红树林杂色曲霉EPS调控CD4+ T 细胞亚群网络治疗哮喘的分子机制研究

Bronchial asthma is a common respiratory disease that is lack of specific therapeutic drugs in clinic and has became a challenge for research and various burdens to the asthmatic patients. At present, it is well known that bias to the Th2 immune response is the major mechanism that contributes to formation of the chronic inflammation of the asthmatic lunges. Thus, reversion of Th2 bias to Th2 is an important strategy for effective asthma treatment. Various study results have demonstrated that imbalance of CD4+ T cell subtype networks, such as Th1, Th2, Th17 and Treg, is presented in the asthmatic patients and contributed to asthma pathogenesis, and that rebalance Th2 immune response to Th1 response shows a promising approach for asthma treatment. Recently, we have isolated two newly reported Paecilomyces lilacinus　PH106 and PH108 from mangrove environment in Hainan and our initial study results showed that treatment of asthmatic model mice with the exopolysaccharide (EPS) from strain PH108 significantly reduced airway inflammation and significantly decreased airway hyperresponsiveness. Moreover, EPS treatment can also reversed Th2 to Th1 immune response and regulated the function of Th17 and Treg. These results indicate that EPS is a potential chemical compound for asthma treatment. To further study the exact anti-asthma molecular mechanism of EPS, in this study, we will establish conventional OVA-induced mouse asthma model and respiratory syncytial virus (RSV) repeatedly infection asthma model to further systematically study the anti-asthma effects and possible side effects induced by EPS. In addition, in order to confirm the relationship between EPS treatment and various CD4+ cell subtypes (especially Th17 and Treg), we will separate Th17, Treg and other immune cell types from the spleen, lymphnodes and lung tissues and analyze their numbers, phenotypes and functions by flow cytometry and other immunological methods. At last, we will use various methods, including molecular biology, immunology and cytobiology, to further study the molecular mechanisms related to why the rebalance of CD4+ cell subtypes (especially Th17 and Treg) cause reversion of Th2 to Th1 immune response after EPS treatment. Our study results will promote EPS to be used as a potential anti-asthma drug in the future.

CD4+T细胞亚群网络失衡是哮喘发病的重要因素，恢复该网络的平衡是缓解哮喘病情的重要手段。研究表明，多糖是良好的调节CD4+网络细胞（包括Th1、Th2、Th17和Treg）的调节剂。申请人团队从海南红树林中分离获得一株未经报道的杂色曲霉真菌，前期研究发现，杂色曲霉胞外多糖(EPS)能够明显减轻哮喘模型小鼠肺部的慢性炎症，逆转Th2/Th1免疫反应，能够调节Th17和Treg的功能，说明杂色曲霉EPS 能够调控CD4+T 细胞亚群网络的平衡。为进一步探讨EPS 调节CD4+T细胞网络的作用机制，我们将采用动物模型了解EPS治疗的效果，同时用流式细胞术、免疫学、分子生物学等生物医学技术在细胞和分子水平上检测相关细胞因子、转录因子等的水平及相应信号转导通路，争取揭开杂色曲霉EPS对CD4+T 细胞亚群网络失衡调节治疗哮喘的分子机制，为开发哮喘治疗的新型药物提供理论依据。

EPS是我们从海南红树林环境真菌中分离的一种胞外多糖，前期研究发现EPS具有明显抗哮喘炎症的作用。体外实验发现，EPS可促进巨噬细胞释放NO并表达其诱导酶iNOS，也能提高巨噬细胞中TNF-α和IL-1β含量。EPS处理后能增强巨噬细胞的吞噬能力。我们还发现NF-κB和MAPK信号通路参与了EPS介导的巨噬细胞的活化，其中TLR4和Dectin-1受体是EPS的受体，EPS通过这两个受体激活巨噬细胞。在OVA小鼠模型中，发现EPS治疗后，小鼠气道高反应性明显下降，支气管灌洗液中的炎症细胞（包括嗜酸性粒细胞）明显减少，肺组织未见明显的炎症细胞（包括嗜酸性粒细胞）侵润，气管中也没有明显的分泌物，接近正常小鼠肺组织的病理改变；对哮喘炎症发病的主要介质IgE、组织胺等进行了检测。用多种技术方法检测发现EPS治疗可以明显下调外周血IgE的水平，分泌IgE的淋巴细胞数量也明显下降，接近于正常组小鼠的水平；用类似的方法检测组织胺的浓度，也发现有相似的结果；我们用ELISA和流式细胞术等技术对EPS抗哮喘炎症作用机理进行了研究，发现EPS治疗后Th2细胞因子分泌明显下调，但Th1细胞因子则明显上升，上升水平接近正常小鼠白细胞的分泌水平；在检测外周血白细胞和肺组织侵润淋巴细胞中的Treg和Th17细胞数量时发现，EPS治疗后外周血Treg细胞数量明显减少，Th17细胞数量则明显增多并接近正常细胞；流式细胞术检测肺组织中的Th17淋巴细胞的表型，发现EPS治疗后的Th17细胞分泌IL-5的细胞数量明显减少，而分泌IFN-γ的细胞数量则明显增加。这些结果说明：EPS具有明显抑制哮喘炎症的治疗作用，其机理是通过改善肺组织的免疫微环境，促使哮喘的Th2型免疫反应转变为正常的Th1免疫反应，从而下调IgE、组织胺等免疫炎症介质的水平；也说明EPS具有转化应用的前景。