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

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10 μg/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。

关键词:沙眼衣原体;pORF5质粒蛋白;氧化应激;凋亡;DJ-1

Abstract:

[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 10 μg/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 si-DJ-1 group (P<0.001, P<0.01). [Conclusion] pORF5 can up-regulate the expression of DJ-1 protein to activate the Nrf2/NQO1 pathway, thereby inhibiting LPS-induced oxidative stress and apoptosis.

Key words:Chlamydia trachomatis;pORF5;oxidative stress;apoptosis;DJ-1