生对改善MS神经损伤有重要意义。脱髓鞘产生的抑制环境影响了少突胶质前体细胞（OPCs）募集及分化，导致髓鞘再生困难。研究表明一种携带生物信息的外泌体通过介导OPCs分子信号调控髓鞘再生。前期研究证实补肾益髓方可促进MS髓鞘再生，初步发现本方增加外泌体跨膜蛋白表达，其处理的外泌体有神经保护作用，推测本方促进髓鞘再生与其干预外泌体介导OPCs功能有关，但分子机制尚不清楚。故本课题利用MS的动物模型EAE小鼠，结合体外OPCs模型，以外泌体为研究重点，采用病理切片、透射电镜、免疫荧光、Western blot、RT-PCR、转录组测序高通量分析等技术，检测了小鼠脑组织、血清外泌体中影响OPCs功能的关键蛋白LIF/LIFR、Sema3A/NP1、Gtx和miRs等信号分子的表达，探讨补肾益髓方干预外泌体介导OPCs分子信号促进髓鞘再生的作用机制。结果显示，补肾益髓方能够降低EAE小鼠的神经功能评分，减轻小鼠脑和脊髓内炎细胞浸润和轴突髓鞘脱失；促进NG2、PDGFR-α、MBP、CNPase、GAP-43、KI67、Nestin、BDNF和TrkB等表达，降低PGP9.5、P-tau、NogoA、NgR等表达，以上结果说明补肾益髓方对EAE小鼠和神经细胞具有明显的神经保护作用，并能减轻神经损伤和促进神经修复。进一步研究发现，本方还能够调节小鼠脑组织、血清外泌体中LIF/LIFR、Sema3A/NP1、Gtx蛋白表达，对16个miRs的调节有显著差异。通过qRT-PCR技术验证，其中let-7i-5p、miR-15a-5p、miR-16-5p、miR-98-5p、miR-146b-5p、miR-182-5p、miR-486-5p表达与测序结果一致。KEGG途径和GO生物过程分析结果发现，外泌体中的10DEmiRNAs与调节神经元分化、细胞对生长因子刺激的反应、细胞迁移、GTPase活性和轴突生成有明显的相关性，并丰富和调节了PI3K-AKT、MAPK、Ffox、cGMP-PKG和Wnt信号通路以及轴突引导。体外研究发现补肾益髓方含药血清及其主要成分梓醇也对神经细胞SY5Y具有保护作用，还能够促进OPCs细胞的增殖、迁移和分化。这些结果为阐释补肾益髓治疗MS的科学内涵提供了客观依据。