AMPK/mTOR对哮喘大鼠气道平滑肌增生的调节及分子机制研究

Airway remodeling is an important pathological feature of asthma. Airway smooth muscle hyperplasia is major manifestation of the airway remodeling. Therefore, inhibition of the airway smooth muscle hyperplasia is important for the prevention and treatment of asthma. Recent studies have shown that mTOR signaling pathway may plays an important role in regulating the hyperplasia response of airway smooth muscle, but the molecular mechanism (ie, factors which regulate mTOR, and downstream target molecules regulated by mTOR) is unclear. According to our preliminary experiments and literature analysis, we propose the following hypotheses : (1) AMPK dysfunction induce hyperplasia of airway smooth muscles,and trigger asthma; (2) AMPK reduce Skp2 expression by inhibiting mTOR function, and suppress UPS-mediated degradation of cell cycle inhibitor protein, p21 and p27, and thereby inhibit the progression of cell cycle and hyperplasia of airway smooth muscle; (3) activation of AMPK or inhibition of mTOR could prevent and therapy the airway remodeling of animal models by regulating mTOR-Skp2-p21/p27, which would be a new therapeutic strategy of asthma. We will use cell model and OVA-induced asthmatic animal models as a platform to validate our hypothesis. This study will provide a theoretical basis and experimental evidence for clinical treatment of asthma.

气道重塑是哮喘的重要病理特征，气道平滑肌增生是气道重塑的主要表现。因此减轻或阻止气道平滑肌增生对防治哮喘有重要临床意义。近年来的研究表明mTOR信号通路可能在调控气道平滑肌增生反应中起重要作用，但其分子机制（即受哪些因子调控或通过调控哪些下游靶分子的表达或活性影响气道平滑肌增生反应）尚不清楚。根据我们的预实验及文献分析，我们提出如下研究假设：1.AMPK功能受损促进气道平滑肌增生，引发哮喘；2.AMPK 通过抑制 mTOR 功能，下调Skp2表达，从而抑制UPS介导的细胞周期抑制蛋白p21, p27的降解，抑制细胞周期的进展和ASMCs的增殖；3.激活AMPK 或抑制mTOR 可作为新的治疗靶点，通过调控mTOR-Skp2-p21/p27防治模型动物的气道重塑。我们将以细胞模型和OVA致敏激发的哮喘动物模型为平台，来验证我们的研究假设。本研究将为哮喘的临床治疗提供理论基础及实验依据。

本研究首先通过测定慢性哮喘动物模型气道平滑肌组织中mTOR活性增强而AMPK活性降低，表明AMPK-mTOR通路在慢性哮喘气道平滑肌增生中起重要作用，随后在原代培养的ASMCs中研究发现，激活AMPK或抑制mTOR通路可抑制其细胞增殖，同时可逆转PDGF对ASMCs所致的增殖；通过shRNA干扰AMPK等基因的表达表明，AMPK负性调控mTOR通路，上调SKP-2基因表达，使P21表达下调而达到抑制ASMCs增殖；最后通过动物实验表明， 二甲双胍和雷帕霉素可通过激活AMPK，或直接抑制mTOR通路，可逆转慢性哮喘动物模型的气道重塑（黏膜上皮杯状化生、纤维组织沉积及气道平滑肌细胞的增殖）。