中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (34): 7318-7325.doi: 10.12307/2025.895

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

普鲁士蓝纳米粒子抗氧化恢复退变髓核细胞线粒体功能

张晓宇，韦善文，方佳炜，倪  莉   

  1. 苏州大学附属第一医院骨科，江苏省苏州市   215006
  • 收稿日期:2024-07-31 接受日期:2024-11-05 出版日期:2025-12-08 发布日期:2025-01-17
  • 通讯作者: 倪莉，博士，副研究员，苏州大学附属第一医院骨科，江苏省苏州市 215006
  • 作者简介:张晓宇，男，1999年生，安徽省合肥市人，汉族，苏州大学附属第一医院在读硕士，主要从事椎间盘退变修复方面的研究。
  • 基金资助:
    博士后科研基金项目(2021M702393)，项目负责人：倪莉；苏州市科技局项目(SKY2023148)，项目负责人：倪莉

Prussian blue nanoparticles restore mitochondrial function in nucleus pulposus cells through antioxidation

Zhang Xiaoyu, Wei Shanwen, Fang Jiawei, Ni Li   

  1. Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Received:2024-07-31 Accepted:2024-11-05 Online:2025-12-08 Published:2025-01-17
  • Contact: Ni Li, MD, Associate researcher, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Zhang Xiaoyu, Master candidate, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Supported by:
    Postdoctoral Science Foundation of China, No. 2021M702393 (to NL); Suzhou Science and Technology Bureau Project, No. SKY2023148 (to NL)

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摘要：


文题释义：

普鲁士蓝纳米粒子：是一种纳米尺度的铁氰化合物，呈深蓝色，具有独特的立方晶体结构。普鲁士蓝纳米粒子结合了普鲁士蓝的化学特性和纳米材料的优势，拥有大比表面积、高反应活性和可调控性质。由于多功能性，普鲁士蓝纳米粒子在环境净化、生物医学、能源存储和催化等多个领域展现出广阔的应用前景。
线粒体功能障碍：是指氧化应激和遗传变异等多种因素引起细胞内的能量产生中心——线粒体无法正常执行其生理功能的状态。在这种情况下，线粒体在能量产生、细胞代谢调节、氧化还原平衡维持等方面的能力受到显著影响，进而导致细胞功能异常和多种疾病的发生。


背景：恢复髓核细胞的正常活性氧水平和线粒体功能、抑制髓核细胞凋亡是延缓椎间盘退变的关键靶点。普鲁士蓝纳米粒子具有类过氧化物酶活性，能够有效清除病理微环境中的活性氧，保护髓核细胞免受氧化应激损伤。

目的：探讨普鲁士蓝纳米粒子延缓大鼠髓核退变的生物学功能和机制。
方法：采用水热法制备普鲁士蓝纳米粒子，表征其微观形貌与粒径大小。采用不同质量浓度(20，40，60，80，100 µg/mL)的普鲁士蓝纳米粒子干预第2代SD大鼠尾椎髓核细胞，24 h后通过CCK-8实验检测细胞增殖；采用60 µg/mL普鲁士蓝纳米粒子干预第2代SD大鼠尾椎髓核细胞，1，3 d后通过活死染色观察髓核细胞活力。取第2代SD大鼠尾椎髓核细胞，观察细胞贴壁后分3组干预：对照组不进行任何干预，脂多糖组加入脂多糖，脂多糖+普鲁士蓝纳米粒子组加入脂多糖与60 µg/mL普鲁士蓝纳米粒子，干预24后分别进行活性氧、线粒体超氧化物、线粒体膜电位检测，干预48 h后分别进行RT-qPCR检测与阿尔新蓝染色。

结果与结论：①透射电镜下可见普鲁士蓝纳米粒子为均匀的纳米立方体，平均粒径为130 nm；②CCK-8检测结果显示20-60 µg/mL普鲁士蓝纳米粒子无明显的细胞毒性，后续实验选择60 µg/mL普鲁士蓝纳米粒子进行细胞干预；活死染色结果显示60 µg/mL普鲁士蓝纳米粒子不影响髓核细胞活力；③与对照组比较，脂多糖组髓核细胞内活性氧、线粒体超氧化物水平升高(P < 0.01)，线粒体膜电位降低(P < 0.01)，Ⅱ型胶原、聚集蛋白多糖mRNA表达降低(P < 0.01)，基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5 mRNA表达升高(P < 0.01)，阿尔新蓝染色阳性区域减少(P < 0.01)；与脂多糖组比较，脂多糖+普鲁士蓝纳米粒子组髓核细胞内活性氧、线粒体超氧化物水平降低(P < 0.01)，线粒体膜电位升高(P < 0.01)，Ⅱ型胶原、聚集蛋白多糖mRNA表达升高(P < 0.01)，基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5 mRNA表达降低(P < 0.01)，阿尔新蓝染色阳性区域增加(P < 0.01)。结果表明，普鲁士蓝纳米粒子通过减轻髓核细胞氧化应激、恢复线粒体功能并维持细胞外基质合成和分解代谢平衡延缓大鼠髓核退变。

