NF-κB信号通路介导羟基红花黄色素A减轻TNF-α诱导的内皮细胞EA.hy926损伤作用研究  

NF-κB Signaling Pathway Mediates the Protective Effect of Hydroxy Safflor Yellow A on TNF-α Induced Endothelial Cell EA.hy926 Injury

作　　者：贺稳 陈晗 朱明明 杨学攀 余曦 施洪飞[1] HE Wen;CHEN Han;ZHU Mingming;YANG Xuepan;YU Xi;SHI Hongfei(Nanjing University of Chinese Medicine,Nanjing Jiangsu China 210023;Nanjing University of Chinese Medicine Hanlin College,Taizhou Jiangsu China 215300)

机构地区：[1]南京中医药大学,江苏南京210023 [2]南京中医药大学翰林学院,江苏泰州215300

出　　处：《中医学报》2023年第2期351-359,共9页Acta Chinese Medicine

基　　金：国家自然科学基金项目(81574044);江苏省高等学校自然科学研究面上项目(18KJB330003)。

摘　　要：目的:探讨羟基红花黄色素A对人重组肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的人脐静脉内皮细胞EA.hy926炎症反应和氧化应激的干预作用及其可能机制。方法:培养原代细胞株EA.hy926,采用不同浓度TNF-α(0μg·L^(-1)、10μg·L^(-1)、20μg·L^(-1)、50μg·L^(-1))干预EA.hy926细胞建立细胞损伤模型,并采用不同浓度羟基红花黄色素A(0.1μmol·L^(-1)、1μmol·L^(-1)、10μmol·L^(-1)、100μmol·L^(-1))进行干预,CCK-8法检测细胞活力;Hoechest染色检测细胞凋亡情况;硝酸还原酶法检测细胞上清液中一氧化氮(nitric oxide,NO)含量;ELISA法检测细胞上清液中内皮素-1(endothelin-1,ET-1)含量;Western Blot法检测E-选择素(E-selectin)、单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)、细胞间黏附因子-1(intercellular cell adhesion molecule-1,ICAM-1)、血管细胞黏附因子-1(vascular cell adhesion molecule-1,VCAM-1)以及核转录因子-κB(nuclear transcription factor-kappa B,NF-κB)信号通路相关蛋白的表达水平;RT-qPCR检测SOD mRNA、CAT mRNA、GSH-px mRNA的表达水平。结果:羟基红花黄色素A能明显提高TNF-α诱导后内皮细胞的活力(P<0.01),抑制细胞凋亡,增加NO释放(P<0.01),并减少ET-1含量(P<0.01),显著降低E-selectin、MCP-1、ICAM-1、VCAM-1、p-IκB-α及细胞核中P65、P50的蛋白表达水平(P<0.05),显著升高细胞中SOD mRNA、CAT mRNA、GSH-px mRNA及IκB-α蛋白表达水平(P<0.05),显著升高细胞胞浆中P65、P50的蛋白表达水平(P<0.05)。结论:羟基红花黄色素A对TNF-α诱导的EA.hy926细胞炎症反应和氧化应激具有改善作用,其机制可能与抑制NF-κB信号通路活化有关。Objective:To investigate the intervention effect and possible mechanism of hydroxy safflor yellow A on the inflammatory response and oxidative stress of human umbilical vein endothelial cells EA.hy926 induced by human recombinant tumor necrosis factor-α(TNF-α).Methods:The primary cell line EA.hy926 was cultured,and different concentrations of TNF-α(0 μg·L^(-1),10 μg·L^(-1),20 μg·L^(-1),50 μg·L^(-1)) were used to intervene EA.hy926 cells to establish a cell injury model,and different concentrations of hydroxy safflor yellow A(0.1 μmol·L^(-1),1 μmol·L^(-1),10 μmol·L^(-1),100 μmol·L^(-1)) were used to intervene,and the cell viability was detected by CCK-8 method.Cell apoptosis was detected by Hoechest staining.Nitric oxide(NO) content in cell supernatant was detected by the nitrate reductase method.Endothelin-1(ET-1) content in cell supernatant was detected by ELISA.E-selectin,monocyte chemoattractant protein-1(MCP-1),intercellular cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),and nuclear factor kappa B(NF-κB) signaling pathway-related proteins were detected by Western Blot.And the expression levels of SOD mRNA,CAT mRNA,and GSH-px mRNA were detected by RT-qPCR.Results:Hydroxy safflor yellow A can significantly improve the viability of endothelial cells induced by TNF-α(P<0.01),inhibit cell apoptosis,increase NO release(P<0.01),and reduce ET-1 content(P<0.01).It also can significantly reduce the protein expression levels of E-selectin,MCP-1,ICAM-1,VCAM-1,p-IκB-α,and P65,P50 in the cell nucleus(P<0.05),significantly increase the expression levels of SOD mRNA,CAT mRNA,GSH-px mRNA and IκB-α protein in cells(P<0.05),and significantly increase the protein expression levels of P65 and P50 in the cell cytoplasm(P<0.05).Conclusion:Hydroxy safflor yellow A has a protective effect on TNF-α-induced inflammatory response and oxidative stress in EA.hy926 cells,and its mechanism may be related to the inhibition of activation of the NF-κB signaling pathway.

关 键 词：羟基红花黄色素A 内皮细胞 炎症反应 氧化应激 NF-ΚB信号通路 

分 类 号：R285.5[医药卫生—中药学]