首页|茶黄素对ox-LDL诱导的THP-1巨噬细胞泡沫化和氧化应激的影响

茶黄素对ox-LDL诱导的THP-1巨噬细胞泡沫化和氧化应激的影响

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[目的]探索茶黄素对氧化型低密度脂蛋白(ox-LDL)诱导的THP-1巨噬细胞泡沫化和氧化应激的影响及机制.[方法]使用50 μmol/L茶黄素、10 μmol/L核因子E2相关因子2(NRF2)抑制剂ML385预处理THP-1来源的巨噬细胞,随后加入100 mg/Lox-LDL刺激细胞24 h建立泡沫细胞模型.通过CCK-8法和LDH释放量检测茶黄素对THP-1巨噬细胞活力的影响.通过RT-qPCR和Western blot检测炎症因子白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)的表达;ELISA法检测炎症因子的释放.采用油红O染色检测细胞内脂质积累,DiL标记氧化型低密度脂蛋白(DiL-ox-LDL)染色检测脂质摄取,DCFH-DA探针检测活性氧(ROS)水平.通过Western blot和RT-qPCR检测脂质摄取、胆固醇外排及氧化应激相关蛋白的表达.[结果]经100 mg/L的ox-LDL处理,THP-1巨噬细胞活力明显降低,脂质摄取、积累明显增加,脂质摄取相关蛋白表达增加,胆固醇外排相关蛋白表达明显减少,炎症与ROS水平明显增加,髓过氧化物酶(MPO)和NADPH氧化酶2(NOX2)表达增加(P<0.05);加入茶黄素后,细胞活力升高,细胞内脂质积累明显减少,脂质摄取相关蛋白的表达明显减少,胆固醇外排相关蛋白表达明显增多,炎症因子IL-6、IL-1β、TNF-α的表达与释放明显降低,ROS水平降低,MPO与NOX2表达减少(P<0.05).茶黄素预处理改变了 NRF2信号通路中NRF2、血红素加氧酶1(HO-1)、Kelch样ECH关联蛋白1(KEAP1)的表达,增加了 NRF2核易位,减缓了细胞内氧化应激.ML385预处理细胞后,NRF2、HO-1、KEAP1和CD36蛋白表达水平明显降低.[结论]茶黄素可以显著抑制THP-1巨噬细胞泡沫化,抑制ox-LDL诱导的THP-1巨噬细胞炎症并通过NRF2/HO-1信号通路减缓了氧化应激.
Effects of theaflavin on ox-LDL-induced foam cell formation and oxidative stress in THP-1 derived macrophages
Aim To investigate the effect of theaflavin on oxidized low density lipoprotein(ox-LDL)-induced foam cell formation and oxidative stress in THP-1 macrophages and its mechanism.Methods THP-1 derived macro-phages were pretreated with 50 μmol/L theaflavin and(or)10 μmol/L nuclear factor erythroid 2-related factor 2(NRF2)inhibitor ML385,then 100 mg/L ox-LDL was added to the cells for 24 h to establish the foam cell model.The effect of theaflavin on THP-1 macrophages viability was evaluated by CCK-8 assay and LDH release.The expression of inflamma-tory cytokines interleukin-6(IL-6),interleukin-1 β(IL-1β),tumor necrosis factor-α(TNF-α)were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot.The release of inflammatory cytokines were detected by enzyme linked immunosorbent assay(ELISA).Intracellular lipid accumulation was detected by Oil red O staining,and lipid absorption was observed by DiL-labeled oxidized low density lipoprotein(DiL-ox-LDL)staining.Re-active oxygen species(ROS)level was detected by DCFH-DA probe.The expression of lipid uptake,cholesterol efflux and oxidative stress-related proteins were detected by Western blot and RT-qPCR.Results Treatment with 100 mg/L ox-LDL significantly decreased cell viability and cholesterol efflux-related protein expressions,increased lipid uptake,ac-cumulation and lipid uptake-related protein expressions,and significantly promoted inflammation and ROS level,as well as the expressions of myeloperoxidase(MPO),NADPH oxidase 2(NOX2)in THP-1 macrophages(all P<0.05).After pretreatment with theaflavin,cell viability was increased,intracellular lipid uptake,accumulation and lipid uptake-related protein expressions were significantly reduced,cholesterol efflux-related protein expressions were significantly increased,the expression and release of IL-6,IL-1β and TNF-α were significantly decreased,ROS level was significantly decreased,and the expression of MPO and NOX2 were decreased(all P<0.05).Pretreatment with theaflavin effectively alleviated intracellular oxidative stress by altering the expression of NRF2,heme oxygenase-1(HO-1)and Kelch-like ECH-associated protein 1(KEAP1)in NRF2 signaling pathway,and enhanced the translocation of NRF2 into the nucleus.After pretreat-ment with ML385,the expression levels of NRF2,HO-1,KEAP1 and CD36 were significantly decreased.Conclusion Theaflavin can significantly inhibit ox-LDL-induced foam cell formation,inflammation,and oxidative stress through NRF2/HO-1 signaling pathway in THP-1 macrophages.

theaflavinfoam cellTHP-1 macrophagesoxidative stress

石萌萌、黄锐、黄子乐、胡军威、肖靖杰、刘艳红、武军驻

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武汉大学泰康医学院(基础医学院),湖北省武汉市 430071

武汉大学中南医院心血管内科,湖北省武汉市 430071

武汉大学人民医院检验科(武汉大学人民医院转化医学研究院),湖北省武汉市 430060

茶黄素 泡沫细胞 THP-1巨噬细胞 氧化应激

国家自然科学基金项目

82300322

2024

中国动脉硬化杂志
中国病理生理学会 南华大学

中国动脉硬化杂志

CSTPCD
影响因子:1.086
ISSN:1007-3949
年,卷(期):2024.32(9)
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