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

作　　者：谭超[1,2,3] 周靖轩 卢倩倩 刘彬彬[1,2,3] 蔡轶 王君 邹黎黎[1,2,3] TAN Chao;ZHOU Jingxuan;LU Qianqian;LIU Binbin;CAI Yi;WANG Jun;ZOU Lili(The First College of Clinical Medical Science,China Three Gorges University,Yichang 443000,Hubei,China;Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,China Three Gorges University,Yichang 443002,Hubei,China;Institute of Infection and Inflammation,Medical College,China Three Gorges University,Yichang 443002,Hubei,China)

机构地区：[1]三峡大学第一临床医学院,湖北宜昌443000 [2]肿瘤微环境与免疫治疗湖北省重点实验室(三峡大学),湖北宜昌443002 [3]三峡大学感染与炎症损伤研究所,湖北宜昌443002

出　　处：《微生物学报》2022年第3期1049-1060,共12页Acta Microbiologica Sinica

基　　金：国家自然科学基金(81903105);宜昌市医疗卫生科研项目(A19-301-15,A19-301-50);天然产物研究与利用湖北省重点实验室(三峡大学)开放课题(NPRD-2018009);三峡大学学位论文培优基金(2020SSPY103)。

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(Staphylococcusaureus,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 oxygenspecies,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(thioredoxinreductase,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10μmol/L依布硒啉与5μmol/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。[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(H2 DCFDA)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.

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 

分 类 号：R965[医药卫生—药理学]