基于“虚-痰-瘀”夙根理论对支气管哮喘气道重塑中诱导纤维异常修复的HMGB1调控机制研究

Fiber abnormal repair caused by the immune inflammation of asthma is an important part of the airway remodeling. HMGB1 is the key medium linking of endogenous immune injury and abnormal repair transformation, and the receptor signal pathway it mediated plays an important role as a bridge in asthmatic airway remodeling. Our previous study shows that the Danlong Dingchuan Decoction with the effects of tonifying Qi, activating blood and reducing phlegm could suppress airway remodeling by inhibiting the expression of TGF- β1，MMP-9 and TIMP-1，and by regulating the balance of MMP-9/TIMP-1 in asthmatic mice, which prompted that Danlong Dingchuan Decoction had a better regulatory role of the intrapulmonary complex immune and inflammatory microenvironment. Based on clinical experience we think that the main pathogenesis of asthma airway remodeling is deficiency of kidney Yang, lingering-root phlegm and blood stasis obsessed in the lung which leading to lung airways displaced to raise. So we put forward the hypothesis that multi-target role in regulating of Danlong Dingchuan Decoction on airway remodeling may be related to blocking HMGB1, the key medium of remodeling, and inhibiting intrapulmonary immune inflammation/ maintenance complex signal molecules caused by it. This study focuses on the core theory "abnormal damage repairing" of airway remodeling’ pathogenesis, with HMGB1 protein as the research target, and experimentally explores the possible mechanisms of Danlong Dingchuan Decoction in the treatment of airway remodeling, so as to provide a more scientific approach to study anti- remodeling drug of Traditional Chinese Medicine.

哮喘免疫炎症导致的气道纤维异常修复是气道重塑的重要组成部分。HMGB1是链接内源性免疫损伤和异常纤维修复的关键介质，其介导的信号通路在哮喘气道重塑中发挥了重要的桥梁作用。我们前期研究显示，具有“益气活血化痰”功效的丹龙定喘汤可抑制哮喘小鼠TGF-β1、MMP-9/TIMP-1的表达而改善哮喘小鼠的气道重塑进程，提示丹龙定喘汤对哮喘肺内复杂的免疫炎症微环境有较好调控作用。基于临床我们认为：哮喘气道重塑的主要病机是肾阳虚衰，酿生“痰瘀夙根”胶结不解，潜伏肺窍，气道失养所致。故提出“丹龙定喘汤治疗气道重塑的多靶点调节作用可能与其阻断促进气道重塑中的重要介质HMGB1，抑制其所激活的肺内免疫炎症/纤维修复信号通路有关”的假说。本研究着眼于哮喘气道重塑的“炎症损伤/异常纤维修复”病理机制，以HMGB1蛋白为研究靶点，探讨丹龙定喘汤在哮喘气道重塑治疗中的作用机制，为抗气道重塑药物的研究提供科学途径。

支气管哮喘是是儿童时期最常见的慢性呼吸道疾病，严重危害儿童身心健康和生活质量。气道慢性炎症和气道重塑是支气管哮喘的两大病理特征，消除气道炎症和阻止气道重塑是治疗哮喘的关键环节。而“免疫炎症损伤/异常纤维修复”是哮喘气道重塑的重要病理机制之一，HMGB1 介导的受体信号通路在炎症免疫损伤过程中发挥重要的桥梁作用。因此，抑制HMGB1在肺内的过度表达，阻断其介导的“免疫炎症损伤/异常纤维修复”信号通路，有可能阻止气道重塑的发生。中医药防治哮喘具有多途径、多环节、多靶点整体调节的优势，故此，研究中医药抗哮喘的有效方法、方药具有广阔的应用前景和巨大的经济、社会效益。中医学认为“气虚夹痰夹瘀”是气道重塑的关键病机，益气活血化痰是其基本治疗思路，以丹龙定喘汤为代表的“益气活血化痰法”治疗哮喘疗效确切。.基于此，本课题将哮喘小鼠分为空白对照组、模型组、强的松组、丹龙定喘汤高剂量组、丹龙定喘汤中剂量组、丹龙定喘汤低剂量组，每组30只，使用OVA致敏造模成功后，分别予醋酸泼尼松及丹龙定喘汤，连续灌胃给药42 d后，分别采用HE及masson染色、免疫组化法（IHC、ELISA）、Western Blot、ELISA法、Q-PCR法分析丹龙定喘汤对气道重塑及肺组织中HMGB1、α-SMA、IL-4、IFN-γ、TGF-β1、RAGE、TLR2、TLR4、TNF-α的影响。另外，选用正常肺成纤维细胞和人肺腺癌A549 细胞为评价载体，采用液相芯片技术检测A549、HLFB 培养液上清TNF-α、IL-6、IL-8、IL-1β变化；Western-blot 法检测A549、HLFB 总蛋白中ERK 1/2、STAT3、NF-κB的表达。.实验结果表明，丹龙定喘汤能有效减少肺组织中HMGB1的表达，且抑制IL-4、TNF-α、IL-6、IL-8、IL-1β释放（P＜0.05），调节RAGE、TLR2分泌（P＜0.05），促进IFN-γ分泌（P＜0.05），减少嗜酸性粒细胞及中性粒细胞炎性因子释放，有效抑制HMGB1介导的“免疫炎症损伤/异常纤维修复”信号通路；并降低肺组织中TGF-β1及α-SMA的水平（P＜0.05），减少气道胶原及基质沉积，减轻气道纤维化，从而改善气道重塑。