https://orcid.org/0009-0000-0369-2004 (张晓宇)；https://orcid.org/0009-0001-0963-8364 (倪莉) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 普鲁士蓝纳米粒子, 氧化应激, 线粒体功能障碍, 髓核, 细胞外基质, 工程化骨科材料

Abstract: BACKGROUND: Restoring the normal level of reactive oxygen species and mitochondrial function of nucleus pulposus cells and inhibiting apoptosis of nucleus pulposus cells are key targets for delaying intervertebral disc degeneration. Prussian blue nanoparticles have peroxidase-like activity, which can effectively remove reactive oxygen species in the pathological microenvironment and protect nucleus pulposus cells from oxidative stress damage.
OBJECTIVE: To investigate the biological functions and mechanisms of Prussian blue nanoparticles in delaying nucleus pulposus degeneration in rats.
METHODS: Prussian blue nanoparticles were prepared by hydrothermal method, and their micromorphology and particle size were characterized. Prussian blue nanoparticles with different mass concentrations (20, 40, 60, 80, and 100 µg/mL) were used to intervene in the caudal nucleus pulposus cells of passage 2 SD rats. Cell proliferation was detected by CCK-8 assay after 24 hours. 60 µg/mL Prussian blue nanoparticles were used to intervene in the caudal nucleus pulposus cells of passage 2 SD rats. The viability of nucleus pulposus cells was observed by live-dead staining after 1 and 3 days. Passage 2 SD rat caudal vertebrae nucleus pulposus cells were obtained and observed for cell adhesion before being divided into three intervention groups. The control group did not receive any intervention. The lipopolysaccharide group was added with lipopolysaccharide. The lipopolysaccharide + Prussian blue nanoparticle group was added with lipopolysaccharide and 60 µg/mL Prussian blue nanoparticles. Reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential were detected 24 hours after intervention. RT-qPCR detection and Alcian blue staining were performed 48 hours after intervention. 
RESULTS AND CONCLUSION: (1) Under transmission electron microscopy, Prussian blue nanoparticles were uniform nanocubes with an average particle size of 130 nm. (2) CCK-8 assay results showed that 20-60 µg/mL Prussian blue nanoparticles had no obvious cytotoxicity, and 60 µg/mL Prussian blue nanoparticles were selected for cell intervention in subsequent experiments. Live-dead staining results showed that 60 µg/mL Prussian blue nanoparticles had no effect on the viability of nucleus pulposus cells. (3) Compared with the control group, the levels of reactive oxygen species and mitochondrial superoxide in nucleus pulposus cells in the lipopolysaccharide group were increased (P < 0.01), the mitochondrial membrane potential was decreased (P < 0.01), the mRNA expressions of type II collagen and aggrecan were decreased (P < 0.01), the mRNA expressions of matrix metalloproteinase 13 and thrombospondin integrin metallopeptidase 5 were increased (P < 0.01), and the positive area of Alcian blue staining was reduced (P < 0.01). Compared with the lipopolysaccharide group, the levels of reactive oxygen species and mitochondrial superoxide in nucleus pulposus cells in the lipopolysaccharide + Prussian blue nanoparticle group were decreased (P < 0.01), mitochondrial membrane potential increased (P < 0.01), mRNA expression of type II collagen and aggrecan increased (P < 0.01), mRNA expression of matrix metalloproteinase 13 and thrombospondin integrin metallopeptidase 5 decreased (P < 0.01), and positive area of Alcian blue staining increased (P < 0.01). The results showed that Prussian blue nanoparticles delayed the degeneration of rat nucleus pulposus by reducing oxidative stress of nucleus pulposus cells, restoring mitochondrial function, and maintaining the balance of extracellular matrix synthesis and catabolism. 

Key words: Prussian blue nanoparticle, oxidative stress, mitochondrial dysfunction, nucleus pulposus, extracellular matrix, engineered orthopedic material

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