槲皮素对铜绿假单胞菌LasB诱导THP-1巨噬细胞应激反应的保护作用  

Quercetin protects THP-1 macrophages against cellular stress response induced by Pseudomonas aeruginosa protease LasB

作　　者：任彦颖 刘心伟 朱芮 有小娟 李登州 何志强[2] 王春霞 杨曦明[3] 李永伟[1] Ren Yanying;Liu Xinwei;Zhu Rui;You Xiaojuan;Li Dengzhou;He Zhiqiang;Wang Chunxia;Yang Ximing;Li Yongwei(The Second Clinical Medical College of Henan University of Chinese Medicine,Zhengzhou 450046,China;Medicine Laboratory Center of Henan Province Hospital of Traditional Chinese Medicine,Henan Key Laboratory of Traditional Chinese Medicine for the Prevention and Treatment of Drug-resistant Bacterial Infections,Zhengzhou Key Laboratory of Pathogenic Microorganisms and Bacterial Resistance Monitoring,Zhengzhou 450002,China;Medicine Laboratory Center,Dongzhimen Hospital of Beijing University of Chinese Medicine,Beijing 100700,China)

机构地区：[1]河南中医药大学第二临床医学院,郑州450046 [2]河南省中医院检验中心,河南省防治耐药菌感染中医药重点实验室,郑州市病原微生物与细菌耐药监测重点实验室,郑州450002 [3]北京中医药大学东直门医院检验中心,北京100700

出　　处：《中华预防医学杂志》2024年第3期337-346,共10页Chinese Journal of Preventive Medicine

基　　金：河南省中医药科学研究专项(2022ZY1078、2023ZY1011);河南省高等学校重点科研项目(22A360008)。

摘　　要：目的:探讨槲皮素对LasB诱导THP-1巨噬细胞凋亡、炎症反应和氧化应激的保护作用,旨在为槲皮素作为毒力抑制剂用于铜绿假单胞菌感染治疗提供借鉴和参考。方法:采用实验性研究。实验菌株为标准菌株,利用重组蛋白表达技术获得LasB蛋白,弹性蛋白刚果红法和荧光标记弹性蛋白法检测LasB酶活性。构建LasB诱导的THP-1巨噬细胞感染模型并使用槲皮素干预,CCK-8比色法检测细胞活力,倒置显微镜观察细胞形态。TUNEL联合Annexin V/PI染色法检测细胞凋亡。RT-qPCR和ELISA检测炎症细胞因子和环氧合酶水平。DCFH-DA荧光染色检测胞内ROS水平。采用单因素方差分析对实验数据进行统计学分析,多重比较采用Tukey检验。结果:成功获得相对分子质量33 000的纯度>90%的具有弹性蛋白水解活性的pLasB蛋白。pLasB(100 μg/ml)刺激下,THP-1巨噬细胞活力显著下降,细胞完整率明显降低,细胞凋亡率显著升高,炎性细胞因子IL-1α、IL-1β、IL-6、IL-10、IL-12和TNF-α水平显著上调,细胞内ROS荧光强度和 COX-2 水平明显增加。而在2.5 μmol/L、5 μmol/L和10 μmol/L浓度槲皮素干预下,THP-1巨噬细胞活力明显改善,细胞凋亡率从18.32%±0.17%分别降低至13.17%±0.20%、11.43%±0.06%和7.74%±0.04%( F=1 679, P<0.05);促炎细胞因子IL-1α、IL-1β、IL-6、IL-12和TNF-α显著下调,抗炎细胞因子IL-10显著上调;细胞内ROS荧光强度( F=86.92, P<0.05)和 COX-2表达水平( F=24.62, P<0.05)均明显下降。 结论:槲皮素能有效干预铜绿假单胞菌LasB诱导的THP-1巨噬细胞凋亡、炎症和氧化应激,可能作为铜绿假单胞菌的毒力抑制剂。ObjectiveTo investigate the protective effect of quercetin against LasB-induced apoptosis,inflammation,and oxidative stress in THP-1 macrophages,providing valuable insights into the use of quercetin as a virulence inhibitor for Pseudomonas aeruginosa infection treatment.MethodsThis was an experimental study.The experimental strain was the standard strain.The LasB protein was obtained utilizing protein recombination technology,while the enzyme activity of LasB was assessed through both the Elastin Congo red assay and fluorescently labelled elastin assay.The LasB-induced THP-1 macrophage infection model was established,and quercetin was utilized for intervention.Cell viability was evaluated via CCK-8 assay,while cell morphology was observed under an inverted microscope.Apoptosis detection involved employing both TUNEL and Annexin V/PI staining.The mRNA expression and protein levels of inflammatory cytokines and COX-2 were determined by RT-qPCR and ELISA respectively.Intracellular ROS levels were quantified using the DCFH-DA fluorescent probe.One-way ANOVA was used for statistical analysis,and Tukey test was used for multiple comparisons.ResultsThe pLasB with a molecular weight of 33000 and acceptable enzymatic activity(purity>90%),was successfully obtained.THP-1 macrophages treated with pLasB at a concentration of 100μg/ml presented significantly decreased viability and integrity rate when compared with the normal control group.Additionally,pLasB promoted apoptosis,up-regulated the levels of inflammatory cytokines IL-1α,IL-1β,IL-6,IL-10,IL-12,and TNF-α,increased intracellular ROS fluorescence intensity,and elevated COX-2 mRNA expression level.Furthermore,the viability of THP-1 macrophages was significantly enhanced under quercetin intervention at concentrations of 2.5μmol/L,5μmol/L and 10μmol/L.The apoptosis rate exhibited a significant reduction from 18.32%±0.17%to 13.17%±0.20%,11.43%±0.06%and 7.74%±0.04%,respectively(F=1679,P<0.05).There was a notable down-regulation of pro-inflammatory cytokines IL-1�

关 键 词：槲皮素 巨噬细胞 凋亡 炎症 氧化应激 

分 类 号：R285[医药卫生—中药学]