中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (30): 6391-9397.doi: 10.12307/2025.915

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

香草酸抑制终板软骨细胞炎症反应和细胞外基质降解

于庆贺1,蔡子鸣2,吴锦涛2,马鹏飞1,张  鑫1,周龙千2,王亚坤2,林晓钦2,林文平1   

  1. 1广州中医药大学附属深圳平乐骨伤科医院脊柱外科,广东省深圳市  518118;2广州中医药大学附属深圳平乐骨伤科医院,广东省深圳市  518118
  • 收稿日期:2024-08-26 接受日期:2024-10-23 出版日期:2025-10-28 发布日期:2025-03-27
  • 通讯作者: 林文平,博士,主任医师,博士生导师,广州中医药大学附属深圳平乐骨伤科医院脊柱外科,广东省深圳市 518118
  • 作者简介:于庆贺,男,1992年生,河北省邢台市人,汉族,2020年南方医科大学毕业,硕士,医师,主要从事脊柱脊髓损伤修复方面的研究。 并列第一作者:蔡子鸣,男,2000年生,江西省南昌市人,汉族,广州中医药大学在读硕士,主要从事中西医结合治疗脊柱疾病的研究。
  • 基金资助:
    国家自然科学基金面上项目(81771323),项目负责人:林文平;广东省自然科学基金面上项目(2021A1515010722,2024A1515010445),项目负责人:林文平;深圳市自然科学基金面上项目(JCYJ20190813112401660),项目负责人:林文平

Vanillic acid inhibits inflammatory response and extracellular matrix degradation of endplate chondrocytes

Yu Qinghe1, Cai Ziming2, Wu Jintao2, Ma Pengfei1, Zhang Xin1, Zhou Longqian2, Wang Yakun2, Lin Xiaoqin2, Lin Wenping1   

  1. 1Department of Spine Surgery, Shenzhen Pingle Orthopedic Hospital, Affiliated Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518118, Guangdong Province, China; 2Shenzhen Pingle Orthopedic Hospital, Affiliated Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518118, Guangdong Province, China
  • Received:2024-08-26 Accepted:2024-10-23 Online:2025-10-28 Published:2025-03-27
  • Contact: Lin Wenping, MD, Chief physician, Doctoral supervisor, Department of Spine Surgery, Shenzhen Pingle Orthopedic Hospital, Affiliated Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518118, Guangdong Province, China
  • About author:Yu Qinghe, MS, Physician, Department of Spine Surgery, Shenzhen Pingle Orthopedic Hospital, Affiliated Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518118, Guangdong Province, China Cai Ziming, Master candidate, Shenzhen Pingle Orthopedic Hospital, Affiliated Hospital of Guangzhou University of Chinese Medicine, Shenzhen 518118, Guangdong Province, China Yu Qinghe and Cai Ziming contributed equally to this article.
  • Supported by:
    National Natural Science Foundation of China, No. 81771323 (to LWP); Natural Science Foundation of Guangdong Province of China, No. 2021A1515010722, 2024A1515010445 (to LWP); Natural Science Foundation of Shenzhen Municipality, No. JCYJ20190813112401660(to LWP) 

摘要:


文题释义:
香草酸:是一种从黄芪、当归等中草药和多种可食用植物中提取的酚类化合物,具有显著的抗炎、抗氧化作用。
终板软骨细胞:是一种位于椎间盘终板软骨中的透明软骨细胞,具有缓冲应力、供应营养的作用。

背景:研究表明,香草酸具有抗炎、抗氧化应激的作用,但它对终板软骨细胞是否有保护作用尚不清楚。
目的:探讨香草酸对炎症微环境下终板软骨细胞的作用及机制。
方法:①从SD大鼠尾椎椎间盘中提取原代终板软骨细胞,通过甲苯胺蓝染色、Ⅱ型胶原蛋白免疫荧光鉴定;②CCK-8法检测白细胞介素1β、香草酸对终板软骨细胞增殖活力的影响,以确定后续处理细胞应用的香草酸浓度;③向完全培养基中添加10 ng/mL白细胞介素1β模拟炎症微环境,用低、中、高质量浓度的香草酸处理终板软骨细胞,通过Western blot、免疫荧光检测炎症标志物和细胞外基质蛋白表达;④Western blot检测核因子κB信号通路蛋白水平。
结果与结论:①贴壁培养的终板软骨细胞形态呈梭形或三角形,甲苯胺蓝染色、Ⅱ型胶原蛋白免疫荧光阳性,证明实验提取的细胞为终板软骨细胞;②CCK-8结果显示,2.5,5,10,20 μg/mL香草酸处理终板软骨细胞24 h无明显增殖抑制效果;与白细胞介素1β组相比,5,10,20 μg/mL香草酸处理24 h的终板软骨细胞活力显著提高(P < 0.05),因此后续选用5,10,20 μg/mL香草酸为低、中、高剂量组处理终板软骨细胞;③与模型组(含10 ng/mL 白细胞介素1β的完全培养基)相比,香草酸低、中、高剂量组终板软骨细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、基质金属蛋白酶13、基质金属蛋白酶3、肿瘤坏死因子α蛋白表达水平均明显降低(P < 0.05);④与模型组相比,香草酸低、中、高剂量组终板软骨细胞中聚集蛋白聚糖、Ⅱ型胶原蛋白的蛋白表达水平显著增加(P < 0.05);⑤与模型组相比,香草酸低、中、高剂量组终板软骨细胞中磷酸化核因子κB和磷酸化核因子κB 抑制蛋白的蛋白表达水平显著降低(P < 0.05);⑥结果说明香草酸可能通过抑制核因子κB信号通路激活减轻白细胞介素1β诱导的大鼠终板软骨细胞的炎症反应和细胞外基质降解。
https://orcid.org/0000-0001-5029-5466(林文平)

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

关键词: 香草酸, 终板软骨细胞, 炎症, 椎间盘退变, 核因子κB, 工程化组织构建

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

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