Penicilazaphilone C作用于调节性T细胞抑制哮喘炎症的分子机制研究

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 Th2 immune response is the major mechanism that contributes to formation of the chronic inflammation of the asthmatic lunges, and that regulatory T cells are major regulator that can reverse the imbalance to the Th2/Th1 immune response. Penicilazaphilone C is a new azaphilone that was firstly isolated by our laboratory from a marine fungus Penicillium sclerotiorum growing in Hainan mangrove environment. Our initial study results showed that treatment of asthmatic model mice with Penicilazaphilone C significantly reduced airway inflammation and significantly decreased airway hyperresponsiveness. Moreover, Penicilazaphilone C treatment can also reversed Th2 to Th1 immune response and increased the number and function of regulatory T cells. These results indicate that Penicilazaphilone C is a potential chemical compound for asthma treatment. To further study the exact anti-asthma molecular mechanism of Penicilazaphilone C, 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 Penicilazaphilone C. In addition, in order to confirm the relationship between Penicilazaphilone C treatment and various immune cells (especially regulatory T cells), we will separate the regulatory T cells and other immune cell types from the spleen, lymphnodes and lung tissues and analyze their numbers, phenotypes and functions by flow cytometry and 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 regulatory T cells cause reversion of Th2 to Th1 immune response after Penicilazaphilone C treatment. Our study results will promote Penicilazaphilone C to be used as a potential anti-asthma drug in the future.

调节性T 细胞(Treg)是控制Th细胞亚群分化的主要效应细胞，参与哮喘发病的调控。我们从海南红树林真菌中分离获得一种新结构活性物质Penicilazaphilone C (PAC)。前期研究发现，PAC能够减轻哮喘小鼠肺部的慢性炎症、逆转Th2/Th1免疫反应、调节Treg的功能。在此前期研究的基础上，我们将进一步建立传统OVA和反复感染合胞病毒二种哮喘模型，用PAC进行治疗并观察治疗效果和副作用。治疗结束时提取脾脏、淋巴结和肺组织分离Treg和其它免疫炎症细胞，采用流式细胞术和免疫学技术检测这些细胞的数量、表型和功能，了解这些细胞与Pc治疗效果之间的关系。最后，利用分子生物学、免疫学、细胞生物学等技术研究PAC通过调节Treg功能治疗哮喘的分子机理，为将PAC作为具有自主知识产权的治疗哮喘新型药物模型分子或先导结构奠定理论基础。本项目研究可能为哮喘提供新的治疗手段，具有良好的应前景。

PenC是本项目负责人团队从生长于海南红树林环境的一株真菌中分离的一种新的化合物。本项目研究结果发现，PenC对巨噬细胞有免疫调节作用，除了诱导巨噬细胞表达NO诱导本酶iNOS进而促使生成和释放NO，也能诱导巨噬细胞表达并大量分泌TNF-α和IL-1β。PenC作于巨噬细胞后能大增强其吞噬异物的活性。PenC激活巨噬细胞的主要通路为NF-κB和MAPK信号通路，这两个通路激活主要通过PenC以配体的形式直接作用于巨噬细胞的的TLR4和Dectin-1受体。PenC通过这两个受体作用进一步激活下游信号分子。在OVA小鼠炎症哮喘模型中，PenC治疗可以下调小鼠的气道高反应性，明显减少支气管灌洗液中的炎症细胞（包括嗜酸性粒细胞）数量，侵润肺组织的炎症细胞（包括嗜酸性粒细胞）也明显减少，气管中的分泌物也比较少，肺组织的病理改变接近正常小鼠的肺组织；本项目采用多种技术手段检测了哮喘炎症发病相关的多种炎症介质，发现PenC治疗小鼠外周血IgE的水平以及分泌IgE的淋巴细胞的数量都明显下降，几乎和正常组小鼠的水平相当；同样，PenC治疗小鼠血中的组织胺浓度和分泌组织胺的淋巴细胞数量也明显减少；我们用WB、ELISA和流式细胞术合并阻断和过表达等技术研究了PenC治疗哮喘的相关机理，发现PenC治疗后Th2细胞及其分泌的细胞因子均明显降低，相反Th1细胞和细胞因子则明显增高，两者下调或增高的水平均几乎和正常小鼠相应细胞的水平相当；我们还发现，外周血炎症细胞和侵润于哮喘肺组织中的淋巴细胞中，经PenC治疗后Treg细胞数量下降，相反Th17细胞数量则上升，PenC治疗小鼠中这两种细胞的数量也几乎接近于正常小鼠的细胞数量；用流式细胞术更进一步检测肺Th17淋巴细胞的表型时发现，PenC治疗小鼠Th17细胞分泌Th2型细胞因子（比如IL－5）明显减少，而Th1型细胞因子（比如TNF-γ）却明显上升。这些结果说明：PenC具有很好的哮喘治疗作用，可以减轻哮喘的慢性炎症，其分子机理是通过改善肺组织的免疫炎症微环境，促使不正常的Th2型免疫反应转变为正常的Th1免疫反应，进而下调IgE、组织胺等免疫炎症介质水平。因此，PenC具有一定的转化应用治疗哮喘的前景。