高糖诱导内质网应激对血管内皮细胞炎症反应的作用

Effects of Endoplasmic Reticulum Stress in Inducing Inflammation in Vascular Endothelial Cell

【目的】探讨高糖通过诱导内质网应激(ERS)引起人脐静脉内皮细胞(EAhy926)炎症反应的作用机制。【方法】人脐静脉内皮细胞给予正常浓度糖(5.5 mmol/L)或高浓度糖(25 mmol/L)干预0、2、4、8、12 h,或ERS诱导剂毒胡萝卜素(TG)0.5μmol/L干预24 h。Western blot检测下列蛋白表达水平:细胞间黏附因子-1(ICAM-1)、肿瘤坏死因子-α(TNFα)等炎症因子;葡萄糖调节蛋白78(GRP78)、磷酸化真核细胞翻译起始因子2α(p-eIF2α)、活化转录因子4(ATF4)等ERS标志蛋白;以及磷酸化信息传递与转录活化因子3(p-STAT3)和总信息传递与转录活化因子3(t-STAT3)。【结果】炎症因子ICAM-1、TNFα,ERS标志蛋白GRP78、p-eIF2α、ATF4以及p-STAT3等蛋白的表达均随高糖干预时间的延长而逐步增加,除GRP78在干预8 h后达到峰值(P<0.05),其余蛋白均在干预12 h后达到峰值(P<0.05)。给予ERS诱导剂TG干预细胞24 h,GRP78、p-eIF2α、ICAM-1、TNFα的表达水平均明显增加,分别是对照组的5.55倍、1.63倍、1.76倍、2.07倍(P<0.05);此外,p-STAT3的表达水平也有所增加,为对照组的2.15倍(P<0.05),但t-STAT3水平无明显变化(P>0.05)。【结论】高糖可诱导内皮细胞发生ERS与炎症反应,ERS诱导剂可诱发细胞的炎症反应,磷酸化的STAT3信号途径可能起到连接ERS与炎症反应的重要作用。

[Objective] To explore the relationship between endoplasmic reticulum stress （ERS） and inflammation in human umbilical vein endothelial cell lines （EAhy926） exposed to high glucose. [Methods] The EAhy926 cells were incubated in normal glucose （NC, 5.5 mmol/L） or high glucose （HG, 25 mmol/L ） for 0, 2, 4, 8, 12 hours, or exposed to ERS inducer thapsigargin （TG, 0.5 μmol/L） for 24 hours. Western blot analysis was employed to assess the protein expression of inflammatory factors： intercellular adhesion molecule-1 （ICAM-1）, tumor necrosis factor-a （TNFa）, ERS markers： glucose-regulated protein 78 （GRP78）, phosphorylated eukaryotic translation initiation faetor-2a （p-elF2a）, activating transcription factor 4 （ATF4）, and phosphorylated signal transducer and activator of transcription 3 （p-STAT3）, total STAT3 （t-STAT3）. [ Results ] The protein expression of inflammatory factors, ERS markers and p-STAT3, were significantly increased in a time-dependent manner after HG treatment. Except the level of GRP78 was peaked at 8 hours （P 〈 0.05）, all the others were peaked at 12 hours （P 〈 0.05）. Moreover, after exposure of EAhy926 ceils to ERS inducer TG, the protein expression of GRP78, p-elF2a ICAM-1, TNFct increased 5.55, 1.63, 1.76, and 2.07 folds of NC group, respectively （all P 〈 0.05 ）, and p-STAT3 protein expression increased 2.15 folds of NC group （P 〈 0.05）, but there was no significant difference in the protein expression of t-STAT3 between NC and TG groups. [ Conclusion ] High glucose could induce ERS and inflammation in EAhy926 cells. ERS inducer also could induce inflammation in EAhy926 cells. Phosphorylated STAT3 pathway may play an important role in bridging ERS and inflammation.