南方医科大学学报

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (3): 614-621.doi: 10.12122/j.issn.1673-4254.2025.03.19

• • 上一篇    

源自蛇毒的蛋白C激活剂通过调控HIF-1α抑制BNIP3活性氧生成保护人脐静脉内皮细胞免受缺氧-复氧损伤

廖茗(), 钟文华, 张冉, 梁娟, 徐文陶睿, 万文珺, 吴超, 李曙()   

  1. 皖南医学院病理生理学教研室，安徽 芜湖 241002
  • 收稿日期:2024-09-10 出版日期:2025-03-20 发布日期:2025-03-28
  • 通讯作者: 李曙 E-mail:1813890002@qq.com;wylishu@wnmc.edu.cn
  • 作者简介:廖 茗，在读硕士研究生，E-mail: 1813890002@qq.com
  • 基金资助:
    安徽省教育厅重大研究项目(KJ2020ZD55);安徽省重点实验室校级开放课题(LAB202208);安徽省大学生创新创业训练计划(S202310368059┫皖南医学院2023年度校中青年科研基金┣WK2023ZQNZ01)

Protein C activator derived from snake venom protects human umbilical vein endothelial cells against hypoxia-reoxygenation injury by suppressing ROS via upregulating HIF-1α and BNIP3

Ming LIAO(), Wenhua ZHONG, Ran ZHANG, Juan LIANG, Wentaorui XU, Wenjun WAN, Chao LI Shu WU   

  1. Department of Pathophysiology, Wannan Medical College, Wuhu 241002, China
  • Received:2024-09-10 Online:2025-03-20 Published:2025-03-28

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摘要：

目的 探究蛇毒天然组分蛋白C激活剂（PCA）抑制ROS生成发挥抗血管内皮细胞损伤作用及机制。 方法 体外培养人脐静脉血管内皮细胞系（HUVECs），建立内皮细胞氧糖剥夺/再复氧（OGD/R）模型，检测PCA和2-ME2（HIF1α抑制剂）单独处理及合用对细胞ROS生成和HIF-1α、BNIP3、Beclin-1 蛋白表达的影响。OGD/R细胞模型转染BNIP3特异性siRNA，分为空载组、干扰组、PCA 组、干扰+PCA 组，检测各组HIF1α、BNIP3、Beclin-1 蛋白表达量及ROS含量。 结果 抑制HIF1α表达研究结果表明，PCA处理后蛋白HIF-1α、BNIP3、Beclin-1表达水平增高（P<0.05）而ROS含量明显降低（P<0.05），2-ME2+PCA组与PCA组相比，蛋白HIF-1α、BNIP3、Beclin-1表达水平降低（P<0.05），而ROS含量增高（P<0.05），与之相反，PCA+DMOG组与PCA组相比，蛋白HIF-1α、BNIP3、Beclin-1表达水平增高（P<0.05），而ROS含量降低（P<0.05）。BNIP3干扰研究结果显示，干扰组+PCA组与干扰组相比，蛋白BNIP3表达增高（P<0.05），HIF-1α没有变化（P>0.05），ROS含量降低（P<0.05）；干扰+PCA 组与 PCA组相比蛋白 BNIP3、Beclin-1 蛋白表达水平降低（P<0.05），而ROS含量都增高（P<0.05）。 结论 蛇毒PCA通过调控HIF-1α上调BNIP3抑制OGD/R诱导HUVECs 产生的ROS，发挥抗损伤作用。

关键词: 内皮细胞氧糖剥夺/再复氧, 蛋白C激活剂, 线粒体自噬, HIF-1α

Abstract:

Objective To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O2) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. Results In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. Conclusion Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.

Key words: oxygen-glucose deprivation/reoxygenation, protein C activator, mitochondrial autophagy, hypoxia-inducible factor-1α