穿山龙调控PI3K/Akt信号通路介导的气道上皮-间质转化在哮喘气道重构中的作用机制研究

Airway remodeling is one of the most popular mechanisms of bronchial asthma, and is the main reason of aggravation and irreversible airflow limitation for asthma patients in later period. While, airway epithelial cells can take part in airway remodeling of asthma through the epithelial - interstitial (EMT), a process whereby fully differentiated epithelial cells undergo transition to a messenchymal phenotype. Recently, PI3K/Akt is described as en important role in EMT process, but it needs more experimental support. Rhizoma Dioscoreae Nipponicae, an effective Chinese traditional medicine with relieving cough and asthma function is widely used in clinic of asthma. Furthermore, previous studies have found that it has inhibitory effect on airway remodeling of asthma, and can influence the expression of some characteristic protein of EMT. Its effective components have some inhibitory effects on PI3K/Akt signal transduction pathway in tumor cells according to recent studies. Thus, we propose that Rhizoma Dioscoreae Nipponicae may suppress aiway remodeling of asthma by interventing PI3K/Akt-mediated EMT. In this study, we intend to futher explore the effect of Rhizoma Dioscoreae Nipponicae on PI3K/Akt and EMT-phenotypic change on tissue level, cellular level and molecular level though some methods such as siRNA-based silence and over expression of encoding genes, phosphorylation detection and specific inhibitor blockage and inducer of critical signaling kinases. This research is expected to provide new evidence for traditional Chinese medicine Rhizoma Dioscoreae Nipponicae in the treatment of asthma.

气道重构是哮喘患者晚期病情加重、气流受限不可逆的主要原因，而气道上皮细胞可通过上皮间质转化（EMT）模式参与哮喘气道重构过程。EMT过程包括特征性蛋白表达及迁移能力改变，研究发现PI3K/Akt信号通路在EMT发生过程中起重要作用，但其机制尚需深入研究。穿山龙作为一种止咳平喘中药被临床用于治疗哮喘并获较好疗效，前期研究已发现其对哮喘气道重构具抑制作用，并能影响部分EMT特征性蛋白表达，且有研究发现穿山龙有效成分在肿瘤细胞中对PI3K/Akt信号转导通路有一定抑制作用。因此，我们提出“穿山龙可通过调控PI3K/Akt介导EMT过程来实现抑制哮喘气道重构的作用”这一假说。本项目拟采用靶基因沉默及过表达、信号通路关键激酶磷酸化水平检测及特异性阻断剂、诱导剂运用等技术，从组织、细胞和分子水平观察并探明穿山龙对哮喘中PI3K/Akt及EMT相关表型转变的影响，从而为穿山龙治疗支气管哮喘提供新靶点。

气道重构是继慢性呼吸道炎症后研究者们最为关注的支气管哮喘的疾病机制，而气道上皮细胞可通过上皮-间质转化（EMT）模式，参与哮喘气道重构发展过程。因此对哮喘EMT的研究及治疗方法具有切实的意义。中药穿山龙可化痰祛瘀，临床已应用于治疗支气管哮喘，我们已进行相关研究，发现其抗炎疗效对哮喘气道重构具有抑制作用，但穿山龙对EMT的改变以及其分子机制和相关信号通路研究有待于阐明。本项目建立慢性哮喘模型，观察中药穿山龙及其主要成分薯蓣皂苷干预下哮喘小鼠肺功能、气道病理改变及EMT标志物基因的变化，并从细胞水平，观察薯蓣皂苷对气道上皮细胞内EMT过程中相关基因表达的影响，此外，通过高通量测序，探索其分子生物学机制及信号通路研究。结果表明穿山龙及其主要成分薯蓣皂苷可抑制慢性哮喘小鼠肺功能下降，减轻慢性哮喘小鼠上皮下胶原沉积、肺泡壁水肿及气道平滑肌增厚等气道重构病理改变，从动物及细胞水平均可下调间质细胞标志基因TGF-β1、α-SMA和MMP-9 mRNA表达，上调上皮细胞标志基因E-cadherin mRNA表达，从而抑制EMT。其机制和下调包括PI3K/AKT信号通路在内的多条信号通路节点有关。本研究进一步阐明了穿山龙的抑制气道重构作用机制，为穿山龙治疗哮喘提供更深入确切的实验依据及进一步研究思路，为哮喘防治提供新思路和药物靶点，具有较高的理论创新性和临床意义。