"肺肠合治法"对肠道菌群失调合并过敏性哮喘TLRs/NF-κB 通路及调控蛋白β-arrestin影响的研究

Asthma is harmful to the health. The studies shown that enteric dysbacteriosis is susceptible to be accompanied by allergic asthma. Based on theory of exterior-interior relationship between lungs and intestines in TCM, allergic asthma due to enteric dysbacteriosis belongs to lungs disease involving intestines, then being sick simultaneously. The accomplished project supported by NSFC (No: 30472127) testified that Fèi Cháng Hé Zhì Fǎ (therapy of treating lungs and intestines simultaneously) could prevent and treat allergic asthma. Project 6 of 973 Program (No: 2009CB522706) working in the progress, lungs and large intestine are exterior and interior related, testified that the flora of lungs and large intestine are dynamical correlated, and Fèi Cháng Hé Zhì Fǎ could improve enteric dysbacteriosis and intestinal inflammation. It's reported that TLR/NF-κB signal pathway participates in occurrence of allergic asthma and intestinal inflammation, while β-arrestin is key regulating protein on upper pathway. Therefore, we put forward the hypothesis that Fèi Cháng Hé Zhì Fǎ could prevent and treat allergic asthma due to enteric dysbacteriosis by recovering the flora ecology, and regulating TLRs/NF-κB signal pathway and the key regulating protein β-arrestin. The study will center on the effect of Shēng Jiàng Sǎn (Ascending and Descending Powder), the representative formula of Fèi Cháng Hé Zhì Fǎ, on relative model animals' mucosal inflammatory factors, sIgA, and flora molecular ecology to testify the effectiveness; and reveal Fèi Cháng Hé Zhì Fǎ influences the action mechanism of correlative gene expression of TLRs/NF-κB signal pathway by regulating the expression of β-arrestin. The study has significant scientific sense on enriching the theory of lungs and large intestine are exterior and interior related, and guiding the reasonable application of Fèi Cháng Hé Zhì Fǎ in clinic.

过敏性哮喘严重危害人类健康，研究发现肠道菌群失调易合并过敏性哮喘。根据中医"肺与大肠相表里"理论，属中医"肺肠合病"范畴。我们已结题自然基金课题证实"肺肠合治法"能防治过敏性哮喘；进行中的973"肺与大肠相表里"项目证实"肺"与"大肠"菌群动态相关，"肺肠合治法"能改善肠道菌群失调及肠道炎症;文献报道TLR/NF-κB信号通路参与过敏性哮喘及肠道炎症，β-arrestin是该通路上游关键调控蛋白。据此，提出工作假说："肺肠合治法"可通过调节TLRs/NF-κB信号通路及其上游关键调控蛋白β-arrestin，防治菌群失调合并过敏性哮喘。我们拟通过"肺肠合治法"代表方"升降散"对模型动物粘膜免疫,菌群分子生态的影响，验证其有效性；揭示其通过影响β-arrestin，调控TLRs/NF-κB信号通路的作用机制。对于丰富"肺与大肠相表里"理论，指导临床合理运用"肺肠合治法"，具有重要科学意义。

“卫生假说”认为生命早期内环境中微生物的多少与免疫耐受形成和变应性疾病的发生相关。过敏性哮喘的发生、发展与早期肠道菌群的紊乱关系密切。肠道菌群失调合并过敏性哮喘属于中医“肺肠合病”，根据中医“肺与大肠相表里”的理论，“肺肠合治”法能防治肠菌群失调并过敏性哮喘。本研究通过饲喂抗生素加白念菌定植复制肠道菌群失调，并运用卵清蛋白诱发哮喘，从整体、细胞、分子水平考察了肺肠合治方对模型的治疗作用，并从肠道菌群生态及TLR2、4/MyD88信号转导角度初步探讨其作用的免疫机制。有效性研究发现“肺肠合治”法治疗作用确切：能改善呼吸功能；调节肠道菌群紊乱；减轻肺、肠组织发生的损伤性病理变化；降低血液及呼吸道Eos等炎细胞数量，下调IgE水平，缓解全身和局部过敏性炎症反应；恢复肺、肠黏液中sIgA含量，恢复肺、肠黏膜免疫功能；调节肺IL-4与IFN-γ及GATA-3 和T-bet mRNA 的表达水平，恢复Th1/Th2失衡。机制研究部分显示，“肺肠合治”法作用机制可能是： 1.显著下调肺组织TLR2、TLR4、MyD88与TRAF6 mRNA，从而抑制肺组织中TLR2、4/MyD88信号转导；但并非通过直接影响通路调控蛋白β-arrestin2实现；2.肠道整体菌群生态多样性并未恢复，但菌群紊乱得到一定程度调节；3.上调肠组织TLR2、MyD88与TRAF6及β-arrestin-2的表达，抑制TLR4表达。据此结果推测，“肺肠合治”法的作用机制，可能与下调肺部TLRs/NF-κB信号通路中关键分子，抑制通路过度活化相关。治疗组肠道菌群生态多样性的丧失，可能与肠道β-arrestin-2高表达及TLR2/MyD88信号转导通路的活化相关。因此，我们认为“肺肠合治”法通过调肺气、通腑气，从多环节、多途径和多靶点对肠道菌群失调合并过敏性哮喘发挥作用；其机制可能在于通过下调肺TLRs/NF-κB信号转导通路中关键信号转导分子，抑制通路过度活化，恢复局部Th1/Th2失衡，缓解炎症反应，调节肺、肠免疫平衡，防治肠道菌群失调合并过敏性哮喘。这对于指导临床科学、合理应用“肺肠合治”法治疗肺肠合病提供了实验室依据；亦对后续“肺与大肠相表里”的中医理论研究的提供了思路。