温肾通督方调控MAIs/NgR介导的NC-MG细胞交互效应促进神经再生的机制研究

Based on the theory of “Struggle between vital and evil”, which describes the mechanism of spinal cord injury (SCI) as “kidney deficiency and stasis in Du Channel”, we applied the “Wenshen Tongdu” formula (Jisuikang) in preliminary research to treat SCI. The results showed that it could effectively alleviate local inflammation, improve the microenvironment and up-regulate neuron growth factors, indicating that it could adjust the function of neuron (NC) and microglia/macrophage (MG) and inhibit the myelin associated inhibitors (MAIs). Based on the researches, which show that NgR, receptor of Nogo-66, is a common receptor of MAIs on NC and MG in local injury region, we put forward a hypothesis: the expression level of MAIs/NgR plays an important role in interaction between NC and MG, which may be influenced by “Wenshen Tongdu” formula. This project focuses on the interaction between NC and MG mediated by MAIs/NgR. By establishing rat model of spinal cord injury and co-culture system of NC-MG in vitro, we will observe the effect of MAIs/NgR on the interaction between NC and MG with CRISPR/Cas9 and double immunofluorescence labeling method, thus analyzing the nature of “Struggle between vital and evil” and further revealing the mechanism of “Wenshen Tongdu” formula on neuron regeneration effect.

根据“正邪交争”理论和脊髓损伤（SCI）后“肾督虚损、瘀阻督脉”的病机，运用温肾通督方（脊髓康）治疗SCI，前期发现其可改善局部炎症微循环，促进神经生长因子表达，同时提示可有效抑制髓磷脂相关抑制因子（MAIs），并影响神经元（NC）、小胶质/巨噬细胞（MG）的功能。研究表明Nogo-66受体NgR是MAIs作用于脊髓损伤区NC和MG两种重要细胞的共同受体，基于此，我们认为MAIs/NgR水平对NC、MG的交互作用可能具有重要的调控作用，研究温肾通督方调控SCI后细胞交互作用机制，将对寻找SCI治疗靶标具有积极意义。本项目着眼于MAIs/NgR介导的细胞交互效应，建立大鼠SCI模型及NC、MG体外共培养体系，采用CRISPR/Cas9、免疫荧光双标等技术，观察MAIs/NgR对共存状态下NC、MG交互作用的影响，分析SCI后正邪交争的可能物质基础，进一步揭示温肾通督方促进神经再生的作用机制。

脊髓损伤（Spinal Cord Injury，SCI）后神经元的再生是骨科学及神经科学领域的研究热点和难点。从正邪入手，多靶点整体调控神经再生的中医药被证实有着独特的治疗优势。我们在文献研究基础上，通过多年的临床实践，将SCI后的正邪交争归纳为“肾督虚损，瘀阻督脉，枢机统率失职”， 基于此以“温肾”、“通督”为治则拟定了“扶正祛邪”大法，构建了温肾通督方（脊髓康）。SCI后神经元表面的Nogo-66受体（NgR）表达上调可使神经元“正气”衰退，而激活的巨噬细胞表面的NgR表达上调却可能增强其吞噬“祛邪”的能力，进而改善局部微环境。为了阐明MAIs/NgR水平在SCI后细胞交互影响中的关键作用，并明确温肾通督方对SCI后NC-MG交互作用的影响，本项目从三个方面进行了系统研究，分别是SCI后神经元及小胶质/巨噬细胞表面NgR的表达情况，MAIs/NgR水平对SCI区神经元、小胶质/巨噬细胞功能的影响及脊髓康的干预效应，脊髓康调控MAIs/NgR促进神经元存活及轴突再生的机制。运用CRISPR/Cas9、免疫荧光双标、病理染色、分子互作等技术进行多层次验证，整理、分析数据后得出SCI后神经元及小胶质/巨噬细胞表面NgR的表达增多，且与损伤时间相关；脊髓康可通过抑制MAIs/NgR的表达改善SCI大鼠的运动功能，在28d具有显著差异，而且脊髓康对神经元NgR的表达具有明显抑制作用；NgR是脊髓康作用的重要靶点，脊髓康可抑制神经元NgR的表达及促进小胶质/巨噬细胞NgR的表达，增强细胞间的交互作用而改善共培养环境。综上，MAIs/NgR介导的神经元-小胶质/巨噬细胞交互效应是影响SCI后神经再生的关键，与SCI“肾督虚损、瘀阻督脉”病机高度一致。NgR是SCI后正邪交争的关键靶点，温肾通督方可通过精准调控NgR的表达以扶正祛邪，加强细胞间交互作用，从而改善微环境，促进神经再生。