基于“肺-肠轴”探讨壮医三十六荡坎蛤散治疗儿童哮喘缓解期的作用机制

Asthma is the most common lung disease caused by chronic airway allergic inflammation, it affects children’s health seriously. Clinically research has confirmed that Zhuang medicine Sanshiliudang Kanhan San can enhance asthma children's constitution and reduce asthma attack times. This topic is to combine the relevant animal experimental research, based on the theory of "lung-gut axis", research the treatment mechanism of Sanshiliudang Kanhan San on asthma remission. In this experiment, the C57BL/6 mice sensitized with OVA were modeled and based on the 16S rRNA gene analysis technique, animal feces were used as specimens to carry out qualitative and quantitative analysis of "lung-gut axis" in mice; using metabonomics technique to analyse the metabolite of gut;using modern molecular biology related techniques, immuno-inflammation markers in biological samples such as bronchoalveolar lavage fluid and plasma were detected, and the status of innate immune cells in the thymus and spleen of rats was examined by flow cytometry. By analyzing the data, we summarized intestinal flora and its metabolite were specifically expressed in model animals during asthma remission, furthermore clarified its association with the body's immunity, initial profoundly that Zhuang’s medicine Sanshiliudang Kanhan San can be regulating to the action mechanism of the "lung-gut axis" which as influence to the body's immune status. This topic is to clarify the mechanism of the treatment of asthma during the remission stage by Zhuang medicine Sanshiliudang Kanhan San and prove the scientific nature of Zhuang medicine on therapy of asthma.

哮喘是一种常见的气道慢性炎症性疾病，严重危害儿童的健康。前期研究提示三十六荡坎蛤散方能够增强哮喘患儿体质，减少哮喘发作。本课题拟基于“肺-肠轴”中肠道微生物免疫作用的相关理论，探讨三十六荡坎蛤散治疗哮喘缓解期的作用机制。本研究拟以OVA致敏C57BL/6纯系小鼠造模，基于16S rRNA基因分析技术，以动物肺脏及粪便为标本，对小鼠肠道菌群进行定性、定量分析；代谢组学相关方法检测肠道内源性代谢产物；运用现代分子生物学技术检测肺泡灌洗液、血浆等标本中的免疫炎症指标，并以流式细胞术检测大鼠胸腺、脾脏等标本固有免疫细胞的状态。通过数据分析，总结哮喘缓解期模型动物特异性表达的肠道菌群及代谢产物，并进一步明确其与机体免疫的关联，初步阐明壮医三十六荡坎蛤散通过调节肠道菌群影响机体免疫状态的作用机制。本课题有助于从改善气道慢性炎症角度明确三十六荡坎蛤散治疗哮喘缓解期的作用机理，佐证壮药治疗哮喘的科学性。

哮喘是一种以慢性气道炎症为特征的异质性疾病，我国患有哮喘的儿童基数庞大，尽管目前我国哮喘患儿病死率不高，但其症状控制效果欠佳，对患儿的生长发育、学习及生活造成了诸多不良影响。因此，寻找有效防治哮喘的方法，是儿科医师亟待解决的问题之一。哮喘的发病机制与气道慢性炎症反应密切相关，研究发现肠道菌群是驱动出生后免疫系统发育成熟和诱导免疫反应平衡的基本因素，肠道菌群及其组分能够被固有免疫细胞和肠上皮细胞的模式识别受体识别，影响其信号转导，从而发挥其对宿主的免疫调节作用。故本课题基于“肺-肠”轴中肠道微生物免疫作用的相关理论，探讨三十六荡坎蛤散治疗哮喘缓解期的作用机制。主要研内容包括：（1）基于TLRs受体信号通路，明确肠道菌群影响免疫状态的作用机制，并阐明三十六荡坎蛤散治疗哮喘潜在的作用机理。（2）揭示壮医方三十六荡坎蛤散主要涉及的代谢通路。（3）基于肺肠轴将肠道菌群与肺部菌群及炎症水平联系起来，从机体免疫角度，阐述中医理论“肺与大肠相表里”的科学依据。结果显示三十六荡坎蛤散可明显改善哮喘行为；明显减轻哮喘小鼠肺组织的病理改变；显著降低血清和BALF中IL-1β，IL-6，TNF-α，HMGB1，NF-κB炎症细胞因子水平，有效改善OVA诱导的哮喘小鼠气道炎症，有效调节 T 淋巴细胞水平，改善哮喘小鼠的免疫功能，显著降低哮喘小鼠肺组织TLR4、MyD88、p-NF-κB p65/NF-κB p65水平，其作用机制可能与HMGB1/TLR4/MyD88信号通路密切相关。Hsd17b2，Plp2，Vnn1，Olfm4，Hsbp1，Gimd蛋白可能是三十六荡坎蛤散基于肺-肠轴理论治疗哮喘的潜在药物靶点。各组小鼠肠道菌群OTU数目、Alpha多样性、Beta多样性、差异物种组成等有较大差异，说明哮喘小鼠伴有肠道菌群紊乱现象。并且可能通过影响肠道菌群对哮喘缓解起作用。细胞实验也验证了三十六荡坎蛤散能提高细胞存活率，降低细胞凋亡率，降低细胞上清液中IL-1β、IL-6、TNF-α、HMGB1、NF-κB含量，显著降低细胞中HMGB1、TLR4、MyD88和NF-κB p65 mRNA表达水平以及细胞中TLR4、MyD88、NF-κB p65、p-NF-κB p65蛋白表达水平。