中国血液净化

中国血液净化 ›› 2019, Vol. 18 ›› Issue (09): 630-634.

• 基础研究 • 上一篇    下一篇

内皮素-1促进人腹膜间皮细胞增殖诱导腹膜血管新生的机制

朱楠1,贾洁爽1,杨满1,谷立杰1,王玲1,袁伟杰1   

  1. 1. 上海交通大学附属第一人民医院肾内科
  • 收稿日期:2019-04-23 修回日期:2019-06-12 出版日期:2019-09-12 发布日期:2019-09-02
  • 通讯作者: 袁伟杰ywj4169@163.com E-mail:ywj4169@163.com
  • 基金资助:
    上海交通大学医工交叉基金(项目编号：YG2016QN27)；上海市卫计委青年项目(项目编号：20164Y0234)；
    中华医学会临床医学科研专项资金-施维雅肾脏病青年研究与发展项目(项目编号：17010080677)；
    国家自然科学基金(项目编号：81800676)

The mechanism of endothelin-1 promoting peritoneal mesothelial cell proliferation-induced peritoneal angiogenesis#br#

  1. 1Department of Nephrology, the First Affiliated Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, China
  • Received:2019-04-23 Revised:2019-06-12 Online:2019-09-12 Published:2019-09-02

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摘要：

【摘要】目的探讨激活内皮素-1(endothelin-1,ET1)导致腹膜血管新生是腹膜损伤的一种潜在的作用机制。方法培养人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)(HMrSV5)，高糖刺激HPMCs，观察ET1 的变化；加入外源性ET1，Western blot 检测细胞外信号调节激酶1/2(ERK1/2)和E26 转录因子-1(Ets-1)磷酸化；分别使用siRNA 和封闭性抗体抑制Ets-1，通过BrdU 测定HPMC 的增殖；最后，通过小管形成实验，使用Image J 软件分析5 个随机选择的区域的总毛细管长度。结果高糖可诱导HMPC 高表达ET1(2.5%，F=5.561，P=0.036；4.25%，F=4.784，P=0.008)；外源性ET1 诱导下，HPMC 中ERK1/2 的磷酸化水平显著上调(F=66.347，P=0.026)；ERK1/2 下游的转录因子Ets- 1 表达显著升高(F=110.614，P=0.031)；阻断MEK-1 显著消除ET1 对ERK 1/2 的激活(F=56.361，P=0.006)；加入ETA和ETB受体抑制剂后，ETAR 抑制剂可明显降低Ets- 1 的表达(F=131.191，P=0.039)。ET1 可诱导HPMCs 增殖(F=4.671，P=0.046)，其可被ET1 拮抗剂减弱(F=5.329，P＜0.001；F=5.362，P＜0.001)；抗体和siRNA 介导的Ets-1 阻断具有相似的抗增殖作用(F=5.638，P＜0.001；F=4.611，P=0.007)。因此，ET1 通过ERK1/2-Ets-1 信号传导途径特异性地促进HPMCs 增殖。另外，ET1 处理的HPMsC 的条件培养基可以显著促进HPMCs 中血管内皮生长因子(vascular endothelial growth factor,VEGF)产生(F=11.614，P＜0.001)和内皮细胞血管生成。结论ET1 通过ERK1/2-Ets-1 信号通路促进HPMCs 增殖，并促进VEGF 产生和内皮细胞血管生成。

关键词: 腹膜透析, 内皮素-1, 细胞外信号调节激酶1/2, E26 转录因子-1, 血管内皮生长因子, 血管生成

Abstract:

【Abstract】Objective To investigate the mechanism of endothelin-1 (ET1) activation leading to the peritoneal mesothelial cell proliferation-induced peritoneal angiogenesis. Methods Human peritoneal mesothelial cells (HPMCs) (HMrSV5) were cultured and underwent high glucose stimulation. The secretion of ET1 from HPMCs was assayed. Exogenous ET1 was added in the medium and phosphorylated extracellular signalregulated kinase 1/2 (ERK1/2) and E26 transformation specific- 1 (Ets- 1) were detected by western blot. Knockdown and blockage of Ets-1 were carried out by siRNA interference and anti-Ets-1 antibody, respectively.
The proliferation of HPMCs was determined by BrdU method. Total capillary length of the five randomly selected regions was analyzed by capillary formation experiments and Image J software. Results High glucose stimulation induced the higher expression of ET1 in HMPCs (2.5% glucose, F=5.561, P=0.036; 4.25% glucose, F=4.784, P=0.008). Exogenous ET1 promoted the increases of phosphorylated ERK 1/2 (F=66.347, P=0.026) and Ets-1 (F=110.614, P=0.031), the downstream molecule of ERK1/2, in HPMCs. Blocking MEK-1 in HPMCs could significantly abolish the activation of ERK 1/2 induced by ET1 (F=56.361, P=0.006). After the addition of ETA and ETB receptor inhibitors to HPMCs, ETA receptor inhibitor was found to reduce the expression of Ets- 1 (F=131.191, P=0.039). ET1 was capable of inducing HPMC proliferation (F=4.671,P=0.046), which could be attenuated by ET1 antagonists (F=5.329, P＜0.001; F=5.362, P＜0.001). Knockdown and blockage of Ets- 1 had similar anti- proliferative effects (F=5.638, P<0.001; F=4.611, P=0.007).These findings suggest that ET1 specifically promotes HPMC proliferation through the ERK1/2-Ets-1 signaling pathway. In addition, HPMCs in conditional media and stimulated with ET1 significantly promoted VEGF production (F=11.614, P＜0.001) and endothelial cell angiogenesis. Conclusion ET1 promotes HPMCs proliferation, VEGF production and endothelial cell angiogenesis via the ERK1/2-Ets-1 signaling pathway.

Key words: Peritoneal dialysis, Endothelin-1, ERK1/2-Ets-1, Angiogenesis

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