Carbon dot Prussian blue nanoenzyme antioxidative stress delays intervertebral disc degeneration

doi:10.12307/2023.177

Abstract

Abstract: BACKGROUND: In recent years, oxidative stress has been found to be an important activation mechanism of intervertebral disc degeneration. Nanoparticles with enzyme-like activity have attracted a lot of attention in the study of anti-oxidant stress, but there is little research on delaying intervertebral disc degeneration.
OBJECTIVE: To construct carbon dot Prussian blue nanoparticles (PEI-600-Fe C-dots Prussian Blue Nanoparticles, CD-PBs), to verify that CD-PBs can eliminate excessive reactive oxygen species, regulate oxidative stress and delay intervertebral disc degeneration in rats by in vitro and in vivo experiments.
METHODS: Based on the hydrothermal method, polyethyleneimine was used as carbon source to prepare carbon dots by mixing with citric acid and FeCl3•6H2O, and then Prussian blue was synthesized in situ on its surface to construct CD-PBs. (1) In vitro experiment: SD rat nucleus pulposus cells were extracted and cultured for three generations. Then, H2O2 solution (control group) and CD-PBs +H2O2 solution (experimental group) were added for co-culture, and the cells cultured alone were used as blank controls. After the cells were treated for 24 hours, the level of intracellular reactive oxygen species and mitochondrial membrane potential were detected by fluorescent probes. The mRNA expression levels of type II collagen, aggrecan, matrix metalloproteinase 3 and tumor necrosis factor α were analyzed by real-time quantitative PCR. (2) In vivo experiment: 30 SD rats were randomly divided into three groups. The blank control group did not do any treatment. In the control group, a model of coccygeal intervertebral disc degeneration was established. In the experimental group, CD-PBs solution was injected into the degenerated intervertebral disc space. At 8 weeks after operation, the morphology of intervertebral disc was observed by pathological section.
RESULTS AND CONCLUSION: (1) In vitro experiments: The intracellular reactive oxygen species level in the control group was higher than that in the blank control group (P < 0.05). The level of intracellular reactive oxygen species in the experimental group was lower than that in the control group (P < 0.05). The mitochondrial fluorescence intensity of the control group was lower than that of the blank control group (P < 0.05). The mitochondrial fluorescence intensity of the experimental group was higher than that of the control group (P < 0.05). Compared with the blank control group, the mRNA expression of type II collagen and aggrecan decreased in the control group (P < 0.05); the mRNA expression of matrix metalloproteinase 3 and tumor necrosis factor α increased (P < 0.05). Compared with the control group, the mRNA expression of collagen type II and aggrecan in the experimental group increased (P < 0.05); the mRNA expression of matrix metalloproteinase 3 and tumor necrosis factor α decreased (P < 0.05). (2) In vivo experiment: Hematoxylin-eosin staining showed that the height of the intervertebral disc space in the control group was significantly reduced; the arrangement of the annulus fibrosus was disordered; the structure of the nucleus pulposus was destroyed, and the cells and matrix were lost in large quantities. In the experimental group, the height of the intervertebral disc space did not decrease significantly; the nucleus pulposus was partially lost, and the structure of the annulus fibrosus was slightly disordered, which was significantly improved compared with the control group. (3) The results showed that CD-PBs could prevent oxidative stress by removing intracellular reactive oxygen species and delay intervertebral disc degeneration.

Key words: intervertebral disc degeneration, carbon dot, nanozyme, Prussian blue, reactive oxygen species, mitochondria, oxidative stress

CLC Number:

Cao Zhipeng, Shi Yu, Li Ke, Lin Wenzheng, Jiang Letao, Bu Wenzhen, Zhu Hai, Du Jianwei, Wang Huihui, Chen Hao. Carbon dot Prussian blue nanoenzyme antioxidative stress delays intervertebral disc degeneration[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(25): 4038-4044.

Figures/Tables 7

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