Pyridoxine对血小板源性一氧化氮生物合成的影响及其作用机制的研究

发布时间：2018-04-25 04:21

本文选题：pyridoxine + 血小板　；参考：《南京医科大学》2010年博士论文

【摘要】： 动脉粥样硬化(Atherosclerosis, AS)是一种以内皮细胞受损、平滑肌细胞侵入血管内膜、血管平滑肌细胞增殖为主的血管重塑的病理过程,其发病过程十分复杂,其确切病因尚未完全阐明。大量的研究证实血小板的激活和聚集在AS的发生发展中发挥了重要作用。一氧化氮(nitric oxide, NO)是内皮产生的最主要的舒张因子,其参与了体内多种生理性活动,例如舒张血管、抑制血管平滑肌细胞的增殖、降低血管的渗透性。同时越来越多的研究证实NO除以上作用外,还能够抑制血小板的聚集,维持血管内环境的平衡,从而预防多种心血管疾病的发生与发展。NO是L-精氨酸在一氧化氮合酶(NOS)催化下的产物,NOS有三种亚型:内皮型(eNOS)、神经型(nNOS)、诱导型(iNOS)。目前认为,血小板上主要存在eNOS和iNOS,其中发挥主要作用的是eNOS。已知PI3K/Akt途径是影响eNOS活性的经典的信号转导通路,但是目前对于血小板上eNOS的活性调控研究并不多。 Pyridoxine是维生素B6的有效的生物成分,参与人体多种代谢过程并发挥了重要的作用,尤其是已经有研究报道,维生素B6能够增加内皮NO的生物合成,并在心血管疾病中发挥治疗作用。现阶段对Pyridoxine的研究发现,其具有降低血液中高同型半胱氨酸浓度的作用,以及抑制血小板聚集等多种功效,对于其机制的研究最初认为Pyridoxine在体内作为一种辅酶参与多种代谢活动,但其在血小板上的作用机制目前尚未有报道。本研究的目的首先是检测维生素B6在体外是否能增加血小板NO的合成,其次研究其发挥这一作用的机制。提取正常人的血小板,观察Pyridoxine是否能抑制聚诱剂诱导的血小板的聚集,及其对血小板上cGMP(NO生物活性检测指标)的水平的影响。用Western blot检测eNOS Ser1177磷酸化水平和蛋白激酶Akt磷酸化水平。HTRF分析血小板上磷脂酰肌醇-3-激酶的活性。研究结果显示,Pyridoxine呈浓度依赖性的抑制由ADP或者凝血酶引起的血小板聚集,当浓度达到2mmol/L时,其抑制血小板聚集的能力不再随着浓度的增加而增加。放射免疫法试验结果显示Pyridoxine能增加NO的生物活性。我们进一步研究发现,Pyridoxine能增加eNOS Ser1177位磷酸化,增加Akt丝氨酸磷酸化,增加PI3K的活性,并且这一作用能够被PI3K的抑制剂LY294002抑制。本研究结果表明,Pyridoxine能够有效抑制ADP或凝血酶诱导的血小板的激活,并增加血小板上NO的生物合成。该作用是通过改善PI3K的活性,增加下游Akt磷酸化,进一步增加eNOS Ser 1177位点磷酸化来实现的。本研究为pyridoxine对于血栓性疾病相关的疾病的治疗和/或预防提供了理论基础。
[Abstract]:Atherosclerotic atherosclerosis (ASS) is a pathological process of vascular remodeling with endothelial cells damaged smooth muscle cells invading the intima and vascular smooth muscle cells proliferating. The pathogenesis of ASS is very complicated and its exact etiology has not been fully elucidated. Numerous studies have confirmed that platelet activation and aggregation play an important role in the development of as. Nitric oxide (no) is the most important relaxation factor produced by endothelium, which is involved in many physiological activities, such as vasodilating, inhibiting the proliferation of vascular smooth muscle cells and reducing the permeability of blood vessels. At the same time, more and more studies have proved that no can inhibit platelet aggregation and maintain the balance of the intravascular environment in addition to the above effects. Therefore, to prevent the occurrence and development of many cardiovascular diseases. No is a product of L-arginine catalyzed by nitric oxide synthase (NOS). There are three subtypes of nitric oxide synthase (NOS): endothelial-type eNOSN, nerve-type nNOSN, and inducible iNOS. At present, eNOS and iNOS exist mainly in platelets, among which eNOSs play a major role. It is known that PI3K/Akt pathway is a classical signal transduction pathway affecting the activity of eNOS, but there are few studies on the regulation of eNOS activity in platelets. Pyridoxine is an effective biological component of vitamin B6, which is involved in many metabolic processes and plays an important role in human body. Especially, it has been reported that vitamin B6 can increase the biosynthesis of no in endothelial cells. And play a role in the treatment of cardiovascular disease. Recent studies on Pyridoxine have shown that it can reduce the concentration of homocysteine in blood and inhibit platelet aggregation. As a coenzyme, Pyridoxine is thought to be involved in many metabolic activities in vivo, but the mechanism of its action on platelets has not been reported. The aim of this study was to investigate whether vitamin B _ 6 could increase platelet no synthesis in vitro and to study the mechanism by which vitamin B _ 6 could play this role. To observe whether Pyridoxine can inhibit platelet aggregation induced by polymeric attractant and its effect on the detection of cGMP(NO bioactivity on platelets. The activity of phosphatidylinositol-3-kinase on platelets was determined by Western blot, eNOS Ser1177 phosphorylation level and protein kinase Akt phosphorylation level. The results showed that Pyridoxine inhibited platelet aggregation induced by ADP or thrombin in a concentration-dependent manner. When the concentration reached 2mmol/L, its ability to inhibit platelet aggregation was no longer increased with the increase of concentration. The results of radioimmunoassay showed that Pyridoxine could increase the biological activity of no. We further found that Pyridoxine could increase the phosphorylation of eNOS Ser1177 site, increase Akt serine phosphorylation, and increase the activity of PI3K, which could be inhibited by LY294002, an inhibitor of PI3K. These results suggest that Pyridoxine can effectively inhibit the activation of platelets induced by ADP or thrombin and increase the biosynthesis of no on platelets. This effect was achieved by improving the activity of PI3K, increasing the phosphorylation of downstream Akt and further increasing the phosphorylation of eNOS Ser 1177 site. This study provides a theoretical basis for the treatment and / or prevention of thrombotic disease-related diseases by pyridoxine.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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