基于HMGB1/TLR4/NF-κB信号通路研究抵当汤及其拆方干预深静脉血栓形成的作用机制

Deep venous thrombosis (DVT) has been widely concerned due to its complicated with fatal pulmonary embolism and persistent sequelae.Previous studies have demonstrated that inflammatory cytokines and NF-κB/IκB signaling pathway play an important role in the evolution of DVT. However, it is not clear that how the release of inflammatory cytokines to play its amplification effect and to activate NF-κB signaling pathway in the development of thrombosis. It is known to all that Di-dang decoction fomulated by a famous ancient physician called Zhang zhong-jing was used to treated DVT, but the compatibility and action target of two groups in Di-dang decoction remain to be further studied. High mobility group box 1 (HMGB1) is a regulatory factor that plays a central role in the whole process of inflammatory response and can affect many aspects of thrombosis. TLR4 is the main receptor. It is speculated that the HMGB1/TLR4/NF-κB signaling pathway may be the key link between inflammation and thrombosis in DVT. The aim of this study was to dynamically observe the effects of Di-dang Decoction and its decomposed recipes on inflammatory factors, HMGB1, TLR4 and NF-κB in DVT animal model and Human umbilical vein endothelial cells through vitro and in vivo experiments, to reveal the key signal pathway of endothelial injury in DVT, and to explore the targets of compatibility of Di-dang Decoction. The study may reveal a new breakthrough in the pathogenesis of DVT, and provide a new theoretical and experimental basis for the prevention and treatment of DVT in Chinese medicine.

深静脉血栓形成（DVT）因可并发肺栓塞及迁延难愈的后遗症而引起广泛关注。前期研究证实炎症因子及NF-κB/IκB信号通路在DVT的病程演变中起重要作用。但炎症因子的释放在血栓的发生发展中如何发挥其扩增效应以及激活NF-κB通路的具体机制尚不清楚。仲景治疗下焦蓄血症的名方抵当汤中两组药对的配伍意义和作用靶点尚需进一步研究。高迁移率族蛋白B1（HMGB1）是在炎症反应全过程起中枢作用的调节因子，TLR4是其主要受体。我们推测HMGB1/TLR4/NF-κB信号通路是DVT中炎症与血栓之间关键的联结纽带。本课题拟通过离体和在体实验动态观察抵当汤及其拆方对DVT动物模型和脐静脉内皮细胞的炎症因子、HMGB1、TLR4和NF-κB等的干预作用，揭示DVT中内皮损伤的关键信号通路，探讨抵当汤中不同药对配伍的作用靶点，该研究可能会在揭示DVT发病机制上有新突破，为中医防治DVT提供新的理论和实验依据。

深静脉血栓形成（DVT）以其高发病率、急性期可并发致死性肺栓塞、慢性期可发生迁延难愈的后遗症而引起广泛关注。目前该病的治疗方式主要以药物治疗和（或）手术治疗为主。中医药基于整体观念和辨证论治，凭借其多靶点作用、合理配伍可以增效减毒等优点，在治疗DVT方面拥有得天独厚的优势。然而，目前中医药治疗DVT的关键靶点和分子信号通路尚未明确。本研究在国家自然科学基金面上项目“基于HMGB1/TLR4/NF-κB信号通路研究抵当汤及其拆方干预深静脉血栓形成的作用机制（NO：81774311）”资助下，以HMGB1/TLR4/NF-κB信号通路为切入点，通过离体和在体实验动态观察仲景名方抵当汤干预DVT的关键机制。研究内容及创新点主要包括：证实了在血栓状态下，HMGB1/TLR4/NF-κB信号通路与其下游的炎症细胞因子共同参与了与凝血和纤溶系统密切相关的DVT的发生与发展过程，在联结 DVT 凝血系统和炎症介质之间发挥关键联结纽带作用，调控该信号通路可能成为干预DVT的重要途径。抵当汤可以通过下调HMGB1/TLR4/NF-κB的炎症信号通路、增加抗凝活性和纤溶功能，保护内皮细胞而达到抑制炎症反应、促进血栓溶解机化的作用。具有泻热化瘀的大黄-桃仁配伍和逐瘀通络的水蛭-虻虫配伍分别在血栓后的不同时相、对该炎症信号通路和凝血途径的不同靶点表现出了不同优势，但具有泻热逐瘀的抵当汤疗效仍优于以上两组。本研究不仅丰富了仲景蓄血证的论治，诠释了中医瘀热互结病机的理论内涵，而且完善了抵当汤多靶点治疗DVT的分子生物学机制，证实了泻热法和逐瘀法的优势干预靶点，为经方抵当汤治疗DVT提供实验数据支持。这对于阐明DVT的发生发展和转归规律，寻找中药治疗DVT的优势靶点，证明中医药治疗DVT的有效性都具有重要的科学意义。