内皮损伤致血栓形成与KLF2分子调控相关性及益气活血方干预作用的研究

Krüppel-like factor 2 has an important role in the protection of the biological function of the vascular endothelium. On the other hand, the patients with syndrome of blood stasis due to qi deficiency tend to suffer from the damage of endothelial cells, and so on. We hypothesize that Yiqihuoxue Decoction may be used for the treatment of endothelial dysfunction and thrombus formation via KLF2 regulatory pathway. First, we focus on the close relationship between KLF2 expression levels and thrombus formation in vitro and in vivo. Then, using reverse transcription polymerase chain reaction and gain- and loss-of-function approaches, we identify the value of KLF2 on the thrombosis events related to the endothelial dysfunction. For the next step, enzyme linked immunosorbent assay is applied for the observation of the expression of cytokine accompanied with the formation of thrombus in mice, and the regulatory role of KLF2 is elucidated. Finally, the anti-KLF2 mAb or soluble KLF2/IgG1 is used respectively to block in vivo the KLF2 pathways in mice, or KLF2 knock-out mice are used, to explore the effects of KLF2 pathway on endothelial dysfunction and thrombus formation, as well as to assess its mechanism underlying the antithrombotic role. On the basis of above, Yiqihuoxue Decoction is added to observe its intervention on the thrombosis because of endothelial dysfunction. In a word, the accomplishment of this grant will be to clarify the mechanism of KLF2 in thrombus formation caused by endothelial dysfunction and to provide a theoretical basis for the clinical application of Yiqihuoxue Decoction in the treatment of thrombotic diseases.

气虚血瘀证患者存在内皮细胞受损等改变，KLF2转录因子对血管内皮具有重要的保护作用，那么用于治疗气虚血瘀证的益气活血方是否通过KLF2调控途径保护血管内皮、抗血栓形成呢？本项目在阐明KLF2表达与体内外血栓形成密切相关的基础上，应用逆转录-多聚酶链合反应、基因获得以及基因敲除技术探讨KLF2表达水平对于内皮功能紊乱致血栓形成事件的价值；应用酶联免疫吸附法检测血栓形成时小鼠体内炎性因子的表达水平，探讨KLF2的调节作用；利用抗KLF2单抗或可溶性KLF2/IgG1封闭小鼠血栓形成模型体内的KLF2通路，或利用KLF2基因敲除小鼠，研究KLF2调控通路的阻断作用对于内皮功能失调及血栓形成的影响，探讨其作用机制，并观察益气活血方的干预作用。本项目的完成将阐明KLF2在内皮功能紊乱致血栓形成机制中的作用，并为临床应用益气活血方治疗血栓性疾病提供理论依。

由炎症引起的血管内皮功能紊乱受转录因子KLF2调控，其高表达时能够抑制致炎因子诱导内皮促血栓因子表达，是研究药物抗内皮炎症反应、抗血栓形成的潜在靶点。益气活血方广泛用于防治心血管疾病，具有潜在的抗炎、抗血栓疗效。研究结果提示，益气活血方在细胞及动物水平均能明显抑制致炎因子（内毒素、凝血酶）诱导的血管内皮炎症反应（炎性因子TNF-α、IL-6、IL-8分泌及粘附因子VCAM-1、ICAM-1表达）和促血栓因子（PAI-1、TF）表达。运用慢病毒过表达/沉默KLF2蛋白表达水平，发现过表达或沉默KLF2引起促血栓因子表达水平降低或升高，提示益气活血方对血管内皮的保护作用具有KLF2依赖性。应用SB203580（p38 MAPK通路抑制剂）、PDTC（NF-κB通路抑制剂）抑制相应通路，发现益气活血方通过调控p38MAPK及NF-κB通路活性发挥抑制内毒素诱导内皮炎症反应、促血栓因子表达的作用。同时，益气活血方可通过升高UCP2表达水平来减少氧化应激引起的心肌细胞凋亡。益气活血方组成之一的红花提取物羟基红花黄色素A通过降低miR-1表达水平减少氧化应激引起的心肌细胞凋。此项目的完成，阐明了KLF2在炎症引起内皮细胞功能紊乱中的调控作用，并为益气活血方临床防治炎症、血栓相关心血管疾病以及心肌缺血性疾病提供了理论依据。项目资助发表论文5篇，其中SCI论文2篇，中文核心论文2篇。申请专利2项。培养硕士生3名（在读）。项目投入经费72.00万元，实际支出63.67万元，结余8.33万元。各项支出与预算基本相符，结余资金将用于支付待发表的论文版面费和后续深入研究。