| 摘要: |
| 【 目的】探究金黄色葡萄球菌外囊泡（EVs）对奶牛乳腺上皮细胞（BMECs）炎症的影响，为系统阐述金 黄色葡萄球菌EVs诱导BMECs炎性损伤的分子机制奠定基础。【 方法】通过差速+超速离心法分离纯化金黄色葡萄 球菌的EVs，利用透射电镜和纳米颗粒跟踪分析对EVs进行鉴定；用DIO荧光染料示踪金黄色葡萄球菌EVs，观察BMECs对金黄色葡萄球菌EVs的内化情况；用0，5，10，20，25 μg/mL EVs处理BMECs，测定EVs对BMECs增殖活力的影响；将20 μg/mL EVs与BMECs共孵育0，1，3 h，通过Western Blot、RT-qPCR测定NLRP3信号通路炎性因子表达量；用NLRP3抑制剂MCC950预处理BMECs 3 h后，用20 μg/mL EVs与BMECs共孵育1 h，Western Blot测定NLRP3信号通路炎性因子表达量。【 结果】成功从金黄色葡萄球菌培养上清液中获得纯化的EVs，其具有双层膜和典型的杯盘状结构，粒径大小为80~320 nm，符合革兰氏阳性菌EVs特征 。EVs可被BMECs内化至胞内。 0~25 μg/mL EVs对BMECs的活性无显著影响。经20 μg/mL EVs处理后，BMECs IL-18、IL-1β、IL-6、TNF-α基因mRNA相对表达量在1 h时均较对照极显著上升（P<0.01），IL-18、IL-1β基因mRNA相对表达量在3 h时也较对照分别极显著和显著升高；Western Blot结果显示，与0 h处理组相比 ，EVs处理1，3 h BMECs NLRP3信号通路中NLRP3、 GSDMD、N-GSDMD、caspase1、IL-18、IL-1β等炎性因子蛋白表达量大多显著或极显著上升；与EVs处理组相比，MCC950＋EVs处理组BMECs NLRP3信号通路中NLRP3、GSDMD、N-GSDMD、c-caspase1、IL-18、IL-1β等炎性因子蛋白表达量均显著或极显著下调。【 结论】金黄色葡萄球菌在正常增殖过程中能分泌EVs，可被BMECs内化。EVs通过NLRP3信号通路诱导BMECs发生炎症反应，表明EVs在金黄色葡萄球菌感染BMECs的过程中有重要作用。 |
| 关键词: 金黄色葡萄球菌 外囊泡 奶牛乳腺上皮细胞 NLRP3信号通路 |
| DOI：10. 13207/j. jnwafu. 2026. 07. 002 |
| 分类号: |
| 基金项目:宁夏回族自治区自然科学基金项目（2023AAC03322）；2023 年宁夏回族自治区青年拔尖人才培养项目（宁人社函〔2024〕106 号）；宁夏回族自治区科技创新领军人才项目（2021GKLRLX10） |
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| Induction of inflammation in mammary epithelial cells of dairy cows by Staphylococcus aureus extracellular vesicles |
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HUANG Qian1, YANG Jiangliu1, QIAO Yarui1, LI Jun1,2, ZHOU Xuezhang1
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1.Key Laboratory of Conservation and Utilization of Western Characteristic Resources，Ministry of Education，School of Life Sciences，Ningxia University，Yinchuan，Ningxia 750021，China;2.Ningxia Traditional Chinese Medicine Development and
Utilization Engineering Technology Research Center，Ningxia Polytechnic University，Yinchuan，Ningxia 750021，China
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| Abstract: |
| 【 Objective】The present study investigates the effects of Staphylococcus aureus extracellular vesicles（EVs） on the inflammation of bovine mammary epithelial cells（BMECs） and aims to lay a foundation for the systematic elucidation of the molecular mechanisms by which S. aureus EVs induce inflammatory damage in BMECs.【MethodThe EVs of Staphylococcus aureus were isolated and purified by differential and ultracen-trifugation，and identified using transmission electron microscopy and nanoparticle tracking analysis. The internalisation of S. aureus EVs was observed by tracking them with DIO fluorescent dye. BMECs were exposed to 0，5，10，20，and 25 μg/mL EVs，determine the effect of EVs on the enhancing vitality of BMECs. BMECs were incubated with 20 μg/mL EVs for 0，1，and 3 hours to assess the expression of NLRP3 signaling pathway-related inflammatory factors by Western blot and RT-qPCR. BMECs were subjected to a 3-hour pre-treatment with the NLRP3 inhibitor MCC950 before being incubated with 20 μg/mL EVs for 1 hour. The expression levels of inflammatory factors in the NLRP3 signalling pathway were determined by Western Blot analysis.【Result】Purified EVs were successfully isolated from the culture medium of S. aureus. These EVs exhibited a double-layered membrane and a typical cup-shaped structure. The particle sizes of the purified EVs ranged from 80 to 320 nm，and these characteristics are consistent with those of Gram-positive bacterial EVs. The EVs were effectively internalized by BMECs. In the concentration range of 0?25 μg/mL，EVs exerted no significant effect on BMECs viability. Following treatment with 20 μg/mL EVs，the relative mRNA expression levels of IL-18，IL-1β，IL-6，and TNF-α genes of BMECs were extremely significantly higher than those in the control group at 1 h（P<0. 01）. Furthermore，the relative mRNA expression levels of IL-18 and IL-1β genes were also extremely significantly and significantly higher than those in the control group at 3 h，respectively. The Western Blot results demonstrated that compared with the 0 h treatment group，the protein expression levels of NLRP3，GSDMD，N-GSDMD，caspase1，IL-18，IL-1β inflammatory factors in the NLRP3 signalling pathway of BMECs in the EVs-treated group were mostly significantly or extremely significantly increased at 1 and 3 hours.Compared with the EVs-treated group，the MCC950＋EVs-treated group exhibited a significant or extremely significant decrease in the protein expression levels of NLRP3，GSDMD，N-GSDMD，c-caspase1，IL-18，IL-1β inflammatory factors in the NLRP3 signalling pathway of BMECs.【Conclusion】Staphylococcus aureus can secrete EVs during normal proliferation. These EVs can be internalised by BMECs and induce an inflammatory response in BMECs via the NLRP3 signalling pathway. This suggests that EVs have an important role in the pro?
cess of S. aureus infection of BMECs. |
| Key words: Staphylococcus aureus extracellular vesicles bovine mammary epithelial cells NLRP3 signalling pathway |
