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