【作者】 吴勇；

【导师】 陈琪； 季勇；

【作者基本信息】 南京医科大学 ， 病理学与病理生理学， 2010， 博士

【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。更多还原

【Abstract】 Atherosclerosis(AS) is a kind of damaged endothelial cells, smooth muscle cells lining blood vessel invasion, vascular smooth muscle cell proliferation mainly of the pathological process of vascular remodelling,and its incidence is complex and exact cause has not been fully clarified. Numerous studies confirm that platelet activation and aggregation in the occurrence and development of AS has played an important role.Nitric oxide (NO), the primary endothelium-derived relaxing factor, participates in variety of biological activities in vivo, such as vasorelaxation , inhibition of vascular smooth muscle cell proliferation, and inhibition of vascular permeability.At the same time a growing number of studies confirmed the role of NO addition to the above, but also can inhibit platelet aggregation to maintain a balanced environment within the blood vessels, thus preventing the development of cardiovascular diseases. NO is synthesized from the amino acid L-arginine by a family of enzymes called the NO synthases(NOS). Among of the three isoforms(eNOS, iNOS, nNOS), it is reported that the isoform present in platelet is eNOS. While much less is known about the mechanisms determining its activity in platelet.There is a classical signaling transduction that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increasing NO production.Pyridoxine, a major component of vitamin B6, palys vital roles in numerous metabolic processes in the human body. Nowadays, it has been associated with some benefits in non-randomised studies, such as lowing blood homocyserine concentration, reducing platelet aggregation. And the feasible mechanism may be attributable to the role of pyridoxine as a cofactor in several enzymatic reactions in atherosclerosis. But the mechanism on platelets is still unclear.The purpose of this study was firstly to determine whether pydidoxine can increase the platelet NO synthesis in vitro, followed by studing its mechanism of this role. The platelets were extracted from the healthy human to observe whether pydidoxine can inhibit their aggregation induced by ADP or Thrombin,and cGMP(an index of bioactive NO) was measured by radioimmunoassay. eNOS Ser-1177-specific phosphorylation and phosphorylation of protein kinase Akt were determined in platelets by western blot .The activity of phosphatidylinositol-3-kinase was analysed by HTRF.Firstly, we detected a marked concentration-dependent effect of pyridoxine on the platelet aggregation induced by ADP or Thrombin. When the concentration of pyridoxine was 2mmol/L, its ability to inhibit platelet aggregation is no longer increased as the concentration increases. RIA results showed that pyridoxine can increase the biological activity of NO. We further found that , pyridoxine can increase eNOS Ser-1177 phosphorylation and Akt serine phosphorylation, it can also increase PI3K activity and this effect can be inhibited by PI3K inhibitor LY294002. The results of this study show that, pyridoxine can effectively inhibit the ADP or Thrombin-induced platelet activation and increased the biosynthesis of NO on platelet. Pyridoxine play this role by improving the activity of PI3K, thereby increasing the downstream Akt phosphorylation, further increase in eNOS Ser-1177 phosphorylation site. This has important potential therapeutic implications for the use of pyridoxine in the prevention and/or treatment of Thrombosis-related disease.更多还原