Nox1/4抑制剂减轻脂多糖致人脐静脉融合细胞的损伤

Nox1/4 inhibitor reduces lipopolysaccharide-induced injury in EA.hy926 cells

目的研究Nox1/4抑制剂对脂多糖致人脐静脉融合细胞EA.hy926损伤的保护作用及相关机制。方法将EA.hy926细胞分为对照组、LPS(脂多糖)组、Nox1/4抑制剂(GKT137831)+LPS组。CCK8法检测细胞活力;试剂盒检测培养液中乳酸脱氢酶(LDH)与一氧化氮(NO)的含量;免疫荧光法检测细胞内活性氧(ROS)的水平;ELISA检测细胞IL-1β、IL-6的分泌情况;Western blot检测细胞内内质网应激标志性蛋白表达水平。结果 LPS组较对照组比较,NO的释放量、ROS生成量以及炎性因子IL-1β和IL-6释放量明显增加(P<0.05);使用Nox1/4抑制剂干预后,ROS产生以及NO、IL-1β和IL-6的释放量明显降低(P<0.05)。与对照组相比,LPS组细胞内GRP78、p-PERK、ATF6和IREα蛋白表达上调,提示内质网应激被激活;Nox1/4抑制剂干预后,内质网应激标志性蛋白表达下降。结论 Nox1/4抑制剂减轻了LPS诱导的EA.hy926细胞内质网应激损伤。其作用机制可能是通过减少细胞内Nox1/4来源的ROS的生成,抑制内质网应激,进而减轻炎性反应,改善内皮功能。

Objective To study the protective effect of Nox1/4 inhibitor on dysfunction of EA.hy926 human umbilical vein fusion cells induced by lipopolysaccharide. Methods EA. hy926 cells were divided into control group, LPS group, Nox1/4 inhibitor(GKT137831)+LPS group. CCK8 assay was used to detect cell viability, LDH and NO content in culture medium and ROS level in cells was detected by immunofluorescence, IL-1β and IL-6 secretion by ELISA, and endoplasmic reticulum stress marker protein expression was detected by Western blot. ResultsThe release of NO, ROS and inflammatory factors IL-1β and IL-6 in LPS group increased significantly(P< 0.05), while the release of NO, IL-1β and IL-6 decreased significantly after Nox1/4 inhibitor intervention(P< 0.05). The expression of GRP78, p-PERK, ATF6 and IREα was up-regulated and endoplasmic reticulum stress was significantly activated in LPS group. The intervention of Nox1/4 inhibitor inhibited the decrease of endoplasmic reticulum stress marker protein expression induced by LPS. Conclusions Inhibition of Nox1/4 attenuates LPS-induced endoplasmic reticulum stress injury in EA.hy926 cells. The mechanism may be that Nox1/4 inhibitor may reduce the production of ROS from Nox1/4,inhibit endoplasmic reticulum stress, alleviate inflammatory response and improve endothelial function.