补阳还五汤对肺纤维化中诱导免疫炎症/纤维修复的高迁移率族蛋白(HMGB1)调控机制研究

Researches confirms that, 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 IPF. Our previous study found that, the Buyang Huanwu Decoction could inhibit the expression of TGF- β1 and Smad3mRNA of pulmonary fibrosis rats, decrease the content of HYP and TNF-α, and block the up-regulated expression of ERK1/2 and p-ERK, which prompted that, multi-targeted molecular pharmacological mechanism of Buyang Huanwu Decoction had a better regulatory role of the intrapulmonary complex immune and inflammatory microenvironment. Based on this, we put forward the hypothesis "Multi-target role in regulating of Buyang Huanwu Decoction on pulmonary fibrosis may be related to blocking HMGB1, the key medium of fibrogenic generation, and inhibiting intrapulmonary immune inflammation/ maintenance complex signal molecules caused by it. This study focuses on the core theory "abnormal damage repairing" of pulmonary fibrosis' pathogenesis, with HMGB1 protein as the research target, and experimentally explores the possible mechanisms of Buyang Huanwu Decoction in the treatment of pulmonary fibrosis from both in vivo and in vitro cytological test, so as to provide a more scientific approach to study anti-fibrosis drug, and provide more ideas for the development of high quality traditional chinese drug against pulmonary fibrosis .

研究证实，HMGB1是链接内源性免疫损伤和异常修复转化的关键介质，其介导的受体信号通路在IPF中发挥了重要的桥梁作用。我们前期研究发现，补阳还五汤可以抑制肺纤维化大鼠TGF-β1、Smad3mRNA表达，降低HYP、TNF-α含量，阻断ERK1/2、p-ERK的表达上调，提示补阳还五汤的多靶点分子药理机制对IPF肺内复杂的免疫炎症微环境有较好的调控作用。基于此，我们提出了"补阳还五汤治疗肺纤维化的多靶点调节作用可能与其阻断了促纤维化生成的关键介质HMGB1，抑制HMGB1所激活的肺内免疫炎症/纤维修复信号分子有关"的假说。本研究着眼于目前肺纤维化发病机制的"损伤异常修复"核心学说，以HMGB1蛋白为研究靶点，从动物体内实验和体外细胞学实验两方面综合探讨补阳还五汤在肺纤维化治疗中的可能机制，为抗肺纤维化药物的研究提供更科学的途径，为开发高质量抗肺纤维化中药新药提供更多的思路。

肺纤维化（Pulmonary Fibrosis，PF）是以肺实质破坏、纤维斑痕和成纤维细胞灶形成、细胞外基质过度沉积为特征的纤维增殖性疾病。目前IPF的病因和发病机制尚不清楚，缺乏有效治疗手段。最近的证据表明，重要的晚期炎症因子高迁移率族蛋白B1（HMGB1），能在细胞核内作为α平滑肌动蛋白(α-SMA)基因的转录活化因子诱导上皮细胞发生间质转化（EMT），成为肺纤维化成纤维细胞灶中肌成纤维细胞的重要来源。在细胞外能启动丝裂原活化蛋白激酶（MAPKs）、核因子κB（NF-κB）等多条信号通路，激活免疫炎症和异常修复。根据IPF的临床特征和病机变化特点，在祖国医学中归属于“肺痿”、“肺痹”等疾病范畴，病因为外邪侵袭、久病伤肺、素体气虚等，产生痰浊、瘀血、热毒等病理产物，引起肺肾亏虚、气虚血瘀、痹阻肺络等病机改变，治疗应以益气活血通络、祛痰滋阴温阳为重点。本研究以HMGB1为靶点，以人肺成纤维细胞（HFL1）、人非小细胞肺癌细胞（A549）和博莱霉素致纤维化大鼠为受试对象，从气虚血瘀的核心病机入手，结合血清药理学，采用益气活血通络的“补阳还五汤”在体内外共同干预的方法，探讨补阳还五汤治疗肺纤维化的分子机制。体外实验结果显示补阳还五汤含药血清干预后，可降低α-SMA、ERK1/2、p-ERK1/2、NF-κBp65、IL-1β、TNF-α、STAT3、p-STAT3 的表达；体内实验结果显示使用不同剂量的补阳还五汤干预后，可不同程度降低HMGB1、RAGE、TLR2、TLR4、α-SMA、IL-1β、TNF-α、和IL-6的水平。补阳还五汤可有效抑制纤维化大鼠肺组织中HMGB1、RAGE和α-SMA的表达并可在体外阻断HMGB1所激活的多个与炎症和纤维化相关的信号通路，减轻免疫损伤，抑制上皮细胞和成纤维细胞向肌成纤维细胞转化，对肺纤维化产生多靶点的调控作用。