基于NF-κB-mTOR通路和自噬基因Atg的支气管哮喘寒饮蕴肺证大鼠气道炎症机理及干预机制研究

Haze is becoming increasingly serious in recent years, and thus the incidence of asthma is rising accordingly. Cell autophagy is a defense mechanism, but at the same time, it can induce apoptosis of lung epithelial cells, thus aggravating asthma. There is a close relationship between the NF-κB-mTOR pathway and cell autophagy. Our previous study shows that “wenyang huayin formula” (warming yang and resolving fluid retention) can relieve asthma by regulating NF-κB pathway and airway remodeling. Thus, we come to the conclusion that this formula can reduce the side effect of cell autophagy by regulating NF-κB-mTOR pathway of the lung tissue, and that it has therapeutic effect on asthmatic airway inflammation. Based on our study, we put forward the hypothesis that “wenyang huayin formula” can help to prevent and treat asthma by regulating the autophagy of NF-κB-mTOR pathway of the lung tissue to influence airway inflammation. By employing the methods of combination of Chinese and Western medicine, we intend to make rat model of bronchial asthma with syndrome of cold fluid retained in lung. Based on the model, we aim to illustrate the biological mechanism of “wenyang huayin formula” influencing airway inflammation by detecting autophagy gene Atg and autophagy protein of lung tissue with Western blot and RT-PCR methods. The study aims to provide a scientific basis for clinic treatment of asthma, which is of significant value both in theory and practice.

近年来雾霾日益严重，哮喘发病率逐年增高。细胞自噬是一种防御机制，但其同时又会诱导肺上皮细胞的凋亡，从而加重哮喘病情。NF-κB-mTOR通路与细胞自噬有直接关系，结合前期研究发现温阳化饮方可以调控NF-κB通路，以减缓哮喘气道重塑，故提出该方防治哮喘可能通过调控肺组织NF-κB-mTOR通路可以解决细胞自噬所带来的副作用，对哮喘气道炎症起到治疗作用。提出“哮喘寒饮蕴肺证气道炎症机理可能与NF-κB-mTOR通路自噬基因Atg有关，温阳化饮法可能通过调控肺组织NF-κB-mTOR通路的自噬影响气道炎症”的假说。本研究拟用中西医结合方法，制造支气管哮喘寒饮蕴肺病证结合大鼠模型，运用 Western blot 法及荧光定量 RT-PCR法检测肺组织中自噬基因Atg和自噬蛋白，阐明温阳化饮方对哮喘寒饮蕴肺证影响气道炎症的生物学机制，为哮喘的临床治疗提供科学依据，将具有重要的理论意义和临床价值。

本课题探讨基于NF-κB-mTOR通路，应用温阳化饮方对哮喘寒饮蕴肺证大鼠细胞自噬的干预机制进行研究。在造模过程中采用卵清白蛋白致敏，饮用冰水、寒冷环境生存、以及水中游泳等寒冷刺激，后期卵清白蛋白雾化，进一步激发哮喘症状，最终成功建立支气管哮喘寒饮蕴肺证大鼠模型。在药物干预方面，选择温阳化饮方、黄芪甲苷、人参皂苷与温阳化饮方相关的中药有效提取物作为研究，以西药雷帕霉素作为对照药，以探讨温阳化饮方对细胞自噬的作用机制。造模成功后结合动物行为学、肺功能、HE染色、Masson染色相关指标对支气管哮喘寒饮蕴肺证大鼠进行模型评价，模型组大鼠的表现与支气管哮喘寒饮蕴肺证的临床表现基本相符合。结合PCR、Western Blot、免疫组化、透射电镜等相关分子生物学检测方法对大鼠细胞自噬情况进行检测显示，四种药物干预组的大鼠较模型组大鼠细胞自噬水平都有不同程度的提高，自噬相关通路mTOR均呈现不同程度的抑制，自噬相关基因的表达也均有不同程度的提高，提示自噬启动后四种药物均不同程度的缓解了哮喘寒饮蕴肺证大鼠气道炎症的情况。但综合结果看来以温阳化饮方组的效果最为显著，温阳化饮方能够抑制mTOR通路的过度激活，还能够抑制上游PI3K-AKT通路间接的抑制mTOR通路激活细胞自噬，并且能够抑制mTOR下游HIF-1α通路，恢复内环境稳态，促进细胞自噬的功能，减少炎症因子的生成，通过细胞自噬及时清除气道内粘液等代谢产物，进而起到缓解哮喘寒饮蕴肺证气道炎症的作用。说明温阳化饮方通过温补肺阳以及肺气的作用，促进体内水液代谢，减少痰饮的产生，恢复气机，对哮喘气道炎症的治疗具有一定的应用价值。