基于自噬/凋亡调控平衡研究淫羊藿女贞子合糖皮质激素对哮喘模型气道重塑的作用机制

Airway remodeling is one of the important causes of recurrent attacks of asthma, and there is no effective means to improve it. The imbalance between autophagy and apoptosis (Yin-yang imbalance according to the theories of traditional Chinese medicine) is an important pathogenesis of airway remodeling. Based on the common understanding for airway remodeling of asthma in Chinese and Western medicine and the pre-work, the scientific hypothesis is put forward that Herba Epimedii, Fructus Ligustri lucidi and glucocorticoid in compatibility may improving airway remodeling in asthma through regulating the balance between autophagy and apoptosis. Rat asthmatic model induced by ovalbumin was used, combined with 2 kinds of important cells (airway epithelial cells and airway smooth muscle cells) in different states during the developing process of airway remodeling. The major study context in this project includes positive and negative regulators in the processes of autophagy and apoptosis (Bcl-2, Bax, P53, Beclin1, mTOR and Caspase-3) and their interrelation network with cross talks, the activity of autophagy and apoptosis and the degree of airway remodeling. Many advanced detection techniques are applied, such as immunocytochemistry, immunofluorescence, immunohistochemistry, western blot, real-time PCR, flow cytometry and transmission electron microscopy. The project aims are to study the molecular mechanism of airway remodeling of asthma, and elucidate the regulation differences of autophagy and apoptosis among the single use of Herba Epimedii, Fructus Ligustri lucidi or glucocorticoid, the compatibility of the two herbs and the compatibility of glucocorticoid and the two herbs from the different levels of overall, cellular and molecular (proteins and genes), respectively, and provide the clinical and experimental evidence for the prevention and treatment of asthma, especially for refractory asthma with the treatment of the integrative medicine. In addition, the study also provides a microcosmic basis for the basic theory of yin-yang balance in Chinese medicine.

气道重塑是哮喘反复发作难以治愈的重要原因，目前缺乏有效的干预手段。自噬/凋亡失衡（中医认为阴阳不足，阴阳失衡）是其发生的重要机制。基于中西医认识及前期临床与实验基础，提出平补阴阳立法的淫羊藿女贞子协同激素调节自噬/凋亡交互应答网络改善气道重塑的科学假说。采用卵蛋白致哮喘大鼠模型结合气道重塑中2种重要细胞（AECs和ASMCs）的不同状态展开动态研究，以自噬/凋亡的正负调控因子（Bcl-2、Bax、P53、mTOR、Beclin1、Caspase-3）及彼此之间的调控对自噬/凋亡活性与气道重塑程度的改变为研究内容，采用病理、IHC、IF、ICC、WB、流式细胞术、qPCR、透射电镜等技术，从整体、细胞和分子（蛋白及基因）等不同水平，比较研究药物配伍对哮喘气道重塑的作用及机制，以期为临床中西医结合防治哮喘，尤其是难治性哮喘提供实验依据。此外，也为中医“阴阳平衡”的基本理论提供微观依据。

气道重塑是顽固性哮喘的重要原因之一，预防和逆转气道重塑对哮喘的防治具有重要的意义。自噬和凋亡是细胞应激后的两种相互关联的机制，与哮喘气道重塑发病机制密切相关。在细胞、蛋白、基因水平的自噬（促进细胞增殖，属“阳”）/凋亡（促进细胞死亡，属“阴”）内控机制可能是中医阴阳的微观基础。本研究在动物水平和细胞水平上观察平补阴阳药对协同激素（GC）对哮喘气道重塑中自噬/凋亡交互应答网络的调控作用。.动物研究采用卵蛋白致敏并激发建立哮喘模型，比较GC单用或合用淫羊藿女贞子对哮喘的防治作用。结果显示，淫羊藿女贞子协同GC促进ASMCs凋亡，抑制AECs凋亡、AECs和ASMCs增殖；下调Bcl-2、Beclin1、LC3B、NF-κB及Xiap表达；上调Bax、Caspase-3、cleaved-Caspase-3、P53、核P53、mTOR、p-mTOR、Caspase-9及Fas表达；降低Bcl-2/Bax比值、LC3-Ⅱ/LC3-I蛋白比值；促进肺组织凋亡，抑制肺组织自噬，调节哮喘大鼠自噬/凋亡异常，改善哮喘大鼠气道重塑。.AECs研究采用16HBE细胞，分别观察药物对正常培养、PLA及RAP刺激的16HBE细胞的影响。结果显示：GC可损伤正常培养的16HBE细胞，但可改善PLA诱导的16HBE细胞损伤。淫羊藿女贞子可通过调控自噬/凋亡交互应答网络，抑制GC及RAP诱导的16HBE细胞损伤，并协同GC改善PLA诱导的细胞屏障功能障碍。.ASMCs研究采用大鼠原代ASMCs细胞，分别观察药物对正常培养、Hist、ZDF、RAP及3-MA刺激的ASMCs的影响。结果显示：GC可抑制正常培养、Hist、ZDF、RAP及3-MA刺激的ASMCs增殖，诱导细胞凋亡与自噬。淫羊藿女贞子可通过调控自噬/凋亡交互应答网络，可减轻GC对正常培养及RAP刺激的ASMCs增殖活性的抑制，恢复正常细胞活力，抑制GC诱导的ASMCs凋亡；对Hist及ZDF刺激的ASMCs，淫羊藿女贞子可协同GC进一步抑制ASMCs增殖。.该研究表明，淫羊藿女贞子可协同GC防治哮喘气道重塑，其作用机制核心在于调节自噬/凋亡交互应答网络，抑制GC诱导的细胞损伤，并协同GC影响不同模型下的细胞状态。本研究阐明了淫羊藿女贞子配伍协同GC防治哮喘的部分作用机制，为中西医合用防治哮喘的临床治疗及新药开发提供科学依据。