20-HETE在非甾体抗炎药致心血管损伤中的作用及机制研究

Non-steroidal anti-inflammatory drugs (NSAIDs) are the common drugs clinically used for the chronic treatment of arthritis. Non-aspirin NSAIDs have all been found to be associated with the increased risks in cardiovascular diseases such as hypertension, myocardial infarct, and stroke. However, the meachanisms underlying NSAIDs-mediated cardiovascular events have not be understood completely. We previously found that (1) chronic administration of rofecoxib resulted in significant increase in plasma level of both 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoconstrictor, and sP-selectin, the biomarker of platelet activation; (2) 20-HETE shortened the tail bleeding time significantly in a murine model; (3) 20-HETE accelerated rodent platelet aggregation time- and dose-dependently. Some NSAIDs have been known to be associated with platelet function disorder and endothelial dysfunction, therefore, we hypothesize that increased circulation level of 20-HETE caused by chronic administration of NSAIDs is involved in the activation of platelet function disorder and endothelial dysfunction, which is the general reason for the NSIADs-mediated cardiovascular events. To test above hypothesis, we propose to investigate the dose- and time-dependent changes in plasma level of 20-HETE caused by NSAIDs administration, which will be correlated to the dose- and time-dependent changes in blood coagulation system (e.g. blood bleeding time, and particular the key biomarkers of platelet function such as sP-selectin, fibrinogen, cAMP, thrombin, t-PA and PAI-1 and key biomarkers of endothelial function such as ICAM-1, VCAM-1, MCP-1 and ADMA) in a animal model. We will further test the dose- and time-dependent effect of 20-HETE on the blood coagulation system by using both an in vitro and an in vivo models. We will finally test the foundings from above studies in associated clinical samples. By correlating the changes in 20-HETE, coagulation biomarkers, and the key signaling pathways, this proposal will bring a comprehensive understanding of the mechanism underlying NSAIDs-mediated cardiovascular events. Based on our findings, novel strategies will be proposed to prevent the risks of cardiovascular events caused by the administration of NSAIDs, creating a safer translational medication.

非甾体抗炎药（NSAIDs）是风湿病患者长期服用的常见药物。已知非阿司匹林类NSAIDs均增加心血管疾病风险，但其致心血管损伤机制不清。前期我们发现：（1）罗非昔布导致小鼠血浆20-HETE和sP-selectin显著升高；（2）20-HETE显著缩短小鼠尾部流血时间；(3) 20-HETE时间和剂量依赖性地促血小板聚集。因sP-selectin是血小板活化标志物，且NSAIDs能引起血小板功能紊乱和内皮细胞功能障碍，故我们认为：由NSAIDs引起的血液20-HETE增高参与激活血小板功能紊乱和内皮细胞功能障碍是其增加心血管疾病风险的重要机制。为此，本项目首先确定非阿司匹林类NSAIDs致20-HETE浓度升高及其与血小板和内皮功能障碍的关系；随后阐明20-HETE损伤血小板和内皮细胞功能的分子机制；最后采用临床样本加以验证。本项目的实施有望为预防NSAIDs致心血管损伤提供新的干预靶点。

非甾体抗炎药（NSAIDs）是风湿病患者长期服用的消炎止痛药。临床研究发现长期服用非阿司匹林类NSAIDs均可增加心血管疾病的风险。NSAIDs增加心血管风险的机制虽然已经有所报道，但并不能统一解释增加心血管风险作用NSAIDs这一类药所共有的副作用的机制。我们前期研究发现长期服用罗菲昔布（Rofecoxib）显著增加小鼠血液20-HETE的浓度，而20-HETE具有显著的心血管毒性。因此我们认为20-HETE增高可能是NSAIDs增加心血管风险的机制。为此：我们首先建立了基于LC-MS/MS的靶向代谢组学平台用以检测包括20-HETE以及其三个主要代谢物20-COOH-ARA，20-OH-PGE2和20-OH-PGF2α在内的近60种多不饱和脂肪酸（PUFAs）的代谢物。对临床样本的检测发现：长期服用NSAIDs患者血浆中20-HETE的浓度略高或基本持平于健康对照，没有统计学意义，但是20-COOH-ARA的浓度显著高于正常对照，而且这些患者全血细胞中血浆中PECAM-1在mRNA水平显著高于健康对照，标志心血管风险的增高。而在小鼠模型的研究中，布洛芬和塞来昔布均能导致小鼠血浆中20-COOH-ARA的显著升高，而20-HETE变化不显著,而且小鼠尾部流血时间与对照小鼠相比显著缩短，血浆PECAM-1浓度显著高于对照小鼠，也标示着心血管风险增加，并且支持临床数据。而且20-HETE和20-COOH-ARA都能增加人脐静脉内皮细胞和动脉内皮细胞的PECAM的表达，20-COOH-ARA的作用强于20-HETE。我们的研究表明：长期服用NSAIDs药物能增加循环系统20-COOH-ARA/20-HETE的浓度，从而增加心血管风险，其机制可能与20-COOH-ARA和20-HETE增加血管内皮细胞的PECAM-1相关。本研究为防治NSAIDs所致心血管损伤提供了一个新的干预靶点。