中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (23): 3636-3640.doi: 10.12307/2021.032

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

牛膝醇提物调控滑膜成纤维细胞外泌体抑制软骨细胞外基质降解

高  坤1，陈大宇1，张  勇1，刘伟东1，孙淑芬1，赖文强1，马笃军1，吴益宏1，林展鹏2，蒋鹰鹭2，余伟吉1   

  1. 1深圳市中医院骨伤科，广东省深圳市  518033；2广州中医药大学第四临床医学院，广东省深圳市  518033
  • 收稿日期:2020-06-12 修回日期:2020-06-17 接受日期:2020-08-17 出版日期:2021-08-18 发布日期:2021-01-26
  • 通讯作者: 余伟吉，主任医师，深圳市中医院骨伤科，广东省深圳市 518033
  • 作者简介:高坤，男，1987年生，山东省济南市人，2013年南方医科大学毕业，博士，医师，主要从事骨与关节损伤的中医药防治研究。
  • 基金资助:
    深圳市科技计划项目(JCYJ20180302173509878)，项目负责人：高坤；深圳市医疗“三名工程”(SZSM201812066)

Achyranthes bidentata alcohol extract inhibits extracellular matrix degradation of the cartilage by regulating synovial fibroblast exosomes

Gao Kun1, Chen Dayu1, Zhang Yong1, Liu Weidong1, Sun Shufen1, Lai Wenqiang1, Ma Dujun1, Wu Yihong1, Lin Zhanpeng2, Jiang Yinglu2, Yu Weiji1   

  1. 1Department of Orthopedics, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, Guangdong Province, China; 2Fourth Clinical School, Guangzhou University of Chinese Medicine, Shenzhen 518033, Guangdong Province, China
  • Received:2020-06-12 Revised:2020-06-17 Accepted:2020-08-17 Online:2021-08-18 Published:2021-01-26
  • Contact: Yu Weiji, Chief physician, Department of Orthopedics, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, Guangdong Province, China
  • About author:Gao Kun, MD, Physician, Department of Orthopedics, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen 518033, Guangdong Province, China
  • Supported by:
    Shenzhen Science and Technology Plan Project, No. JCYJ20180302173509878 (to GK); Medical  “Three Projects” of Shenzhen, No. SZSM201812066

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摘要：

文题释义：
外泌体：体内体液中广泛存在的由细胞分泌的胞外囊泡，直径在30-120 nm，具有传递信号分子介导细胞与细胞间信号交流的功能。研究证实，外泌体能够通过自身携带的活性蛋白酶在物理上或功能上破坏软骨基质退化的信号传递。
软骨基质平衡：软骨基质由胶原蛋白、蛋白聚糖、弹性蛋白、纤维连接蛋白和整合素等组成，正常状态下保持代谢平衡，能够为联结组织的结构稳态提供机械及化学支持。

