慢性间歇性缺氧激活内质网应激PERK-eIF2α-ATF4通路损伤HUVECs胰岛素信号转导

Endothelial insulin signaling is impaired by chronic intermittent hypoxia-induced endoplasmic reticutum stress in human umbilical vein endothelial cells

目的:探讨慢性间歇性缺氧(CIH)对人脐静脉内皮细胞(HUVECs)胰岛素转导通路的影响,并观察内质网应激在其中的作用。方法:建立CIH的HUVECs细胞模型,采用Western Blot方法在HUVECs中观察CIH对基础和胰岛素刺激(100μmol)后内皮型一氧化氮合酶(e NOS)和蛋白激酶B(AKT)的磷酸化水平的影响;观察CIH对HUVECs内质网应激关键蛋白表达水平的影响;应用内质网应激抑制剂牛磺熊去氧胆酸(TUDCA,100μM)预孵育,检测CIH状态下HUVECs胰岛素信号转导通路变化。结果:与常氧组相比,CIH可显著升高基础状态下HUVECs中AKT的和e NOS的磷酸化水平(P<0.05)。给予100 nmol/L胰岛素刺激后,常氧组HUVECs中AKT和e NOS的磷酸化水平与胰岛素未刺激组相比显著升高(均P<0.05);CIH干预48h和72h后,胰岛素刺激后HUVECs中AKT和e NOS磷酸化水平与基础状态无明显变化。与常氧组相比,CIH可时间依赖性升高HUVECs中蛋白激酶R样内质网激酶(PERK)、真核起始因子2α(e IF2α)的磷酸化水平以及活化转录因子4(ATF4)的蛋白表达水平(均P<0.05)。TUDCA预孵育可逆转上述CIH诱导的HUVECs胰岛素信号转导损伤。结论:CIH激活内质网应激PERK-e IF2α-ATF4通路,进而损伤HUVECs的AKT-e NOS胰岛素信号转导通路。

Objective ：We studied the effects of chronic intermittent hypoxia （CIH） on insulin signaling in human umbilical vein endothelial cells （HUVECs） and the possible role of endoplasmie retieulum （ER） stress in it. Methods： HUVECs were pre-treated with ERS inhibitor taurodeoxyeholic acid （TUDCA） and treated with CIH. Basal and insulin-induced phosphorylation of AKT and endothelial nitric oxide synthase （eNOS） and protein expression of ER stress signaling were measured by Western Blot. Results： In basal condition, CIH enhanced the ratio of phospho （ P）-AKT/ AKT and P-eNOS/eNOS compared with normoxic group （ All P 〈 0. 05）. In insulin-stimulated condition, phosphorylation of AKT and eNOS was reduced by CIH, which was coupled to increased protein expression of activating transcription factor 4 （ATF4） and phosphorylation of protein kinase R-like ER kinase （PERK） and eukaryotic translation initiation factor 2α （eIF2α） （ All P 〈 0. 05） ; these phenomena were reversed by pre-treated TUDCA. Conclusion： Our data show that CIH impairs insulin- dependent activation of the AKT/eNOS pathway in HUVECs possibly through ER stress pathway, which can be attenuated by TUDCA.