重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞铁死亡的信号通路分析

doi:10.11843/j.issn.0366-6964.2025.11.029

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5706-5720.doi: 10.11843/j.issn.0366-6964.2025.11.029

重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞铁死亡的信号通路分析

马月¹(), 苗宇航¹^,²(), 丁涛¹^,², 辛杰¹^,², 马文妍¹^,², 李雅楠¹^,²^,*(), 周学章¹^,²^,*(), 杜军¹^,²^,*()

1. 宁夏大学生命科学学院, 银川 750021
2. 西部特色生物资源保护与利用教育部重点实验室, 银川 750021

收稿日期:2024-10-08 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 李雅楠,周学章,杜军 E-mail:2459892015@qq.com;myh6943@126.com;liyanan@nxu.cn;zhouxuezhang@nxu.edu.cn;dujun@nxu.edu.cn
作者简介:马月(2001-)，女，回族，宁夏吴忠人，本科生，主要从事生物科学基础研究，E-mail: 2459892015@qq.com
苗宇航(1999-)女，山西长治人，硕士生，主要从事动物病原生物学研究，E-mail: myh6943@126.com
第一联系人：
马月与苗宇航为同等贡献作者
基金资助:
2024年大学生创新创业训练计划国家级推荐项目(G202410749023)；宁夏自然科学基金项目(2024AAC03118)；国家自然科学基金(32160044)

Signaling Pathway Analysis of Ferroptosis Induced by Recombinant Candida krusei 14-3-3 Protein in Bovine Mammary Epithelial Cells

MA Yue¹(), MIAO Yuhang¹^,²(), DING Tao¹^,², XIN Jie¹^,², MA Wenyan¹^,², LI Yanan¹^,²^,*(), ZHOU Xuezhang¹^,²^,*(), DU Jun¹^,²^,*()

1. College of Life Science, Ningxia University, Yinchuan 750021, China
2. Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China, Yinchuan 750021, China

Received:2024-10-08 Online:2025-11-23 Published:2025-11-27
Contact: LI Yanan, ZHOU Xuezhang, DU Jun E-mail:2459892015@qq.com;myh6943@126.com;liyanan@nxu.cn;zhouxuezhang@nxu.edu.cn;dujun@nxu.edu.cn

摘要/Abstract

摘要：

旨在研究重组克柔念珠菌14-3-3(rCK14-3-3)蛋白诱导奶牛乳腺上皮细胞(MAC-T)铁死亡的特征，为揭示克柔念珠菌毒力因子损伤奶牛乳腺上皮细胞的机制提供理论依据。采用CCK-8法筛选rCK14-3-3蛋白对MAC-T的最适作用浓度和时间；将奶牛乳腺上皮细胞分为空白对照组、阳性对照组(10 μmol·L^-1 Erastin)、试验组Ⅰ(不同浓度rCK14-3-3蛋白(25、50、75 μg·mL^-1))、试验组Ⅱ(不同浓度rCK14-3-3蛋白(25、50、75 μg·mL^-1)+铁死亡抑制剂Ferrostatin-1(20 μmol·L^-1))、试验组Ⅲ(不同浓度rCK14-3-3蛋白(25、50、75 μg·mL^-1)+Nrf2特异性抑制剂ML385(25 μmol·L^-1))，透射电镜检测细胞铁死亡形态学特征；Western blot检测rCK14-3-3诱导MAC-T铁死亡Nrf2-SLC7A11/GPX4信号通路蛋白表达水平；免疫荧光法检测谷胱甘肽过氧化物酶4(GPX4)、活性氧(ROS)及线粒体膜电位(JC-1)的表达水平；比色法检测MAC-T中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽(GSH)和亚铁离子(Fe²⁺)的含量。结果发现，与空白对照组相比，阳性对照组、试验组Ⅰ出现明显铁死亡特征；阳性对照组、试验组Ⅰ Nrf2、GPX4、SLC7A11蛋白表达水平及GSH含量显著下降(P < 0.05)，ROS、MDA、Fe²⁺含量显著上升，JC-1含量显著下降(P < 0.05)，而与试验组Ⅰ相比，试验组Ⅱ Nrf2、GPX4、SLC7A11蛋白表达水平及GSH含量显著上升(P < 0.05)，ROS、MDA、Fe²⁺含量显著下降，JC-1含量显著上升(P < 0.05)；试验组Ⅲ Nrf2、GPX4、SLC7A11蛋白表达水平及GSH含量显著下调(P < 0.05)，ROS、MDA、Fe²⁺含量显著上升，JC-1含量显著下降(P < 0.05)。本研究表明，rCK14-3-3蛋白可通过Nrf2-SLC7A11/GPX4信号通路诱导MAC-T发生铁死亡，为今后靶向干预铁死亡防治念珠菌性奶牛乳房炎提供了数据支持。