背景：牛膝抑制软骨基质降解、促进膝关节软骨细胞修复的机制目前尚不清楚。外泌体在膝关节滑膜液中广泛存在，其携带大量的细胞因子，发挥特定的生物学功能，对基质金属蛋白酶具有调节作用。推测牛膝有可能通过外泌体发挥对膝骨关节炎的治疗作用。
目的：探讨牛膝醇提物能否调控滑膜成纤维细胞外泌体抑制软骨细胞外基质降解。
方法：①培养正常兔滑膜成纤维细胞，分别应用PBS、100 mg/L牛膝醇提物或10 mmol/L氨基葡萄糖进行体外干预，提取细胞培养上清液中的外泌体，记为EXOs-PBS、EXOs-牛膝醇、EXOs-氨基葡萄糖；②构建实验兔膝骨关节炎模型，关节腔分别注射EXOs-PBS(空白对照组)、EXOs-牛膝醇(实验组)、EXOs-氨基葡萄糖(阳性对照组)，1 次/周，连续注射5 次；治疗前后测定关节活动度，计算关节粘连的改善度；抽取膝关节滑膜液，利用酶联蛋白免疫法检测基质金属蛋白酶1，3，9水平；提取关节软骨基质，利用DMB染色试剂盒检测软骨基质糖胺聚糖含量。
结果与结论：①成功提取滑膜成纤维细胞来源外泌体；②实验组兔关节粘连的改善度为(74.60±8.05)%，明显高于空白对照组(54.00±6.96)% (P < 0.05)；实验组兔膝关节滑膜液内基质金属蛋白酶1，3水平均明显低于空白对照组(P < 0.05)；实验组兔关节软骨基质内糖胺聚糖含量为(330.83±26.71) mg/L，明显高于空白对照组(258.21±41.76) mg/L(P < 0.05)；实验组上述指标结果与阳性对照组基本持平(P > 0.05)；④提示：牛膝醇提物能干预滑膜成纤维细胞外泌体的功能，改善膝骨关节炎模型兔的膝关节活动度，降低滑膜液内基质金属蛋白酶1，3的表达水平，升高糖胺聚糖水平，抑制细胞外基质的降解，保护关节软骨。

https://orcid.org/0000-0003-1400-286X (高坤) 

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: 牛膝, 骨性关节炎, 滑膜, 外泌体, 兔, 模型

Abstract: BACKGROUND: Achyranthes bidentata can inhibit extracellular matrix degradation of the cartilage and promote the repair of articular chondrocytes. Exosomes widely exist in the synovial fluid of the knee joint and carry a large number of cytokines to play specific biological functions and regulate matrix metalloproteinase. Achyranthes bidentata is speculated to play a role in the treatment of knee osteoarthritis through exosomes
OBJECTIVE: To investigate whether Achyranthes bidentata alcohol extract can regulate exosomes of synovial fibroblasts to inhibit extracellular matrix degradation of the cartilage.
METHODS: Synovial fibroblasts were cultured and treated with PBS, 100 mg/L Achyranthes bidentata alcohol extract or 10 mmol/L glucosamine. Exosomes were extracted from the cell supernatant, and labeled as EXOs-PBS, EXOs-Achyranthes bidentata, and EXOs-glucosamine. Rabbit models of knee osteoarthritis were constructed, and EXOs-PBS (blank control group), EXOs-Achyranthes bidentata (experimental group), and EXOs-glucosamine (positive control group) were injected into the articular cavity of the rabbit models, respectively, once a week for 5 consecutive weeks. The joint mobility was measured before and after treatment. Degree of improvement of joint adhesion was calculated. Synovial fluid of the knee joint was extracted to detect matrix metalloproteinases 1, 3, and 9 using enzyme-linked immunosorbent assay. Articular cartilage matrix was extracted to detect cartilage matrix glycosaminoglycan content using DMB staining kit.
RESULTS AND CONCLUSION: The exosomes of synovial fibroblasts were successfully extracted, and the improvement of joint adhesion in the experimental group was (74.60±8.05)%, which was significantly higher than that in the blank control group [(54.00 ±6.96)%, P < 0.05). The levels of matrix metalloproteinases 1 and 3 in the knee joint fluid of model rabbits in the experimental group were significantly lower than those in the blank control group (P < 0.05). The content of glycosaminoglycan in articular cartilage matrix of model rabbits in the experimental group was (330.83±26.71) mg/L, which was significantly higher than that in the blank control group [(258.21±41.76) mg/L, P < 0.05). The above results of the experimental group were basically the same as those of the positive control group (P > 0.05). Achyranthes bidentata alcohol extract can treat exosomes of synovial fibroblasts, alleviate knee joint mobility of knee osteoarthritis rabbits, reduce the levels of matrix metalloproteinases 1 and 3 in synovial fluid of the knee joint, inhibit the degradation of extracellular matrix and protect the articular cartilage.

Key words: Achyranthes bidentata, osteoarthritis, synovium, exosomes, rabbit, model

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