NLRP3炎性体在MPP不同病变阶段的表达及清热止咳方药的干预作用

Mycoplasma pneumoniae（MP） is an atypical bacterial pathogen without a cell wall. MP causes MPP and outside the lung symptoms, and is closely related to asthma, pulmonary fibrosis pathogenesis.NLRP3 is one of the NOD like receptors NLRP subfamily members. NLRP3 inflammasome play an important role in detecting and clearing of invading pathogens as well as intiating the innate immune responses. Activation or dysfunction of NLRP3 inflammasome is closely related with a variety of infectious pneumonia. Whether or not mycoplasma pneumoniae could activate the NLRP3 inflammasome has not yet been reported in the literature. Therefore, the expression of NLRP3 inflammasome in MPP different pathological stages is expected to become a new way to study drug for treatment of MPP.TCM formulas for clearing-heat and relieving-cough is a kind of compound traditional Chinese herb which can treat MPP. Multicenter trial study showed that total efficiency of TCM formulas for clearing-heat and relieving-cough which treated in mycoplasma pneumoniae pneumonia is better than that of azithromycin. The project, based on our previous research, was aimed to investigate ROS,the NLRP3 inflammasome and its down signaling proinflammatory cytokines of dynamic change in whole animal and cell levels and also to investigate the intervention action of TCM formulas for clearing-heat and relieving-cough in MPP different pathology stages by using the techologies of RT-PCR and immunoblotting, which will provide new ideas for the development of Chinese Traditional Medicine with clear mechanisms of action against MPP.

肺炎支原体（MP）是一类无细胞壁结构的非典型性细菌，可引起肺炎及肺外症状，与哮喘、肺纤维化等疾病的发病关系密切。NLRP3是NOD样受体NLRP亚家族成员之一，NLRP3炎性体在识别和清除入侵病原体和激活机体固有免疫反应中发挥重要作用，其激活与失调与多种感染性肺炎关系密切。MP是否可以激活NLRP3炎性体？激活途径是否与ROS有关，尚未见文献报道。因此研究NLRP3炎性体在肺炎支原体肺炎（MPP）不同病变阶段的表达有望成为研究治疗MPP药物的新途径。清肺止咳方药是治疗MPP的中药复方，多中心临床试验研究表明其治疗MPP的痊愈率和总显效率优于阿奇霉素。本项目旨在前期研究的基础上，采用RT-PCR、免疫印迹等技术，在整体动物和细胞水平深入探讨ROS、NLRP3炎性体及下游促炎症细胞因子在MPP不同病变阶段的动态变化及清热止咳方药的干预作用，为开发出作用机制明确的抗MPP中药新药提供新的思路。

本研究采用FQ-PCR、Western-blot、酶联免疫吸附、流式细胞术等手段和在体及细胞水平的肺炎支原体感染模型，考察肺炎支原体感染后NLRP3炎性体的动态变化及清热止咳方药的干预作用。体内试验结果发现，NLRP3炎性体在MP感染BALB/c小鼠的发病初期、急性发病期、稳定期表达上调，恢复期趋于正常；清热止咳方药在急性发病期，稳定期可下调其表达。体外试验研究发现：MP感染早期可诱导RAW264.7细胞NLRP3炎性体活化，即NLRP3炎性体参与了MP的早期感染；动态观察结果证实，MP可能通过刺激RAW264. 7 细胞生成ROS而激活NLRP3炎性体；清热止咳方药含药血清可下调MP感染RAW264. 7 细胞NLRP3炎性体及IL-1β表达异常，可能是其抗MPP的作用机制之一。上述结果证实了我们提出的NLRP3炎性体及其下游促炎症因子参与了MPP的发生和发展，此过程可能与ROS有关的假说，为MPP的防治提供一个新靶点。也为中医药治疗MPP的作用机制研究提供了方向。