[伊布卡林改善缺氧/复氧处理的人脐静脉内皮细胞衍生的微泡损伤的胸主动脉环对乙酰胆碱的舒张反应]

[Iptakalim ameliorates relaxation to acetylcholine in thoracic aortic rings impaired by microvesicles derived from hypoxia/reoxygenation-treated HUVECs].

作者信息

Kun-Wei Zhang, Shao-Xun Wang, Ye-Yi L I, Su Wei, Man Shang, Chao Liu, Miao Liu, Yi-Lu Wang, Qian Zhu, Yan-Na W U, Jun-Qiu Song, Yan-Xia Liu

机构信息

Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.

出版信息

Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2016 Jun 8;32(6):481-486. doi: 10.13459/j.cnki.cjap.2016.06.001.

DOI:10.13459/j.cnki.cjap.2016.06.001

PMID:29926612

Abstract

OBJECTIVE

To investigate the effect of Iptakalim (Ipt) preventing injury of endothelial microvesicles (EMVs) derived from hypoxia/reoxygenation (H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism.

METHODS

H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope (TEM). Thoracic aortic rings of rats were incubated with 10-10 mol/L Ipt and co-cultured with 10 g/ml H/R-EMVs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) was recorded in vitro. The nitric oxide (NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinas (Akt) and phosphorylated Akt (p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting.

RESULTS

H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles (100-1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner (<0.05, <0.01). The expression of total eNOS (t-eNOS) and total Akt (t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-eNOS and p-Akt increased after treated with Ipt (<0.01).

CONCLUSIONS

Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-eNOS and p-Akt expression.

摘要

目的

研究埃他卡林（Ipt）对缺氧/复氧（H/R）处理的人脐静脉内皮细胞（HUVECs）来源的内皮微泡（EMVs）损伤大鼠胸主动脉环舒张功能的影响，并探讨其潜在机制。

方法

建立H/R损伤模型以从HUVECs释放H/R-EMVs。通过超速离心从条件培养基中分离HUVECs来源的H/R-EMVs。使用透射电子显微镜（TEM）对H/R-EMVs进行表征。将大鼠胸主动脉环与10-10 mol/L Ipt孵育，并与10 μg/ml H/R-EMVs共培养4小时，然后在体外记录其对乙酰胆碱（ACh）的内皮依赖性舒张反应。使用Griess试剂测量ACh处理的大鼠胸主动脉环中一氧化氮（NO）的产生。通过蛋白质免疫印迹法检测大鼠胸主动脉环中内皮型一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS，Ser-1177）、丝氨酸/苏氨酸激酶（Akt）和磷酸化Akt（p-Akt，Ser-473）的表达。

结果

H/R处理的HUVECs诱导产生H/R-EMVs，并通过超速离心分离。经TEM观察，分离出的H/R-EMVs呈现为被膜包围的小圆形囊泡（100-1000 nm）。H/R-EMVs显著损害大鼠胸主动脉环由ACh诱导的舒张。与单独的H/R-EMVs处理相比，Ipt处理可使大鼠胸主动脉环的舒张和NO产生呈浓度依赖性增加（<0.05，<0.01）。Ipt或H/R-EMVs对总eNOS（t-eNOS）和总Akt（t-Akt）的表达无影响。然而，Ipt处理后p-eNOS和p-Akt的表达增加（<0.01）。