3,5-二羟基-4-甲氧基苯甲醇减轻人脐静脉内皮细胞缺氧/复氧损伤的作用及其机制  

3,5-Dihydroxy-4-methoxybenzyl alcohol attenuates hypoxic/reoxygenation injury of human umbilical vein endothelial cells

作　　者：杨宗绵 周梦军 张浴玲[1] 黄丹梅[1] 张扬[2] 黄乃淇 张艳美[1] YANG Zongmian;ZHOU Mengjun;ZHANG Yuling;HUANG Danmei;ZHANG Yang;Nai-Kei WONG;ZHANG Yanmei(Department of Pharmacy,Shantou University Medical College,Shantou 515041,China;Key Laboratory of Tropocal Marine Bio-resources and Ecology,Chinese Academy of Science,Guangzhou 510006,China)

机构地区：[1]汕头大学医学院药学系,广东汕头515041 [2]中国科学院热带海洋生物资源与生态重点实验室,广东广州510006

出　　处：《中国病理生理杂志》2024年第11期2067-2073,共7页Chinese Journal of Pathophysiology

基　　金：广东省自然科学基金资助项目(No.2019A1515012101);广东省科技专项资金项目(No.210728156901566)。

摘　　要：目的:探讨多酚类化合物3,5-二羟基-4-甲氧基苯甲醇(DHMBA)对人脐静脉内皮细胞(EA.hy926细胞)缺氧/复氧(H/R)损伤的作用及其机制。方法:采用酸性缺氧液结合厌氧工作站构建H/R模型。将EA.hy926细胞分为对照组、H/R组、H/R+不同剂量DHMBA组、H/R+抗氧化剂依达拉奉组、H/R+活性氧(ROS)促生成剂寡霉素A+DHMBA组。采用CCK-8法检测细胞活力;ELISA法检测细胞内肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)水平;Western blot法检测内皮型一氧化氮合酶(eNOS)和核因子κB(NF-κB)p65蛋白的磷酸化水平;激光共聚焦显微镜检测细胞内一氧化氮(NO)含量;二硝基苯甲酸显色法测定谷胱甘肽(GSH)氧化还原平衡;流式细胞术检测细胞内总ROS水平;硝基四紫唑显色法测定细胞乳酸脱氢酶(LDH)漏出;划痕实验检测细胞的迁移能力。结果:DHMBA在125~1000μmol/L的浓度下无明显细胞毒性。在H/R刺激的EA.hy926细胞中,DHMBA可以提高细胞活力,抑制NF-κB磷酸化,减少炎症因子TNF-α和IL-6含量,增加eNOS蛋白磷酸化水平及NO含量;DHMBA能通过抑制H/R诱导的EA.hy926细胞ROS超载,恢复细胞内GSH和其氧化态(GSSH)的比例,减少LDH漏出,提高细胞迁移能力。结论:DHMBA可以减轻H/R所致的人脐静脉内皮细胞氧化应激、炎症反应、细胞损伤和功能障碍,这与其降低细胞内ROS水平有关。AIM:To explore the effect of polyphenolic compound 3,5-dihydroxy-4-methoxybenzyl alcohol(DHMBA)on hypoxia/reoxygenation(H/R)injury of human umbilical vein endothelial cells(EA.hy926 cells)and its po-tential mechanisms.METHODS:To construct an H/R model,the EA.hy926 cells were cultured in an acidic hypoxia buffer while in an anaerobic workstation.The cells were divided into control,H/R,H/R+different doses of DHMBA,H/R+edaravone(antioxidant)and H/R+reactive oxygen species(ROS)inducer oligomycin A+DHMBA groups.Cell viability was measured by CCK-8 assay,and tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels in cells were mea-sured by ELISA.Phosphorylation of endothelial nitric oxide synthase(eNOS)and nuclear factor-κB(NF-κB)p65 were measured by Western blot.Intracellular NO levels were determined by laser confocal microscopy.Glutathione(GSH)/glu-tathione disulfide(GSSG)oxidation balance was determined by the dinitrobenzoic acid chromogenic method.Intracellular ROS levels were measured by flow cytometry.Lactate dehydrogenase(LDH)leakage was determined using nitro blue tet-razolium staining.Scratch assays were performed to assess cell migration.RESULTS:DHMBA exhibited no significant cytotoxicity between 125 and 1000μmol/L.In H/R-injured human umbilical vein endothelial cells,DHMBA improved cell survival,inhibited phosphorylation of NF-κB p65,reduced the content of TNF-αand IL-6,and increased phosphory-lation of eNOS and NO levels.DHMBA also suppressed ROS overload and restored the ratio between GSH and oxidized GSH,decreased in LDH leakage and increased cell migration in H/R-injured human umbilical vein endothelial cells.CONCLUSION:DHMBA can alleviate H/R-induced oxidative stress,inflammation,cellular damage,and dysfunction,which are associated with the ability of DHMBA to inhibit ROS production in human umbilical vein endothelial cells.

关 键 词：3 5-二羟基-4-甲氧基苯甲醇 血管内皮细胞 缺氧/复氧损伤 氧化应激 内皮功能障碍 

分 类 号：R965[医药卫生—药理学] R543[医药卫生—药学] R363.2