硫化氢上调WDR26减轻内皮细胞缺氧复氧损伤

Hydrogen sulfide upregulates WDR26 and alleviates hypoxia/reoxygenation injury in endothelial cells

摘要:

目的]探讨硫化氢(H₂S)是否通过上调WD重复蛋白26(WDR26)表达来减轻内皮细胞缺氧复氧(H/R)损伤。 [方法]体外培养人脐静脉内皮细胞(HUVEC),缺氧(5%O₂、5%CO₂)16 h/复氧(20%O₂、5%CO₂)3 h造成H/R损伤。采用慢病毒转染技术构建WDR26基因敲低和过表达的HUVEC模型,并通过比色法检测丙二醛(MDA)含量及CCK-8检测细胞存活率；采用免疫荧光法检测细胞中活性氧(ROS)含量；采用Western blot检测WDR26和凋亡相关蛋白的表达；采用ELISA检测培养上清液中乳酸脱氢酶(LDH)、细胞间黏附分子1(ICAM-1)和内皮素1(ET-1)含量。 [结果]外源性H₂S供体NaHS能够增加H/R内皮细胞存活率,降低ROS和MDA含量,进而减轻内皮细胞损伤以及氧化应激反应(P＜0.05或P＜0.01)。NaHS可使WDR26蛋白表达增高(P＜0.05)。过表达WDR26能够增加H/R内皮细胞存活率,降低LDH含量,减少凋亡相关蛋白Caspase-3、p53和Bax的表达,进而减轻内皮细胞损伤,缓解细胞凋亡；反之,抑制WDR26表达则会出现相反的结果(P＜0.05或P＜0.01)。NaHS和/或过表达WDR26能够增加细胞存活率,减少培养上清液中LDH、ICAM-1和ET-1含量,进而改善内皮细胞功能；过表达/抑制表达WDR26可增强/削弱其保护作用(均P＜0.05)。 [结论]H₂S可减轻内皮细胞H/R损伤,其作用部分依赖于上调WDR26表达。

Abstract:

Aim To investigate whether hydrogen sulfide (H₂S) alleviates endothelial cell hypoxia/reoxygenation (H/R) injury by upregulating the expression of WD repeat protein 26 (WDR26).Methods Human umbilical vein endothelial cells (HUVEC) were cultured in vitro under hypoxic conditions (5%O₂,5%CO₂) for 16 h followed by reoxygenation (20%O₂,5%CO₂) for 3 h to induce H/R injury. HUVEC models with WDR26 gene knockdown and overexpression were constructed using lentiviral transfection technology. The content of malondialdehyde (MDA) was detected by colorimetric method and the cell survival rate was detected by CCK-8; the content of reactive oxygen species (ROS) in the cells was detected by immunofluorescence method; the expressions of WDR26 and apoptosis-related proteins were detected by Western blot; the contents of lactate dehydrogenase (LDH), intercellular adhesion molecule-1 (ICAM-1) and endothelin-1 (ET-1) in the cultured supernatant were detected by ELISA. Results The exogenous H₂S donor NaHS could increase the survival rate of H/R endothelial cells, reduce ROS and MDA contents, and thereby alleviating endothelial cell damage and oxidative stress response (P＜0.05 or P＜0.01). NaHS could increase the expression of WDR26 protein (P＜0.05). Overexpression of WDR26 could increase the survival rate of H/R endothelial cells, reduce LDH content, decrease the expression of apoptosis related proteins Caspase-3, p53 and Bax, thereby reducing endothelial cell damage and alleviating cell apoptosis; on the contrary, inhibiting WDR26 expression would result in the opposite effect (P＜0.05 or P＜0.01). NaHS and/or overexpression of WDR26 could increase cell survival rate, reduce LDH, ICAM-1 and ET-1 in the cultured supernatant, and thereby improve endothelial cell function; overexpression/inhibition of WDR26 could enhance/weaken its protective effect (all P＜0.05). Conclusion H₂S can alleviate H/R injury of endothelial cells, and its effect is partly dependent on upregulation of WDR26.