解剖学报

解剖学报 ›› 2023, Vol. 54 ›› Issue (5): 546-552.doi: 10.16098/j.issn.0529-1356.2023.05.007

• 细胞和分子生物学 • 上一篇    下一篇

切应力通过Pim1/Akt调节人脐静脉内皮细胞内皮型一氧化氮合酶Ser633和Ser1177位点磷酸化

  杜大鹏1,2  孙玉2 张敏2 王汉琴1,2*    

  1. 1.湖北医药学院基础医学院解剖学教研室，湖北 十堰 442000; 2.湖北医药学院附属随州医院转化医学研究中心，湖北 随州 441300
  • 收稿日期:2022-06-28 修回日期:2023-01-09 出版日期:2023-10-06 发布日期:2023-12-25
  • 通讯作者: 王汉琴 E-mail:hanqin.wang@hbmu.edu.cn
  • 基金资助:
    国家自然科学基金资助项目;湖北医药学院研究生科技创新项目;湖北医药学院“十四五”湖北省高等学校优势特色学科群（现代医学）项目

Shear stress regulateing the phosphorylation of endothelial nitric oxide synthase Ser633 and Ser1177 in human umbilical vein endothelial cells through Pim1/Akt pathway  

 DU  Da-peng1,2  SUN  Yu2  ZHANG  Min2  WANG  Han-qin1,2*   

  1. 1.Department of Anatomy, Basic Medical College, Hubei University of Medicine, Hubei Shiyan 442000, China;  2.Center for Translational Medicine, Suizhou Hospital, Hubei University of Medicine, Hubei Suizhou 441300, China
  • Received:2022-06-28 Revised:2023-01-09 Online:2023-10-06 Published:2023-12-25
  • Contact: Han-Qin WANG E-mail:hanqin.wang@hbmu.edu.cn

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摘要：

目的  探讨不同模式的血流切应力对人脐静脉内皮细胞（HUVECs）Pim1表达的影响，以及对内皮型一氧化氮合酶（eNOS）Ser1177和Ser633位点磷酸化的调控作用。 方法  体外原代培养的HUVECs，采用平行平板流动腔系统给HUVECs分别施加15 dyn/cm2层流切应力（LSS）和（0.5±4）dyn/cm2振荡切应力（OSS），Western blotting法检测Pim1、Akt和Akt Ser473磷酸化、eNOS、eNOS Ser1177和Ser633位点磷酸化蛋白表达。用特异性小干扰RNA(siRNA)技术分别敲低HUVECs中Pim1和Akt进行干预。  结果  与OSS刺激相比较，LSS显著上调HUVECs中Pim1蛋白表达水平（P<0.01），同时，Akt Ser473磷酸化、eNOS Ser633和Ser1177位点磷酸化蛋白表达增加（P<0.05或 P<0.01）； siPim1和Pim1特异性抑制剂SMI-4a干预后，LSS诱导的Pim1蛋白表达显著被抑制（P<0.05）， 同时Akt Ser473磷酸化、eNOS Ser633和Ser1177位点磷酸化蛋白表达均降低（P<0.05或P<0.01）。敲低Akt，LSS诱导的Akt Ser473磷酸化蛋白表达显著被抑制（P<0.05），同时也显著抑制LSS上调的eNOS Ser633和Ser1177磷酸化蛋白表达（P<0.05），而对LSS上调的Pim1表达无影响（P>0.05）。  结论  切应力可以通过 Pim1/Akt 信号通路调节HUVECs eNOS Ser633和Ser1177位点磷酸化。 

关键词: 切应力, Pim1, 内皮细胞, 内皮型一氧化氮合酶, 免疫印迹法, 人 

Abstract:

 Objective  To explore the effect of different modes of blood flow shear stress on the Pim1 expression in human umbilical vein endothelial cells (HUVECs) and the regulation of phosphorylation at Ser1177. and Ser633 of endothelial nitric oxide synthase (eNOS).     Methods  HUVECs were isolated from fresh human umbilical cord. The parallel plate flow chamber system was used to load 15 dyn/cm2 laminar shear stress (LSS) and (0.5 ± 4) dyn/cm2 oscillatory shear stress (OSS) on HUVECs. Western blotting was used to evaluate the protein levels of Pim1, phosphorylated Akt at Ser473, and phosphorylated eNOS at Ser633 and Ser1177. Small interfering RNA (siRNA) was used to knockdown of Pim1 and Akt.     Results  LSS obviously induced Pim1 protein expression in HUVECs (P<0.01) compared with OSS stimulation. LSS also significantly increased Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser633 and Ser1177 (P<0.05 or P<0.01). Pim1 silencing, or treatment with SMI-4a, an inhibitor of Pim1, abolished the above effects of LSS-stimulated HUVECs (P<0.05 or P<0.01). Knockdown of Akt also prevented the LSS-induced Akt phosphorylation at Ser473 and eNOS phosphorylation both at Ser1177 and Ser633 (P<0.05 or P<0.01). However, Akt knockdown did not alter the expression of Pim1 induced by LSS.     Conclusion  Shear stress can regulate the phosphorylation of eNOS Ser633 and Ser1177 in HUVECs through the Pim1/Akt signaling pathway.  

Key words: Shear stress, Pim1, Endothelial cell, Endothelial nitric oxide synthase, Western blotting, Human

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