Furukawa Kazuo, Fuse Ichiro, Iwakura Yuriko, Sotoyama Hidekazu, Hanyu Osamu, Nawa Hiroyuki, Sone Hirohito, Takei Nobuyuki
Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Asahimachi, Niigata, 951-8585, Japan.
Department of Hematology, Endocrinology and Metabolism, Niigata University Faculty of Medicine, Niigata, Japan.
Cardiovasc Diabetol. 2017 Feb 8;16(1):20. doi: 10.1186/s12933-017-0505-y.
Brain-derived neurotrophic factor (BDNF) exerts beneficial effects not only on diabetic neuropathies but also on cardiovascular injury. There is argument regarding the levels of serum BDNF in patients with diabetes mellitus (DM). Because BDNF in peripheral blood is rich in platelets, this may represent dysregulation of BDNF release from platelets. Here we focused on advanced glycation end products (AGEs), which are elevated in patients with DM and have adverse effects on cardiovascular functions. The aim of this study is to elucidate the role of AGEs in the regulation of BDNF release from human platelets.
Platelets collected from peripheral blood of healthy volunteers were incubated with various concentrations of AGE (glycated-BSA) at 37 °C for 5 min with or without BAPTA-AM, a cell permeable Ca chelator, or PP2, a potent inhibitor of Src family kinases (SFKs). Released and cellular BDNF were measured by ELISA and calculated. Phosphorylation of Src and Syk, a downstream kinase of SFKs, in stimulated platelets was examined by Western blotting and immunoprecipitation.
AGE induced BDNF release from human platelets in a dose-dependent manner, which was dependent on intracellular Ca and SFKs. We found that AGE induced phosphorylation of Src and Syk.
AGE induces BDNF release from human platelets through the activation of the Src-Syk-(possibly phospholipase C)-Ca pathway. Considering the toxic action of AGEs and the protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes. Chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM.
脑源性神经营养因子（BDNF）不仅对糖尿病性神经病变有有益作用，对心血管损伤也有作用。关于糖尿病（DM）患者血清BDNF水平存在争议。由于外周血中的BDNF在血小板中含量丰富，这可能代表血小板释放BDNF的调节异常。在此，我们关注晚期糖基化终产物（AGEs），其在DM患者中升高且对心血管功能有不良影响。本研究的目的是阐明AGEs在调节人血小板释放BDNF中的作用。
将从健康志愿者外周血中采集的血小板与不同浓度的AGE（糖化牛血清白蛋白）在37℃孵育5分钟，同时加入或不加入细胞可渗透的钙螯合剂BAPTA-AM或Src家族激酶（SFKs）的强效抑制剂PP2。通过酶联免疫吸附测定法（ELISA）测量并计算释放的和细胞内的BDNF。通过蛋白质印迹法和免疫沉淀法检测刺激后血小板中Src和SFKs的下游激酶Syk的磷酸化情况。
AGE以剂量依赖性方式诱导人血小板释放BDNF，这依赖于细胞内钙和SFKs。我们发现AGE诱导Src和Syk磷酸化。
AGE通过激活Src-Syk-（可能是磷脂酶C）-钙途径诱导人血小板释放BDNF。考虑到AGEs的毒性作用和BDNF的保护作用，可以推测AGE诱导的BDNF释放是糖尿病早期的一种生物防御系统。在DM进展过程中，AGEs的长期升高可能导致血小板中BDNF的耗竭或下调。
