MicroRNA-155介导黄芪多糖调控EAE小鼠神经小胶质细胞M1/M2极化表型平衡的分子机制研究

Multiple sclerosis (MS) is one of the chronic diseases of the central nervous system frequently happened in young people mediated by autoimmune reaction, which hardly threatens human health. The rate of incidence is rising year by year. So far, the pathogenesis has not been understood. It lacks of effective treatment in clinic. This project is based on the previous research achievements in our laboratory. We found that Astragalus polysaccharides (APS) can effectively suppress experimental autoimmune encephalomyelitis (EAE), the animal model of MS. And the mechanisms are related to its role in regulating microglia activation in EAE model. Recently, more and more studies have been shown that the balance of microglia M1/M2 polarized phenotype plays an important role in the development and the pathogenesis of MS/EAE. MicroRNA-155 (miR-155), as an epigenetic regulator, is a key molecule that regulates the balance of microglia M1/M2 polarized phenotype in MS/EAE. So, in this study we will investigate the potential molecular mechanisms of APS in regulating the balance of microglia M1/M2 polarized phenotype in EAE mice. Using the theories and methods of modern immunology and molecular biology, the influence of APS on the expression of the surface markers of both M1 and M2 microglia will be observed in vivo and in vitro. Furthermore, the effects of APS on the expression of miR-155 in M1 and M2 microglia will be also determined. After transferring the miR-155 mimic and the miR-155 inhibitor to microglia in culture system, the influence of APS on the balance of microglia M1/M2 polarized phenotype will be observed. The molecular mechanisms of the regulatory effects of APS in treating EAE are deeply investigated from the cellular, gene, and protein level both in vivo and in vitro. The biological mechanisms of the treatment to multiple sclerosis by traditional Chinese medicine will be further clarified. It will provide the evidence for the potential effective therapy by traditional Chinese medicine in autoimmune diseases in the central nervous system. There is not any related research report published up to now. As a further study and continuation of our previous work, it is actually a research project of innovation and multidisciplines. It has important scientific significance to the clinical treatment of neurological diseases and immunological disorders. It is also much helpful to the drug development and the translational research of traditional Chinese medicine. Therefore, this project would greatly promote the modernization and internationalization of traditional Chinese medicine.

多发性硬化MS是中枢神经系统自身免疫病，严重危害人类健康，发病机制尚不清楚，临床缺乏有效的治疗方法。本研究团队在前期研究中发现，黄芪多糖对MS动物模型--实验性自身免疫性脑脊髓膜炎EAE具有显著治疗作用，能有效调控神经小胶质细胞活化。在此基础上，本项目进一步探讨黄芪多糖对EAE小鼠神经小胶质细胞M1/M2极化表型平衡的调控，拟运用分子生物学和免疫学理论和方法，在体内和体外分别观察黄芪多糖对神经小胶质细胞M1/M2极化表型标志分子表达的影响；采用细胞转染技术和阻断实验，以表观遗传分子miR-155为靶点，从细胞、分子及整体水平，探讨黄芪多糖对神经小胶质细胞M1/M2极化表型平衡的调控及其分子机制，藉以阐明黄芪多糖治疗中枢神经系统自身免疫病的新靶点。本项目是前期工作的延续和深入，具有鲜明的创新性，至今尚未见报道，对于神经系统自身免疫病的治疗和新药研发、促进中医药的转化研究，具有重要的科学意义。

多发性硬化（Multiple sclerosis，MS）是中枢神经系统(CNS)常见的炎性脱髓鞘疾病，缺乏有效治疗方法。小胶质细胞在疾病状态下呈现两种不同的细胞亚群：即经典活化的M1型与交替活化的M2型。M1型小胶质细胞产生多种促炎细胞因子，参与CNS神经功能障碍，M2型小胶质细胞产生抗炎细胞因子，参与抑制CNS神经炎症。因此，抑制小胶质细胞M1型炎性状态，促进其向M2表型的转变，已成为治疗神经炎症相关疾病的有效策略。黄芪多糖（APS）具有免疫调节等作用，本研究前期实验表明，APS可抑制小胶质细胞活化介导的神经炎症。因此，在前期的研究基础上，本项目进一步探究APS对小胶质细胞M1/M2极化表型的调控作用及其分子机制。首先，通过体内实验观察APS对MS动物模型EAE小鼠小胶质细胞M1型标记分子iNOS、IL-12，M2型标记分子Arg1、CD206的影响，以及对miRNA-155表达的影响。在体外实验中，采用脂多糖（LPS）诱导BV-2小胶质细胞M1极化模型，观察APS干预前后对M1、M2型标记分子的影响，以及对miRNA-155表达的影响。此外，将BV-2小胶质细胞分别过表达、抑制miRNA-155表达后，检测APS对小胶质细胞M1、M2极化表型标记分子表达的影响。结果：体内实验显示，APS可抑制EAE小鼠症状评分。APS可抑制EAE小鼠M1型标记分子的表达，促进M2型标记分子的表达。APS可抑制EAE小鼠中枢神经系统miRNA-155的表达。在体外实验中，APS可抑制BV-2小胶质细胞M1型标记分子的表达，促进M2型标记分子的表达。同时，发现APS可抑制LPS诱导的小胶质细胞中miRNA-155的表达。进一步通过miRNA-155细胞转染高表达和阻断实验，发现APS对小胶质细胞M1/M2极化表型的调控可能与抑制miRNA-155的表达有关。本研究综合了免疫学、分子生物学、表观遗传学等研究方法，从分子、细胞、动物等多水平探讨了黄芪多糖对小胶质细胞M1/M2极化表型的调控作用及机制研究，为APS作为治疗中枢神经系统神经炎症疾病的有效药物提供了新的证据，并提供了新的作用途径与治疗策略。