补肾配伍化痰活血方药对多发性硬化小鼠轴突再生修复微环境的调控机制研究

The axonal damage in central nervous system is an important reason of the progressive neurologic dysfunction in multiple sclerosis (MS) patients. Therefore, it is important to promote the regeneration and repair of damaged axons. For the basic pathogenesis of kidney deficiency accompanying with phlegm and stasis in MS, Erhuang capsule was developed with the role of tonifying kidney combining with resolving phlegm and activating stasis. This formula had efficacy significantly in treatment of MS clinically. The experimental study has found that this formula can reduce axonal injury and promote axon regeneration and repair in experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. Its role was related with increasing of the neurotrophic factor of BDNF and reducing of inhibitory factor of Nogo A, suggesting that this formula can improve axonal microenvironment to promote axonal regeneration and repair, but the regulatory pathway is unclear..In this study, the integrative and collaborative mechanisms and their differences in improving the microenvironment of axonal regeneration and repair on the formulas of tonifying kidney combining with resolving phlegm and activating stasis, tonifying kidney and resolving phlegm and activating stasis will be explored focusing on the signaling pathways of neurotrophic factor of Trk/PI3K/PLCγ and inhibitory factor of RhoA/ROCK in the axonal microenvironment, used by the mice with EAE, and SY5Y cells model, by the technology of immunofluorescence, molecular biology and cell culture, taking Erhuang capsule as a carrier. It will provide strong theoretical and experimental evidences by the therapeutic methods of tonifying kidney combining with resolving phlegm and activating stasis based on the syndrome differentiation treatment of MS.

中枢神经轴突的损伤是多发性硬化（MS）患者神经功能障碍进行性加重的重要原因，促进损伤轴突的再生修复具有重要意义。我们针对MS肾虚痰瘀的基本病机，研制了具有补肾化痰活血作用的二黄胶囊，在临床治疗MS中取得较好疗效。实验研究发现本方能够减轻MS动物模型-实验性自身免疫性脑脊髓炎（EAE）轴突损伤及促进轴突的再生修复，其作用与上调神经营养因子BDNF及降低抑制因子Nogo A有关，提示本方能够改善轴突微环境以促进轴突再生修复，但其调节通路还不清楚。因此，本课题采用EAE小鼠模型及SY5Y细胞模型，利用免疫荧光、分子生物学、细胞培养等技术，以神经营养因子Trk/PI3K/PLCγ信号通路和抑制因子RhoA/ROCK信号通路为研究重点，以二黄胶囊为载体，探讨补肾化痰活血、补肾、化痰活血方药调控上述通路，改善MS轴突微环境的整合、协同作用机制及其差异性，为补肾化痰活血法辨证治疗MS提供理论和实验依据

多发性硬化（multiple sclerosis, MS）是一种中枢神经系统（central nervous system, CNS）自身免疫反应介导的炎性神经退行性病变。中枢神经损伤是MS患者神经功能障碍进行性加重的重要原因，促进神经再生修复具有重要意义。研究发现，神经再生微环境遭到破坏后主要有神经营养因子缺乏和抑制因子生成增多，从而神经生长和再生能力受限。因此，促进内源性修复机制活化，调控轴突神经营养因子和抑制因子及其相关信号通路，促进神经再生，是解决MS中枢神经损伤修复的关键问题。.我们针对MS肾虚痰瘀的基本病机，研制了具有补肾化痰活血作用的补肾益髓（旧称二黄）胶囊，在临床治疗MS中取得较好疗效。实验研究发现本方能够减轻MS动物模型实验性自身免疫性脑脊髓炎（EAE）轴突损伤及促进轴突的再生修复，但其调节通路还不清楚。因此，本课题采用EAE小鼠模型及神经细胞损伤模型，利用免疫荧光、分子生物学、细胞培养等技术，以神经营养因子PI3K/Trk/PLCγ信号通路和抑制因子RhoA/ROCK信号通路为研究重点，以补肾益髓胶囊为载体，探讨补肾配伍化痰活血方药调控上述通路，改善MS轴突微环境的整合、协同作用机制及其差异性，为补肾化痰活血法辨证治疗MS提供理论和实验依据。本课题通过整体动物和细胞实验的研究发现，补肾益髓方及其拆方补肾和化痰活血方对神经细胞损伤模型和EAE小鼠具有明显的神经保护作用，均能够改善EAE小鼠的病理变化，降低神经功能评分，升高损伤的神经细胞的生存率，下调APP、p-Tau以减轻神经损伤，上调MAP-2、NF200、GAP-43以促进神经修复。其机制主要与上调神经营养因子BDNF/TrkB及其信号通路PI3K/Akt，下调抑制因子NogoA/NgR及其信号通路RhoA/ROCK有关。补肾益髓方在神经保护及调节上述神经再生信号通路方面优势明显。补肾方与补肾益髓方作用相似，但化痰活血方不及前二者。这些结果为阐明肾藏精主骨生髓理论及补肾化痰活血法的科学内涵，也为创制新药提供了实验依据。