关键词: 重组克柔念珠菌14-3-3蛋白, 奶牛乳腺上皮细胞, 核因子E2相关因子2-溶质载体家族7A11/GPX4信号通路, 铁死亡

Abstract:

The aim of this study was to investigate the characteristics of ferroptosis induced by recombinant Candida krusei 14-3-3 (rCK14-3-3) protein in bovine mammary epithelial cells (MAC-T), providing a theoretical basis for revealing the mechanism of damage to bovine mammary epithelial cells by Candida krusei virulence factors. The optimal concentration and action time of rCK14-3-3 protein on MAC-T were screened by CCK-8 method. MAC-T cells were divided into blank control group, positive control group (10 μmol·L^-1 Erastin), experimental group Ⅰ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1)), experimental group Ⅱ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1) +ferroptosis inhibitor Ferrostatin-1 (20 μmol·L^-1)), and experimental group Ⅲ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1) +Nrf2-specific inhibitor ML385 (25 μmol·L^-1)). The morphological characteristics of ferroptosis were detected by transmission electron microscopy. The expression levels of Nrf2-SLC7A11/GPX4 signaling pathway proteins induced by rCK14-3-3 in MAC-T were detected by Western blot. The expression levels of glutathione peroxidase 4 (GPX4), reactive oxygen species (ROS), and mitochondrial membrane potential (JC-1) were detected by immunofluorescence. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and ferrous ions (Fe²⁺) in MAC-T were detected by colorimetry. The results showed that compared with the blank control group, the positive control group and experimental group Ⅰ showed obvious ferroptosis characteristics. The expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content in the positive control group and experimental group Ⅰ were significantly decreased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly increased, and the content of JC-1 was significantly decreased (P < 0.05). Compared with experimental group Ⅰ, the expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content in experimental group Ⅱ were significantly increased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly decreased, and the content of JC-1 was significantly increased (P < 0.05). In experimental group Ⅲ, the expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content were significantly decreased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly increased, and the content of JC-1 was significantly decreased (P < 0.05). This study demonstrated that rCK14-3-3 protein could induce ferroptosis in MAC-T through the Nrf2-SLC7A11/GPX4 signaling pathway, providing data support for future targeted intervention in ferroptosis to prevent and treat Candida-induced bovine mastitis.

Key words: recombinant Candida krusei 14-3-3 protein, bovine mammary epithelial cells, nuclear factor E2-related factor 2-solute carrier family 7A11/GPX4 signaling pathway, ferroptosis

中图分类号:

S858.23

马月, 苗宇航, 丁涛, 辛杰, 马文妍, 李雅楠, 周学章, 杜军. 重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞铁死亡的信号通路分析[J]. 畜牧兽医学报, 2025, 56(11): 5706-5720.

MA Yue, MIAO Yuhang, DING Tao, XIN Jie, MA Wenyan, LI Yanan, ZHOU Xuezhang, DU Jun. Signaling Pathway Analysis of Ferroptosis Induced by Recombinant Candida krusei 14-3-3 Protein in Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5706-5720.

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重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞铁死亡的信号通路分析

Signaling Pathway Analysis of Ferroptosis Induced by Recombinant Candida krusei 14-3-3 Protein in Bovine Mammary Epithelial Cells

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