Abstract: BACKGROUND: Research has shown that vanillic acid has anti-inflammatory and anti-oxidative stress effects, but it is unclear whether it has a protective effect on endplate chondrocytes.
OBJECTIVE: To explore the effect and mechanism of vanillic acid on endplate chondrocytes under inflammatory microenvironment.
METHODS: (1) Primary endplate chondrocytes were isolated from the intervertebral disc of SD rats and identified by toluidine blue staining and collagen II immunofluorescence. (2) The CCK-8 assay was employed to detect the effects of interleukin-1β and vanillic acid on the proliferation activity of endplate chondrocytes, in order to determine the concentration of vanillic acid for subsequent cell treatment. (3) An inflammatory microenvironment was simulated by adding 10 ng/mL interleukin-1β to the culture medium, and the endplate chondrocytes were treated with low, medium, and high mass concentrations of vanillic acid. The expression levels of inflammatory markers and extracellular matrix proteins were detected by western blot assay and immunofluorescence. (4) The expression of nuclear factor κB signaling pathway-related proteins was detected by western blot assay.
RESULTS AND CONCLUSION: (1) The morphology of endplate chondrocytes in adherent culture was pike or triangular in shape, positive for toluidine blue staining and immunofluorescence for collagen II, indicating that the experimentally extracted cells were endplate chondrocytes. (2) The CCK-8 assay results showed that treatment with 2.5, 5, 10, and 20 μg/mL vanillic acid for 24 hours did not significantly inhibit the proliferation of endplate chondrocytes. Compared with the interleukin-1β group, the viability of endplate chondrocytes treated with 5, 10, and 20 μg/mL vanillic acid for 24 hours was significantly increased (P < 0.05). Therefore, 5, 10, and 20 μg/mL vanillic acid was selected as the low, medium, and high dose groups for subsequent treatment of endplate chondrocytes. (3) Compared with the model group (complete medium containing 10 ng/mL interleukin-1β), the expression of NOD-like receptor thermal domain associated protein 3 (NLRP3), matrix metalloproteinase 13, matrix metalloproteinase 3, and tumor necrosis factor alpha protein in the endplate chondrocytes of the low, medium, and high doses of vanillic acid groups were significantly reduced (P < 0.05). (4) Compared with the model group, the protein expression of aggrecan and collagen II in the endplate chondrocytes of the low, medium, and high dose groups of vanillic acid significantly increased (P < 0.05). (5) Compared with the model group, the protein expression of phospho-nuclear factor κB and phospho-inhibitor of nuclear factor κB in the endplate chondrocytes of the low, medium, and high dose groups of vanillic acid was significantly reduced (P < 0.05). (6) The above results indicate that vanillic acid may alleviate the inflammatory response and extracellular matrix degradation induced by interleukin-1β in rat endplate chondrocytes by inhibiting the activation of the nuclear factor κB signaling pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: vanillic acid, endplate chondrocytes, inflammation, intervertebral disc degeneration, nuclear factor κB, engineered tissue construction