畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (1): 311-322.doi: 10.11843/j.issn.0366-6964.2024.01.029

• 基础兽医 • 上一篇    下一篇

钙结合蛋白S100A4对BCG感染THP-1细胞自噬的调控作用

刘悦阳1, 李梦媛1,2, 聂雪伊1, 马亚博1, 侯雨欣1, 马伯利1, 杨易1, 徐金瑞1*   

  1. 1. 宁夏大学西部特色生物资源保护与利用教育部重点实验室, 银川 750021;
    2. 宁夏医科大学回医药现代化教育部重点实验室, 银川 750010
  • 收稿日期:2023-05-24 出版日期:2024-01-23 发布日期:2024-01-24
  • 通讯作者: 徐金瑞,主要从事动物病原生物学研究,E-mail:xujr@nxu.edu.cn
  • 作者简介:刘悦阳(1999-),女,宁夏中卫人,硕士生,主要从事动物病原生物学研究,E-mail:3213899698@qq.com
  • 基金资助:
    宁夏自然科学基金项目(2023AAC03201;2023AAC03100)

The Regulation of Calcium-binding Protein S100A4 on Autophagy in THP-1 Cells Infected with Bacillus Calmette-Guérin

LIU Yueyang1, LI Mengyuan1,2, NIE Xueyi1, MA Yabo1, HOU Yuxin1, MA Boli1, YANG Yi1, XU Jinrui1*   

  1. 1. Key Laboratory of Conservation and Utilization of Characteristic Biological Resources in Western China, Ministry of Education, Ningxia University, Yinchuan 750021, China;
    2. Key Laboratory of Modernization of Hui Medicine, Ministry of Education, Ningxia Medical University, Yinchuan 750010, China
  • Received:2023-05-24 Online:2024-01-23 Published:2024-01-24

摘要: 本研究旨在探究牛结核分枝杆菌减毒株卡介苗(Bacillus Calmette-Guérin,BCG)感染人单核巨噬细胞THP-1后,钙结合蛋白S100A4对细胞自噬的调控作用。以感染复数为10∶1的BCG感染THP-1细胞不同时间,用Western blot检测S100A4和LC3Ⅱ的表达,以确定最佳感染时间,并采用透射电镜观察自噬体数量变化。在BCG单独感染或与S100A4小干扰RNA共处理THP-1细胞12 h后,采用qRT-PCR检测S100A4及自噬相关因子——微管相关蛋白轻链3II (microtubule-associated protein light chain 3 Ⅱ, LC3Ⅱ)、自噬相关蛋白7(autophagy-related gene 7, Atg7)、Beclin-1在mRNA水平上的表达,采用Western blot检测S100A4、LC3Ⅱ、Atg7、Beclin-1在蛋白水平的表达。利用mRFP-GFP-LC3检测自噬流,激光共聚焦显微镜观察细胞中mRFP-LC3和mRFP-GFP-LC3点状聚集。结果显示:在BCG感染THP-1细胞不同时间后,S100A4和LC3Ⅱ在蛋白表达水平随感染时间的延长先上升后下降,在12 h时表达最高(P<0.001),且自噬体数量明显增多。在mRNA水平上,与siNC组相比,siNC+BCG感染组S100A4、LC3Ⅱ、Atg7、Beclin-1的mRNA表达水平显著上调(P<0.05),当BCG和siS100A4共处理后,S100A4、LC3Ⅱ、Atg7、Beclin-1在mRNA水平的表达显著(P<0.05)或极显著(P<0.01,P<0.001)下调;在蛋白水平上,与未感染组相比,S100A4、LC3Ⅱ、Atg7、Beclin-1的蛋白表达量显著增多(P<0.05),BCG和siS100A4共处理后,S100A4、LC3Ⅱ、Atg7、Beclin-1的蛋白表达量均极显著减少(P<0.001);与未感染组相比,BCG组自噬体的数量极显著增多(P<0.01),siS100A4+BCG组与siNC+BCG组相比,自噬体的数量显著减少(P<0.05)。S100A4对BCG感染巨噬细胞诱导的自噬具有调控作用,S100A4能够促进BCG诱导的THP-1细胞自噬。

关键词: 结核分枝杆菌, 细胞自噬, S100A4, THP-1细胞

Abstract: The purpose of this study was to explore the regulatory effect of calcium-binding protein S100A4 on autophagy of human monocyte-macrophage THP-1 cells infected with Bacillus Calmette-Guérin (BCG). BCG infected THP-1 cells at different times (MOI=10). The expressions of S100A4 and LC3 proteins were detected by Western blot, and the number of autophagosomes was observed by transmission electron microscopy (TEM). After BCG infection alone or co-treatment with S100A4 small interfering RNA for 12 h, the mRNA expression of S100A4 and autophagy-related factors, LC3Ⅱ(microtubule-associated protein light chain 3 Ⅱ), Atg7 (autophagy-related gene 7), and Beclin1, were detected by qRT-PCR. The protein expressions of S100A4, LC3Ⅱ, Atg7, and Beclin-1 were detected by Western blot. Autophagy flow was detected by mRFP-GFP-LC3, and spot aggregation of mRFP-LC3 and mRFP-GFP-LC3 was observed by laser confocal microscopy. After BCG was infected with THP-1 cells at different times, the protein expression levels of S100A4 and LC3Ⅱ firstly increased and then decreased with the extension of infection time, and the expression levels were the highest at 12 h (P<0.001), and the number of autophagosomes increased significantly. mRNA levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 in the siNC+BCG infection group were significantly up-regulated compared with those in the siNC group (P<0.05), S100A4, LC3Ⅱ, Atg7, and Beclin-1 were significantly expressed at mRNA level after BCG and siS100A4 treatment (P<0.05) or very significantly (P<0.01, P<0.001) down-regulated; Protein levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 increased significantly compared with an uninfected group (P<0.05), the protein expression levels of S100A4, LC3Ⅱ, Atg7, and Beclin-1 were significantly reduced after BCG and siS100A4 treatment (P<0.001); Compared with the uninfected group, the number of autophagosomes in BCG group was significantly increased (P<0.01), the number of autophagosomes in siS100A4+BCG group was significantly reduced compared with siNC+BCG infection group (P<0.05). S100A4 has a regulatory effect on the autophagy of macrophages induced by BCG infection, and S100A4 can promote the BCG-induced autophagy of THP-1 cells.

Key words: Mycobacterium tuberculosis, autophagy, S100A4, THP-1 cells

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