从哮喘气道平滑肌增厚探讨防风-乌梅"相制为用"的机制

It is well demonstrated that airway remodeling serves as a crucial factor that leads to the chronic process and high mortality of asthma. The pathological changes in ASMCs are the important structural foundations of airway smooth muscle hypertrophy, and also play a significant role in airway remodeling. In a previous study we found that the couplet medicines, Fangfeng and Wumei, prevent the thickening of airway smooth muscle, but the detailed molecular mechanisms still remain elusive. Based on this, it is hypothesized that through regulating the PAR2-related molecules of ASMCs involved in airway remodeling, Fangfeng and Wumei inhibits smooth muscle cell proliferation, which is associated with the theory of "the combination of restrictions". In the current study, Fangfeng and Wumei is applied to the treantment of "expelling wind and astringing the lung" for acute asthma attack. With the utilization of methods, including realtime fluores-cence quantitative PCR, FCM, shRNA and others, the main research contents include analyzing: the pathological alterations, induced by Fangfeng and Wumei, of ASMCs in asthma model; the influence of targeting intervention of ASMC PAR2 expression on the regulation of smooth muscle accumulation in the asthmatic airway by "Fangfeng and Wumei"; and the expression of PAR2 pathway related molecules of ASMCs in asthma model with the administration of this couplet medicines. The aim of this study is to identify the molecular targets and mechanisms underlying the inhibitive role of "pungent evaporation and sour astringe" of Fangfeng and Wumei in smooth muscle thickening, further uncover the scientific essence within the theory of "combination of restrictions" in Guominjian, and provide a platform for the study on formulating rules of prescriptions.

气道重塑是引起哮喘病程慢性化、死亡率居高不下的重要原因。ASMC病理变化是哮喘气道平滑肌增殖的结构基础，又是气道重塑的核心环节。前期研究发现药对防风-乌梅具有抑制气道平滑肌增厚作用，但未阐明两药具体配伍机制。基于此提出"防风-乌梅配伍抑制哮喘气道重塑中调控ASMC细胞PAR2相关分子干预平滑肌增厚是'相制为用'的机制"的假说。本研究基于哮喘发作期"祛风敛肺"的治法，选用过敏煎的君臣药防风-乌梅配伍，围绕ASMC病理改变，采用实时荧光定量PCR、FCM、shRNA等技术，从防风-乌梅配伍对哮喘模型ASMC的调控研究；靶向干预ASMC 细胞PAR2表达对防风-乌梅配伍调控哮喘平滑肌增厚的影响；防风-乌梅配伍对哮喘模型ASMC细胞PAR2通路相关分子的调控等三个方面，探索"辛散酸收"防风-乌梅配伍抑制平滑肌增厚的靶点及具体机制，揭示过敏煎中"相制为用"科学内涵，为方剂配伍规律研究奠定基础。

本课题围绕ASMC病理变化，探索辛散酸收配伍“防风-乌梅”抑制气道平滑肌增厚的靶点和作用机制，揭示过敏煎中“相制为用”的科学内涵。通过体内外研究“防风-乌梅”配伍对哮喘模型ASMC细胞的调控作用，发现防风-乌梅配伍可降低ASMC增殖、迁移能力，抑制细胞表型转换，影响细胞周期，降低CyclinD1、bcl-2 mRNA及蛋白表达，升高P27kip1、caspase-3 mRNA及蛋白表达，提示防风-乌梅配伍通过延缓平滑肌增厚达到抑制气道重塑目的。通过靶向干预PAR2靶点发现，防风-乌梅配伍前后可降低细胞增殖、迁移能力，抑制细胞表型转换，阻滞细胞周期进程，降低CyclinD1、bcl-2 mRNA及蛋白表达，升高P27kip1、caspase-3 mRNA及蛋白表达，降低ASMC细胞VEGF、TGF-β、TNF-α、IL-6、IL-8的表达及MMP-9/TIMP-1的比值，减少炎性细胞浸润，改善气道重塑。研究结果提示，防风-乌梅配伍抑制气道重塑中PAR2不是唯一靶点，与中药多成分、多靶点相符合。通过研究PAR2信号通路分子，发现防风-乌梅配伍对ASMC细胞PAR2信号通路Ras、MEK1/2、ERK1/2、c-fos有一定调控作用，使用Ras抑制剂干预HBSMC后，仍能表达Ras、ERK1/2，调控 ASMC细胞周期，抑制细胞迁移，影响气道重塑。研究提示防风-乌梅抑制平滑肌增厚与PAR2信号有关，但非单一途径，可能是多信号途径共同实现。另外，本研究中发现防风-乌梅配伍后，在不同的靶点（指标）表现出 “相制为用”或 “相须为用”的效应。提示防风-乌梅配伍后可能在不同的靶点、不同的层次发挥不同的效应。该研究为进一步深入研究方剂配伍机制和规律研究奠定了基础。