中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (20): 3195-3201.doi: 10.12307/2023.405

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

荣筋拈痛方调控软骨基质代谢防治膝骨关节炎

赵忠胜1，2，郑若曦1，林  洁1，陈  俊3，叶锦霞3，付长龙3，吴广文1，3   

  1. 1福建中医药大学中西医结合研究院，福建省福州市  350122； 2重庆市中医院骨科，重庆市  400021；3福建省中西医结合老年性疾病重点实验室，福建省福州市  350122
  • 收稿日期:2022-02-28 接受日期:2022-06-10 出版日期:2023-07-18 发布日期:2022-11-19
  • 通讯作者: 吴广文，副研究员，硕士生导师，福建中医药大学中西医结合研究院，福建省福州市 350122；福建省中西医结合老年性疾病重点实验室，福建省福州市 350122
  • 作者简介:赵忠胜，男，1990年生，甘肃省康乐县人，汉族，2020年福建中医药大学毕业，博士，主治医师，主要从事中西医结合防治骨关节病的基础与临床研究。
  • 基金资助:
    国家自然科学基金面上项目(82074465)、福建省自然科学基金面上项目(2020J01750)，项目负责人：吴广文；重庆市自然科学基金面上项目(cstc2021jcyj-msXMX0861)，项目负责人：赵忠胜

Rongjin Niantong Fang for treating knee osteoarthritis by regulating cartilage matrix metabolism

Zhao Zhongsheng1, 2, Zheng Ruoxi1, Lin Jie1, Chen Jun3, Ye Jinxia3, Fu Changlong3, Wu Guangwen1, 3   

  1. 1Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou  350122, Fujian Province, China; 2Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, China; 3Fujian Key Laboratory of Integrative Medicine on Geriatrics, Fuzhou 350122, Fujian Province, China
  • Received:2022-02-28 Accepted:2022-06-10 Online:2023-07-18 Published:2022-11-19
  • Contact: Wu Guangwen, Associate researcher, Master’s supervisor, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; Fujian Key Laboratory of Integrative Medicine on Geriatrics, Fuzhou 350122, Fujian Province, China
  • About author:Zhao Zhongsheng, MD, Attending physician, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 82074465 (to WGW); General Project of Fujian Natural Science Foundation, No. 2020J01750 (to WGW); General Project of Chongqing Natural Science Foundation, No. cstc2021jcyj-msXMX0861 (to ZZS)

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摘要：


文题释义：

长链非编码RNA：是全长大于200个核苷酸的非编码RNA，占非编码RNA的80%，缺乏翻译成蛋白的能力，可通过染色质重塑、竞争性内源性RNA、mRNA稳定和支架蛋白募集等多种机制影响基因的转录和转录后表达。
基质金属蛋白酶：是一大类具有相似结构的蛋白酶，构成了细胞外基质降解最重要的蛋白水解系统，基质金属蛋白酶在软骨基质降解中起重要作用。

背景：前期研究发现荣筋拈痛方能有效抑制软骨基质降解。长链非编码RNA GAS5可作为竞争性内源性RNA吸附miR-21-5p调控基质金属蛋白酶的表达，进而影响关节软骨基质代谢。但荣筋拈痛方是否通过长链非编码RNA GAS5/miR-21调控软骨基质代谢，发挥防治骨关节炎的作用机制有待深入研究。
目的：从长链非编码RNA GAS5/miR-21调控软骨基质合成与分解代谢角度，探讨荣筋拈痛方治疗SD大鼠膝骨关节炎的作用机制。
方法：SPF级8周龄雄性SD大鼠60只，随机分为4组，即空白组、模型组、治疗组及对照组，每组15只。后3组采用改良Hulth法建立大鼠膝骨关节炎模型。造模后2周，空白组和模型组予生理盐水，治疗组予荣筋拈痛方汤剂，对照组予盐酸氨基葡萄糖胶囊水溶液，每天灌胃一次，共干预12周。药物干预结束后取材，采用苏木精-伊红染色、改良番红O-固绿染色观察软骨组织显微结构形态变化，实时荧光定量PCR法检测长链非编码RNA GAS5、miR-21的表达及基质代谢因子mRNA的表达，Western blot法检测软骨组织中基质代谢因子蛋白的表达。
结果与结论：①软骨显微结构观察，模型组软骨4层结构紊乱，排列不规律，潮线及黏合线不规则或消失，软骨细胞增生肥大且排列紊乱，软骨下骨增生，胶原、蛋白多糖大量丢失；治疗组和对照组软骨4层结构、潮线可辨，软骨细胞排列较规律，软骨下骨结构较完整，胶原、蛋白多糖部分丢失；②与空白组相比，模型组软骨组织中长链非编码RNA GAS5及基质金属蛋白酶3、基质金属蛋白酶9、基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5 mRNA表达量升高(P < 0.01或P < 0.05)，治疗组、对照组与模型组相比表达量降低(P < 0.01或P < 0.05)；模型组软骨组织中miR-21及组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖mRNA表达量降低(P < 0.01或P < 0.05)，治疗组、对照组与模型组相比表达量升高(P < 0.05)；③与空白组相比，模型组软骨组织中组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖蛋白表达降低(P < 0.01或P < 0.05)，治疗组和对照组中表达升高(P < 0.01或P < 0.05)；模型组软骨组织中基质金属蛋白酶3、基质金属蛋白酶9、基质金属蛋白酶13、血小板反应蛋白解整合素金属肽酶5蛋白表达升高(P < 0.01或P < 0.05)，治疗组和对照组中表达降低(P < 0.01或
P < 0.05)；④提示荣筋拈痛方下调膝骨关节炎大鼠软骨组织中长链非编码RNA GAS5、基质金属蛋白酶3、基质金属蛋白酶9等的表达，上调miR-21、组织金属蛋白酶抑制因子3、Ⅱ型胶原和聚集蛋白聚糖的表达，影响软骨基质分解和合成代谢，减少软骨基质中胶原、蛋白多糖的丢失，延缓软骨基质降解，减轻软骨组织形态结构的破坏。
https://orcid.org/0000-0002-1470-0396(赵忠胜)

