基于Ezrin蛋白的针刺抗哮喘舒张气道平滑肌生物学机制研究

This project is based on a clinically proven method of acupuncture for treating asthma and the previous achievement in biological mechanism research. Previous studies have shown that metallothionein 2 (MT-2) was one of the most important anti-asthmatic factors of acupuncture and it relaxed airway smooth muscle via transgelin-2 (TG2) receptor. However, the molecular mechanism of contraction and relaxation of airway smooth muscle by TG2 receptor is still not clear. It was found that MT-2 significantly increased the level of ezrin phosphorylation via TG2 receptor by using a protein antibody array. The strong interaction between ezrin and TG2 was subsequently confirmed by co-immunoprecipitation and surface plasmon resonance. Therefore, ezrin was thought to play a key role in the transduction of TG2 receptor signaling pathway. This project intends to study the effect of acupuncture on ezrin expression and phosphorylation, to observe the effect of ezrin on the respiratory function, the cellular stiffness, the traction force and relaxation of airway smooth muscle and the corresponding acupuncture regulation. This study will identify the molecular mechanism of ezrin in contraction and relaxation of airway smooth muscle mediated by TG2 receptor, which may provide a new scientific basis for the biological mechanism of acupuncture in treating asthma.

本项目建立在临床有效的针刺抗哮喘方法和生物学机制研究有所发现的基础上。利用基因组学技术发现金属硫蛋白2(MT-2)是重要的针刺抗哮喘效应因子，可通过膜受体肌动蛋白结合蛋白2(TG2)舒张气道平滑肌。然而TG2介导的气道平滑肌收缩舒张分子机制尚不清楚。利用蛋白磷酸化芯片发现TG2激活后可显著增强气道平滑肌细胞Ezrin蛋白磷酸化，经免疫共沉淀和表面等离子共振技术证实TG2和Ezrin可形成稳定蛋白复合体。我们推测Ezrin可结合TG2受体并在下游信号传导通路中起关键作用。本项目拟研究针刺对Ezrin表达、磷酸化影响，检测Ezrin在哮喘呼吸功能、气道平滑肌细胞刚度、牵张力、收缩舒张等生物学作用和针刺调节效应，阐明Ezrin蛋白在TG2受体介导的气道平滑肌收缩舒张调节机制中作用，以期为针刺抗哮喘效应生物学内涵阐释提供新的科学依据。

本项目系统研究了“三穴五针”法针刺对动物哮喘模型Ezrin蛋白表达和第567位苏氨酸(T567)、第478位酪氨酸(Y478)等关键磷酸化位点影响；构建Ezrin低表达气道平滑肌细胞，利用电阻法、胶原法和细胞牵张力显微测量技术分别检测Ezrin对气道平滑肌收缩舒张功能影响；构建Ezrin基因气道平滑肌条件敲除小鼠哮喘模型，深入研究Ezrin对呼吸功能、肺部炎症影响和针刺调节作用。项目内容均按计划执行并完成。主要成果表达方面，本项目共发表课题标注学术论文5篇，其中SCI收录论文2篇。其中项目负责人作为第一作者，应邀在Trends in Biochemical Sciences杂志(2019;44(10):885-96, IF=16.889, 本项目为第一标注)发表评述，系统回顾针刺抗哮喘靶标Transgelin-2结构特点和Ezrin等下游连接蛋白在哮喘等疾病中相互作用关系。项目负责人作为第一兼通讯作者应邀在Frontiers in Cell and Developmental Biology(2021;9:819300, IF=6.081)评价Ezrin等肌动蛋白结合蛋白在进化、结构和疾病新功能中的重要性作用和地位，为进一步明确以Ezrin为代表的肌动蛋白结合蛋白在针刺抗哮喘调控中关键作用奠定了理论基础。项目负责人入选2项市级人才计划，参加国内外重要学术交流5次，团队获国家实用新型专利授权1项，项目主要参与者2名硕士研究生顺利毕业。