紫草素与依布硒啉对金黄色葡萄球菌的协同作用

The synergistic effect of shikonin and ebselen against Staphylococcus aureus

摘要:

【目的】探讨紫草素（shikonin，SKN）协同依布硒啉（ebselen，EbSe）抗金黄色葡萄球菌（Staphylococcus aureus，S. aureus）的作用及机制。【方法】分光光度法测定紫草素-依布硒啉抗金黄色葡萄球菌活性；碘化丙啶（propidium iodide，PI）染色检测紫草素-依布硒啉杀菌效果；透射电子显微镜观察紫草素-依布硒啉对金黄色葡萄球菌细胞形态的影响。通过荧光探针二氯荧光素二乙酸酯（2'，7'-dichlorodihydrofluorescein diacetate，H₂DCFDA）检测紫草素-依布硒啉上调细菌胞内活性氧（reactive oxygen species，ROS）产量的能力；利用5，5'-二硫代双（2-硝基苯甲酸）[5，5'-dithiobis-（2-nitrobenzoic acid），DTNB]实验检测紫草素-依布硒啉对硫氧还蛋白还原酶（thioredoxin reductase，TrxR）活性的影响。通过加入还原剂二硫苏糖醇（DL-dithiothreitol，DTT）清除ROS进行挽救实验，复测相关指标，对上述结果进行验证。收集临床多重耐药金黄色葡萄球菌（multidrug-resistant Staphylococcus aureus，MDRS），通过分光光度法测定紫草素-依布硒啉对MDRS的抑菌活性。【结果】依布硒啉与紫草素均对金黄色葡萄球菌有抗菌活性，且10 μmol/L依布硒啉与5 μmol/L紫草素可发挥显著的协同抑菌活性（P<0.000 1）。紫草素-依布硒啉可协同抑制TrxR活性（P<0.01），同时显著上调金黄色葡萄球菌胞内ROS产量（P<0.001）。通过回补实验发现，DTT可挽救紫草素-依布硒啉对金黄色葡萄球菌的损伤。紫草素-依布硒啉对MDRS有明显的抑菌活性（P<0.000 1）。【结论】紫草素-依布硒啉可通过协同靶向TrxR，诱导细菌胞内产生大量ROS，介导金黄色葡萄球菌死亡。

关键词:金黄色葡萄球菌;依布硒啉;紫草素;硫氧还蛋白还原酶;活性氧

Abstract:

[Objective] To explore the synergistical effect and mechanism of the shikonin and ebselen against S. aureus.[Methods] We used spectrophotometry assay to determine the antibacterial effect of shikonin-ebselen on S. aureus, propidium iodide (PI) staining to detect the bactericidal effect of shikonin-ebselen, and the transmission electron microscope to observe the S. aureus morphological changes after the shikonin-ebselen treatment. We used the 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) staining to detect the effect of shikonin-ebselen on bacterial ROS production, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to detect the effect of shikonin-ebselen on the thioredoxin reductase (TrxR) activity. Reactive oxygen species (ROS) was eliminated by adding ROS scavenger DL-dithiothreitol (DTT), and we further detected the relevant indicators. We isolated the multidrug resistant Staphylococcus aureus (MDRS) strains, and the antibacterial effects of shikonin-ebselen against MDRS were determined by spectrophotometry.[Results] We found shikonin and ebselen both have inhibitory effects on S. aureus, and 5 μmol/L shikonin and 10 μmol/L ebselen could work synergistically against bacteria (P<0.000 1). Shikonin-ebselen can significantly inhibit the activity of TrxR (P<0.01). Meanwhile, shikonin-ebselen can significantly induce the production of ROS in S. aureus (P<0.01). DTT can rescue the S. aureus from the shikonin-ebselen treatment. In addition, shikonin-ebselen also has a significant antibacterial effect on MDRS (P<0.000 1).[Conclusion] Shikonin-ebselen can execute antibacterial effect on S. aureus through inducing the production of a large amount of ROS by co-targeting the TrxR.

Key words:Staphylococcus aureus;ebselen;shikonin;thioredoxin reductase;reactive oxygen species