关键词: 荣筋拈痛方, 膝骨关节炎, 长链非编码RNA GAS5, miR-21, 软骨基质代谢

Abstract: BACKGROUND: Preliminary studies have found that Rongjin Niantong Fang can effectively inhibit the degradation of cartilage matrix. Long non-coding RNA GAS5 can act as a competitive endogenous RNA that adsorbs miR-21-5p to regulate the expression of matrix metalloproteins, thereby influencing the metabolism of articular cartilage matrix. However, whether Rongjin Niantong Fang regulates cartilage matrix metabolism through long non-coding RNA GAS5/miR-21 and plays a role in preventing and treating osteoarthritis needs to be further studied.
OBJECTIVE: To explore the mechanism by which Rongjin Niantong Fang treats knee osteoarthritis by long non-coding RNA GAS5/miR-21 pathway regulating cartilage matrix synthesis and catabolism. 
METHODS: Sixty male SPF-grade Sprague-Dawley rats aged 8 weeks were randomized into four groups (n=15 per group): blank group, model group, treatment group and control group. Knee osteoarthritis models were established using the modified Hulth method in the latter three groups. The blank group and the model group were intragastrically administered with normal saline, the treatment group was intragastrically administered with water extract of Rongjin Niantong Fang, and control group was intragastrically administered with glucosamine hydrochloride capsules, once a day for 12 weeks. After treatment, microstructure and morphological changes of cartilage were observed by hematoxylin-eosin staining and safranin O-fast green staining. The mRNA expression of long non-coding RNA GAS5, miR-21, and matrix metabolic factors in cartilage was detected by real-time PCR. The protein expression of matrix metabolic factors in cartilage was detected by western blot. 
RESULTS AND CONCLUSION: In the model group, the four layers of the cartilage were disordered and arranged irregularly, the tide line and adhesion line were irregular or disappeared, chondrocytes proliferated and arranged disorderly, the subchondral bone proliferated, and a large amount of collagens and proteoglycans were lost. In the treatment and control groups, the four layers of the cartilage and tide line were recognizable, chondrocytes arranged regularly, the subchondral bone structure was relatively intact, and part of collagens and proteoglycans were lost. Compared with the blank group, the expression levels of long non-coding RNA GAS5, matrix metalloproteinases 3, 9, 13, and a disintegrin and metalloproteinase with thrombospondin motif-5 mRNAs and proteins in cartilage were significantly increased in the model group (P < 0.01 or P < 0.05), while the expression levels of miR-21, tissue inhibitors of metalloproteinase 3, type II collagen, and Aggrecan proteins and mRNAs in cartilage were significantly decreased in the model group (P < 0.01 or P < 0.05). Compared with the model group, the expression trends of these genes and proteins in cartilage were reversely changed in the treatment and control groups (P < 0.01 or P < 0.05). To conclude, Rongjin Niantong Fang can reduce the loss of collagen and proteoglycan in cartilage matrix, delay the degradation of cartilage matrix, and reduce the damage of cartilage morphology and structure by down-regulating the expression of long non-coding RNA GAS5, matrix metalloproteinases 3, 9, 13, and a disintegrin and metalloproteinase with thrombospondin motif-5, and up-regulating the expression of miR-21, tissue inhibitors of metalloproteinase 3, type II collagen, and Aggrecan in cartilage of knee osteoarthritis rats.

Key words: Rongjin Niantong Fang, knee osteoarthritis, long non-coding RNA GAS5, miR-21, cartilage matrix metabolism 

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