沙眼衣原体pORF5蛋白通过上调DJ-1激活Nrf2/NQO1通路调控细胞氧化应激与凋亡  

Chlamydia trachomatis plasmid-encoded protein pORF5 up-regulates DJ-1 expression to activate the Nrf2/NQO1 pathway in the regulation of oxidative stress and apoptosis

作　　者：龚思露 周辉[2] GONG Silu;ZHOU Hui(Medical Laboratory Center,The Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410005,Hunan,China;Medical Laboratory and Pathology Center,The First Hospital of Hunan University of Chinese Medicine,Changsha 410007,Hunan,China)

机构地区：[1]湖南中医药大学第二附属医院医学检验中心,湖南长沙410005 [2]湖南中医药大学第一附属医院医学检验与病理中心,湖南长沙410007

出　　处：《微生物学通报》2025年第3期1062-1072,共11页Microbiology China

基　　金：湖南省自然科学基金(2022JJ40324);湖南省卫生健康委卫生科研课题(202011000475,W20243080)。

摘　　要：【背景】pORF5质粒蛋白是沙眼衣原体(Chlamydia trachomatis,Ct)的重要毒力因子,具有抑制细胞氧化应激和凋亡的作用。【目的】探讨pORF5是否通过上调DJ-1蛋白激活核因子红细胞2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/醌氧化还原酶1[NAD(P)H:quinone oxidoreductase1,NQO1]信号通路调控脂多糖(lipopolysaccharide,LPS)诱导的细胞氧化应激与凋亡。【方法】用不同浓度的pORF5质粒蛋白以不同时间刺激HeLa细胞,Western blotting检测DJ-1蛋白表达情况;采用活性氧(reactive oxygen species,ROS)试剂盒、流式细胞术检测pORF5对LPS诱导的细胞氧化应激和凋亡的影响;利用小干扰RNA(small interfering RNA,siRNA)干扰HeLa细胞DJ-1蛋白表达,用荧光显微镜观察细胞内ROS荧光强度,用试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量变化;用Nrf2特异性抑制剂(ML385)预处理细胞1 h,pORF5单独或与LPS共刺激细胞后,分析凋亡相关蛋白Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、B细胞淋巴瘤因子2(B-cell lymphoma-2,Bcl-2)蛋白表达水平;利用Western blotting分析干扰DJ-1蛋白表达对Nrf2/NQO1信号通路的影响。【结果】pORF5可上调DJ-1蛋白表达,并且最佳刺激浓度和时间分别为10μg/mL和18 h;pORF5质粒蛋白可显著减弱LPS诱导的ROS荧光强度及细胞凋亡率(P<0.001);si-DJ-1组中ROS荧光强度增强,MDA含量增多(P<0.001),SOD活性下降(P<0.001);ML385能抑制pORF5诱导的Nrf2、NQO1蛋白表达(P<0.001),上调促凋亡蛋白Bax表达(P<0.05),下调抗凋亡蛋白Bcl-2的表达(P<0.01);si-DJ-1组中Nrf2及NQO1的表达明显降低(P<0.001,P<0.01)。【结论】pORF5质粒蛋白可通过上调DJ-1蛋白激活Nrf2/NQO1信号通路抑制LPS诱导的细胞氧化应激与凋亡。[Background]The plasmid-encoded protein pORF5 is a pivotal virulence factor of Chlamydia trachomatis(Ct),capable of inhibiting oxidative stress and apoptosis.[Objective]To investigate whether pORF5 up-regulates the expression of DJ-1 protein to activate the nuclear factor erythroid 2-related factor 2(Nrf2)/NAD(P)H:quinone oxidoreductase 1(NQO1)pathway in the regulation of lipopolysaccharide(LPS)-induced cellular oxidative stress and apoptosis.[Methods]HeLa cells were treated with different concentrations of pORF5 for different time periods.Western blotting was employed to determine the expression level of DJ-1.The reactive oxygen species(ROS)assay kit and flow cytometry were employed to examine the effects of pORF5 on LPS-induced oxidative stress and apoptosis.The expression of DJ-1 protein in HeLa cells was down-regulated by siRNA-DJ-1,and the cellular ROS fluorescence intensity was observed under a fluorescence microscope.Corresponding assay kits were used to measure the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in each group.After pretreatment with the Nrf2 inhibitor ML385 for 1 h,HeLa cells were stimulated with pORF5 alone or combined with LPS,and the expression levels of the apoptosis-related proteins B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)were determined.Western blotting was employed to evaluate the effect of down-regulating the expression of DJ-1 on the Nrf2/NQO1 pathway.[Results]pORF5 up-regulated the expression of DJ-1,and the up-regulatory effect was the strongest after treatment with 10μg/mL pORF5 for 18 h.pORF5 reduced LPS-induced ROS fluorescence intensity and cell apoptosis rate(P<0.001).The si-DJ-1 group showed increased ROS fluorescence intensity and MDA content but decreased SOD activity(P<0.001).ML385 inhibited pORF5-induced expression of Nrf2 and NQO1(P<0.001),up-regulated the expression of the pro-apoptotic protein Bax(P<0.05),down-regulated the expression of the anti-apoptotic protein Bcl-2(P<0.01).The expression of Nrf2 and NQO1 was down-regulated in the

关 键 词：沙眼衣原体 pORF5质粒蛋白 氧化应激 凋亡 DJ-1 

分 类 号：R378[医药卫生—病原生物学]