基于Ras信号通路调控气道平滑肌增殖探讨黄芩苷抗哮喘气道重建的作用机制

Scutellaria baicalensis was called capital drug of clearing-heat. Baicalin, that is flavonoid components in Scutellaria baicalensis, is cardinal components of clearing-heat. Clearing lung-heat is a primary mean used by asthma Chinese medicine treatment. Airway remodeling is crucial pathological features of asthma, exploring new methods and drugs to prevent and reverse the airway remodeling is the key to asthma research in the future. Over proliferation of Airway smooth muscle cells (ASMCs) is a key to airway remodeling, Ras signaling pathway play important role in the regulation of ASMCs proliferation. Based on validity of Baicalin intervening airway remodeling, this research analyze the difference of protein expression in Ras signaling pathway between normal and airway remodeling of asthmatic mice; between Baicalin intervention and non intervention in lung of mice, using microarray technology of protein. Then above mentioned result was verified in gene and protein level, using lung and ASMCs of mice. Positive protein which Ras signal pathway regulate airway remodeling in asthma, and Baicalin regulate Ras signal pathway are comfirmed. This project aims to further ascertain the role of Ras signal pathway in airway remodeling in asthma, and study the regulatory effect of Baicalin on Ras signal pathway by construction of positive protein high/low expression cell model, in order to provide new scientific basis for the mechanism research of the anti-asthmatic effect of traditional Chinese medicine.

黄芩为清肺热的要药。黄芩黄酮类成分黄芩苷是其主要的清热组分。清肺法是哮喘中医治疗的主要治则。气道重建是哮喘的重要特征，探索气道重建发生的机理和防治药物是哮喘研究的关键之处。气道平滑肌细胞(ASMCs)过度增殖是哮喘气道重建的关键病理改变，Ras信号通路在调控ASMCs增殖中发挥着重要作用。本研究在黄芩苷干预哮喘气道重建有效的基础上，以哮喘气道重建小鼠模型为研究对象，运用蛋白芯片技术，筛选正常小鼠、哮喘气道重建模型小鼠、黄芩苷干预三种状态下Ras通路的差异表达蛋白；利用肺组织和ASMCs，在基因和蛋白层面对上述蛋白芯片结果进行验证，明确Ras通路调控哮喘气道重建和黄芩苷调节Ras通路的效应蛋白；构建Ras通路效应蛋白高/低表达细胞模型，阐释黄芩苷调控Ras信号通路抑制ASMCs增殖，从而缓解哮喘气道重建的作用机理，为中医药抗哮喘机制研究提供新的科学依据。

黄芩为清肺热的要药。黄芩黄酮类成分黄芩苷是其主要的清热组分。清肺法是哮喘中医治疗的主要治则。气道重建是哮喘的重要特征，探索气道重建发生的机理和防治药物是哮喘研究的关键之处。气道平滑肌细胞(ASMCs)过度增殖是哮喘气道重建的关键病理改变，Ras/ERK信号通路在调控ASMCs增殖中发挥着重要作用。本研究在黄芩苷干预哮喘气道重建有效的基础上，进一步研究黄芩苷通过调控Ras/ERK信号通路干预气道平滑肌增殖，从而缓解哮喘小鼠气道重建的作用机理。结果显示黄芩苷能够明显降低哮喘气道重建小鼠的气道阻力，改善气道璧增厚、气道黏液分泌及气道周围胶原沉积，降低气道重建相关细胞因子的表达。利用蛋白芯片分析、肺组织及气道平滑肌细胞增殖模型验证，表明黄芩苷调控Ras/ERK信号通路干预哮喘小鼠气道重建的效应蛋白为PKC、A-Raf、MEK2、ERK、Mnk1、Elk1。选取Ras/ERK信号通路中的A-Raf构建低表达细胞模型，分析得出shRNA-低表达细胞模型中，A-Raf下游的Ras/ERK信号通路效应蛋白（MEK2、ERK、Elk1、MNK1）总蛋白及其磷酸化位点蛋白（p-Mek2-Tyr394、p-ERK3-Ser189、p-Elk1-Ser383、p-MNK1-Thr385）表达量均降低，同时低表达细胞模型增殖水平较对照组降低，提示阻断Ras/ERK信号通路能够抑制细胞增殖，黄芩苷可能通过该机制抑制ASMCs增殖从而改善哮喘气道重建。通过以上系列研究，初步阐明黄芩苷调控Ras/ERK信号通路干预哮喘小鼠气道重建的作用机理，为中医药抗哮喘机制研究提供科学依据。