基于树突状细胞Notch通路调节Th17/Treg轴研究“补肾生髓法”治疗多发性硬化的机制

Multiple sclerosis (MS) is an autoimmune demyelinating disease in the central nervous system, which is an important cause of disability in young adults. MS usually used glucocorticoid treatment but have many side effects. MS has complex etiology. Unbalance of CD4+T cell differentiation, especially Th17/Treg imbalance is an important immunological mechanism of MS. Dendritic cells (DCs) regulation Notch pathway is a key regulator of CD4+T cell differentiation. Traditional Chinese Medicine believes that " kidney deficiency " is the key of the pathogenesis of MS. Our previous study found that "tonifying kidney to promote brain method" can maintained Th17/Treg balance and reduce inflammatory response in MS model. However, whether these effects were regulated by Notch pathway is not clear. For this purpose, this study using immunohistochemistry, flow cytometry, protein level analysis, nucleic acid detection, from animal models in vivo, and cell culture in vitro fully confirmed that "tonifying kidney to promote brain method" can regulate Notch pathway and control Th17/Treg axis balance in MS. This method can be used for the treatment of MS. The aims of this project is provide experimental basis in order to clarify the treatment mechanism of "tonifying kidney to promote brain method", and establish a theoretical basis for clinical application.

多发性硬化 （MS）是一种中枢神经系统自身免疫性脱髓鞘疾病，是导致青年人群劳动能力丧失的重要原因，常用的激素等治疗手段效果欠佳，且有一定副作用。MS的病因复杂，CD4+T细胞的分化偏移尤其是Th17/Treg轴失衡是MS的重要免疫学机制，而树突状细胞调控Notch通路，是调节CD4+T细胞的分化的关键。中医认为“肾虚”是MS发病的基本病机,本课题组前期研究发现“补肾生髓法”具有维持MS模型Th17/Treg轴平衡、减轻炎症反应的功效，但尚未明确这一效应是否基于调控Notch通路实现。为此，本研究拟采用免疫组化、流式细胞检测、蛋白分析、核酸表达等多种方法，从动物模型体内实验、细胞培养体外实验全面论证“补肾生髓法”通过树突状细胞调控Notch通路，调节Th17/Treg轴平衡，可以用于治疗MS，旨在为阐明“补肾生髓法”治疗MS的作用机制提供实验依据，并为临床应用提供理论基础。

多发性硬化 (MS)是一种以脱髓鞘为主要病理特征的中枢神经系统自身免疫性疾病，多为复发-缓解病程，是导致青年人群劳动能力丧失的重要原因，病因尚不明确，常用的激素等治疗手段效果欠佳，且有一定副作用。本课题组在临床研究中发现“补肾生髓法”可以降低MS的复发频率，降低激素副作用的功效。本研究基于临床发现“补肾益髓法”及其代表方剂六味地黄汤的具体作用机制，以常见的MS模型EAE小鼠为体内研究平台，以树突状细胞DCs和Th0细胞培养为体外研究平台，证明六味地黄汤具备DCs的Notch通路，抑制Th0转化为Th17，促进Treg细胞分化，减少促炎因子生成，减轻EAE残疾评分，保护炎症状态下的髓鞘和轴索损伤。研究发现高剂量的六味地黄汤能够显著抑制DCs的Notch通路，调控Th17/Treg轴的分化，下调Th17细胞相关转录因子表达，上调Treg细胞相关转录因子表达，改善EAE模型的残疾评分，减少中枢系统炎症细胞浸润，减轻髓鞘损伤和髓鞘脱失。体外实验发现高浓度六味地黄汤含药血清可以抑制DCs的Notch通路，调节Th0分化，调控Th17/Treg轴，减少致炎因子释放，增高抑炎因子释放，具备MS的治疗作用。在扩展研究中发现六味地黄汤还可以调节小胶质细胞活化，增加抑炎胶质细胞反应，减少促炎胶质细胞活化，促进缓解期髓鞘修复，为临床应用提供进一步使用的依据。