当归补血汤预防肝硬化门静脉血栓的益气活血治法特点及其保护血管内皮损伤的作用机制研究

Portal vein thrombosis is a common complication of liver cirrhosis, which can aggravate the development of portal hypertension, which is closely related to the injury of vascular endothelial cells and the activation of protein C system. Based on the effective experimental results and clinical experience of Tonifying qi and activating blood circulation of Danggui Buxue Tang, we put forward the following hypothesis: Danggui Buxue Tang can prevent and treat liver cirrhosis with portal vein thrombosis, with the characteristic of Tonifying qi and activating blood circulation treatment, through affecting the vascular endothelial cells injury from oxidative damage and protein C system activation. First, the grant will replicate CCl4 induced factor V Leiden mice liver cirrhosis portal vein thrombosis model, observe the effect of the three kinds of preparations(Danggui Buxue Tang with Astragalus and Angelica three proportions of 3:3, 5:1, 1:5) on liver function，blood coagulation function, especially the changes of portal vein thrombosis. Then we choose the better compatibility proportion Danggui Buxue Tang to investigate the cellular and molecular pharmacological mechanism around the NF-kB signaling pathway in endothelial cell. In order to clarify the effect and compatibility features of Danggui Buxue Tang on the prevention and treatment of liver cirrhosis portal vein thrombosis, provide a theoretical basis for clinical effective prevention and treatment of portal vein thrombosis.

门静脉血栓是肝硬化常见并发症，可加重门静脉高压症发展，与血管内皮细胞损伤及蛋白C系统活化密切相关，中医病机多表现为气虚血瘀。基于益气活血的当归补血汤良好治疗肝硬化的实验结果及临床经验，提出以下假说：当归补血汤可防治肝硬化门静脉血栓，具有益气活血配伍特点，主要通过影响血管内皮细胞氧化损伤及蛋白C系统活化而发挥作用。首先，复制CCL4诱导factor V Leiden小鼠肝硬化门静脉血栓模型，采用黄芪当归3种比例配伍(3:3,5:1,1:5)的当归补血汤干预，以抗凝药华法林为对照，观察肝功能、凝血功能、尤其是门静脉血栓变化；继而选择配比疗效较优的当归补血汤，围绕内皮细胞的NF-kB信号通路，探讨该方的细胞分子药理机制。从而明确当归补血汤防治肝硬化门静脉血栓的治法配伍特点及主要作用机制，为临床有效防治肝硬化门静脉血栓提供理论依据。

门静脉血栓是肝硬化的常见并发症，可直接加重门静脉高压症，加速肝纤维化、肝硬化进展，与血管内皮损伤密切相关。首先尝试采用门静脉主干部分结扎复合CCl4构建肝硬化门静脉血栓大鼠模型，动态观察4周、6周、8周及10周大鼠模型是否兼具门静脉血栓与肝硬化的双重特点；进而采用上述复合模型存活大鼠，随机分为当归补血汤高、低剂量组、阳性药（利伐沙班）对照组，另设正常对照组，自造模第5周开始药物干预6周，观察不同剂量当归补血汤对大鼠门静脉血栓、门静脉血流速度，PT、INR、APTT、抗凝血酶III、D-二聚体、PLT等凝血功能指标，肝纤维化相关指标，以及vWF、Rab3B/Rab27A、eNOS表达；SOD活性与MDA含量；NF-κBmRNA水平等血管内皮损伤相关机制的影响。收集与比较部分曙光医院肝硬化有/无门静脉血栓两组患者资料，分析肝硬化并发门静脉血栓的独立危险因素，根据四诊信息归纳其中医证候特点；进一步随访中药干预组与未干预组的临床预后，归纳其中医治法特点。结果：①门静脉主干部分结扎模型复合CCl4染毒10周构建肝硬化门静脉血栓大鼠模型成立，该模型具有门静脉血栓和肝硬化的双重特点；②当归补血汤具有抗门静脉主干部分结扎复合CCl4模型大鼠肝纤维化，增加门静脉血流，改善凝血功能的作用；其部分作用机制与改善内皮损伤、抗脂质过氧化、下调NF-κB表达有关。③当归补血汤对CoCl2诱导的内皮细胞损伤有一定的保护作用，且其作用机制与抗脂质过氧化，改善内皮细胞舒缩功能，抑制TNFα表达有关，但对促凝/抗凝相关分子无明显影响。④肝硬化并发PVT的中医证候特点符合气虚血瘀证表现，益气活血中药可改善肝硬化并发PVT患者肝功能及门静脉血栓再通率；D二聚体、脾脏长径是其独立危险因素。