基于COX信号通路探索参附注射液调控TXA2/PGI2平衡防治血栓闭塞性脉管炎的分子机制

Thromboangiitis obliterans (TAO) is a chronic nonatherosclerotic segmental occlusive vasculitis with arterial lumen thrombosis, which most commonly involves the small- and medium-sized arteries and veins of the extremities and usually leads to gangrene and tissue loss. After a century, the puzzle of TAO has not yet been solved and the trigger of the disease is still controversial. It is reported that thrombosis plays a prominent role in the pathogenesis of TAO. Prostacyclin (PGI2) and thromboxane A2 (TXA2) are the two most important prostanoids modulating vascular thromboresistance. The balance between endothelial PGI2 and TXA2 generation is thought to regulate a wide array of vascular processes including thromboresistance. Vascular endothelial cell (VEC) is exposed to blood and its damage is the basic factor triggering the pathological changes of TAO. A hypercoagulable state of blood in TAO rooted in the injuried VEC, from which higher TXB2 (a stable product of TXA2) and lower 6-K-PGF1 (a stable product of PGI2) levels, and TXA2/PGI2 imbalance occurred and lead to thrombosis and TAO.This theory is demonstrated by many researches including our recent reports. It is well known that the rate limiting step in PGI2 and TXA2 generation is metabolism of arachidonic acid to the prostaglandin intermediary H2 (PGH2) by the cyclooxygenase-1(COX1) and COX2. PGH2 is subsequently converted to PGI2 and TXA2 by prostacyclin synthase (PGIS) and thromboxane synthase(TXAS), respectively.However, few literatures involved the relations between TXA2/PGI2 imbalance and those prostanoid synthesis synthase in TAO. Shenfu injection (SF) which originated from Shenfu Tang, a traditional Chinese formulation, is mainly composed of the extract of radix ginseng and radix aconitum carmichaeli root. Our findings firstly indicated that SF promotes TXA2 / PGI2 balance, improves the antithrombotic function of VEC and reduces thrombosis in TAO rat. But it was largely unknown whether the protective effects of SF related to its influences on COX1, COX2, PGIS and TXAS in TAO. Here, using several agonists and/or antagonists of these synthases in COX signaling pathway, we will conduct some experiments such as in vivo rat TAO model,femoral artery rings and in vitro TAO model of VEC to focus on elucidating the following topics: 1. the correlation between the imbalance of TXA2/ PGI2 ratio and the alterations of TXAS, COX1, COX2 gene and protein expressions, distributions and activities in TAO models,together with femoral vasorelaxation; 2.whether the effects of SF regulating the balance of TXA2/ PGI2 ratio involved in its influences on the gene and protein expressions, distributions and activities of TXAS, PGIS and COX1, and COX2, as well as on the vasorelaxation of femoral artery.These works if completed will further clarify the pathogenesis of TAO and the protective mechanism SF against TAO. It will provide new ideas and new targets for traditional Chinese medicine to clinically prevent and treat TAO disease.

血栓闭塞性脉管炎（TAO）是以中小动静脉节段性非化脓性炎症和动脉腔内血栓形成为特征的慢性闭塞性疾病。TAO病程中血液高凝状态在于血管内皮细胞损伤引起血栓素（TXA2）升高和前列环素（PGI2）降低，而TXA2/PGI2失衡致血栓形成是TAO发病主因。我们最近发现参附注射液(SF)能促进TXA2/PGI2平衡保护TAO大鼠血管抗血栓功能，减少血栓形成。本课题拟从整体动物-血管环-细胞分子水平，基于COX信号通路干预TXA2和PGI2合成，探索：①TAO病程中TXA2/PGI2失衡与其合成酶TXAS、PGIS及上游限速酶COX1和COX2表达、分布和活性改变相关性及对股动脉张力的影响；②SF调控TXA2/PGI2平衡与其影响TXAS、PGIS、COX1和COX2表达、分布和活性的相关性及调节血管张力作用。阐释TXA2/PGI2失衡致TAO发病及SF调控机制。为临床防治TAO提供新靶点和新思路。

血栓闭塞性脉管炎（TAO）是一种以中小动静脉节段性、非化脓性炎症和动脉腔内血栓形成为特征的慢性进行性闭塞性疾病,是严重威胁患者健康的顽症重疾。其发病机制尚未完全清楚。目前认为血栓形成是TAO发病机制中的关键因素，导致TAO基本病理改变的始动环节肯定是血管内皮细胞(,VEC)损伤。其中TXA2/PGI2这一凝血-抗凝机制的严重失衡被认为是血栓形成的病理生理学基础，提示血浆中TXA2和PGI2的平衡失调是导致TAO的主要原因。参附注射液(SF)是红参和附子的提取物,是中医“回阳救逆”古方-参附汤经分离提取灭菌制成。我们前期研究表明SF可能通过抑制血小板聚集，提高血管抗血栓功能，减少TAO模型大鼠血栓形成，改善其病变体征。但SF减少TXA2、增加PGI2和维持TXA2/PGI2这一凝血-抗凝平衡的作用机制尚不清楚。本项目制作在体TAO模型、LPS诱导原代培养HUVECs损伤模型，从整体和细胞分子水平上，进一步研究TAO发病机理及SF防治TAO的作用机制。结果发现：1）SF防治TAO与其减轻血管内皮病理损伤、改善血液流变学和抑制TXA2/PGI2异常有关。2）TAO模型大鼠TXA2含量增加与其合成酶TXAS、以及上游限速酶COX1、COX2基因和蛋白表达上调有关；3)TAO模型大鼠PGI2含量减少与其合酶PGIS及上游限速酶COX1、COX2的基因和蛋白表达下调有关。4）SF减少TXA2涉及其下调受损伤的股动脉中TXAS、COX1和COX2的基因和蛋白表达、分布；5)SF增加PGI2主要与其下调受损伤的股动脉中PGIS、COX1和COX2的基因和蛋白表达有关。6）SF能拮抗VEC的脂质过氧化反应保护血管内皮细胞的结构和功能，是其防治TAO的作用机制之一。7）成功进行了原代人脐静脉内皮细胞HUVECs的培养，并初步建立LPS诱导HUVECs损伤的离体TAO模型：包括细胞形态学、细胞生存率、细胞凋亡、膜脂质过氧化和细胞上清液中TXA2/PGI2比值变化特点；并明确了SF减轻LPS诱导原代培养HUVECs的损伤与其对这些指标改变有关。这些成果进一步阐释了TXA2/PGI2失衡导致TAO的发病机理，以及SF保护VEC抗血栓能力、促进TXA2/PGI2这一凝血-抗凝平衡防治TAO的作用机制。为临床防治TAO这一顽症重疾提供新思路、新靶点和新途径。