EGCG通过Nrf2/HO-1信号通路延缓内皮细胞衰老的实验研究
Experimental study on EGCG delaying endothelial cell senescence via Nrf2/HO-1 signaling pathway
张颖杰 1王绫欢 2马焱 1王天虎 1谷政慧 1房之沂 2沈晓影 3元媛 3田磊 3曹丰3
作者信息
- 1. 100853 北京市,解放军医学院&解放军总医院第二医学中心心血管内科
- 2. 南开大学医学院临床医学系
- 3. 国家老年疾病临床医学研究中心
- 折叠
摘要
目的 探讨表没食子儿茶素没食子酸酯[(-)-epigallocatechin-3-gallate,EGCG]对延缓多柔比星(doxorubicin,Dox)诱导的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)衰老的作用及机制.方法 2023年4～12月于解放军总医院第二医学中心老年医学研究所体外培养HUVEC,分为对照组、Dox(2 μmol/L)组、Dox+EGCG(50 μmol/L)组、Dox+EGCG+ML385(Nrf2特异性抑制剂,2.5 μmol/L)组,采用β-半乳糖苷酶(β-gal)染色检测细胞衰老,活性氧(reactive oxygen species,ROS)染色检测细胞内ROS含量,超氧化物歧化酶(SOD)活性检测试剂盒及丙二醛(MDA)含量检测试剂盒检测SOD活力和MDA含量,CCK-8实验检测细胞活力以及Western blot检测细胞内衰老相关蛋白(P21和P53)、核因子e2相关因子2(nuclear factor-E2-related factor 2,Nrf2)、血红素加氧酶 1(heme oxygenase 1,HO-1)蛋白水平.结果 浓度(10,30,50)μmol/L的 EGCG处理HUVEC 48 h不影响细胞活力,而浓度(70,100,300)μmol/L 的 EGCG预处理后细胞活力下降,因此选择50 μmol/L作为后续使用浓度.与对照组比较,Dox组β-gal阳性率和ROS水平显著增加(P＜0.05),P21和P53蛋白表达增高(P＜0.05),加入EGCG后β-gal阳性率以及ROS水平明显降低(P＜0.05),SOD活性提高(P＜0.05),MDA含量下降(P＜0.05),同时P21和P53蛋白表达量降低(P＜0.05),Nrf2和HO-1显著增加(P＜0.05).ML385组可逆转EGCG的细胞抗衰老效应,表现为Nrf2和HO-1表达量降低(P＜0.05),而P21和P53蛋白表达量增加(P＜0.05).结论 EGCG可能通过激活Nrf2/HO-1通路延缓Dox诱导的HUVEC衰老效应.
Abstract
Objective To investigate the effects and mechanisms of(-)-epigallocatechin-3-gallate(EGCG)on doxorubicin(Dox)-induced senescence in human umbilical vein endothelial cells(HUVEC).Methods Between April and December 2023,HUVEC were cultured in vitro at the Geriatric Research Institute of the Second Medical Center,PLA General Hospital.The cells were divided into four groups:the control group,the Dox group(2 μmol/L doxorubicin),the Dox+EGCG group(50 μmol/L epigallocatechin gallate)and the Dox+EGCG+ML385 group(with 2.5 μmol/L ML385,a specific Nrf2 inhibitor).Cellular senescence was assessed usingβ-galactosidase(β-gal)staining;intracellular reactive oxygen species(ROS)levels were measured using ROS staining;kits for superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were used to assess SOD activity and MDA levels;cell viability was evaluated using CCK-8 assay;levels of senescence-related proteins(P21 and P53),nuclear factor-E2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)were detected by Western blot.Results Treatment with EGCG at concentrations of 10,30,and 50 μmol/L for 48 h did not affect the cell viability,while cell viability decreased after pretreatment with EGCG at concentrations of 70,100,and 300μmol/L,thus 50 μmol/L was selected as the subsequent concentration.Compared with the control group,the Dox group showed a significant increase in β-gal positivity and ROS levels(P＜0.05),elevated expression of P21 and P53 proteins(P＜0.05).After the addition of EGCG,β-gal positivity and ROS levels were significantly reduced(P＜0.05),but SOD activity increased(P＜0.05)and MDA content decreased(P＜0.05).Additionally,the expression levels of P21 and P53 proteins were reduced(P＜0.05),while Nrf2 and HO-1 levels were significantly increased P＜0.05).The ML385 group reversed the anti-senescence effect of EGCG,manifested by decreased expression of Nrf2 and HO-1(P＜0.05)and increased expression of P21 and P53 proteins(P＜0.05).Conclusion EGCG may delay dox-induced senescence in HUVEC by activating the Nrf2/HO-1 pathway.
关键词
表没食子儿茶素没食子酸酯/人脐静脉内皮细胞/衰老/Nrf2/HO-1Key words
(-)-epigallocatechin-3-gallate/Human umbilical vein endothelial cells/Senescence/Nrf2/HO-1引用本文复制引用
基金项目
国家自然科学基金重大研究计划集成项目(92249301)
科技部重点专项(2022YFC3602400)
中央保健科研课题(2020ZD05)
出版年
2024
NETL
NSTL
万方数据