中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (28): 4448-4455.doi: 10.12307/2023.522

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

荣筋拈痛方对膝关节软骨细胞外基质代谢的作用及机制

赵忠胜1，2，陈振沅1，黄云梅1，张  铃1，吴广文3，陈  俊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中医骨伤及运动康复教育部重点实验室，福建省福州市  350122
  • 收稿日期:2022-05-31 接受日期:2022-08-13 出版日期:2023-10-08 发布日期:2023-01-29
  • 通讯作者: 陈俊，副教授，福建中医药大学中西医结合研究院，福建省福州市 350122；中医骨伤及运动康复教育部重点实验室，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，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)，项目负责人：吴广文；福建省自然科学基金面上项目(2020J01750)，项目负责人：吴广文

Role and mechanism of Rongjin Niantong Fang for extracellular matrix metabolism of chondrocytes

Zhao Zhongsheng1, 2, Chen Zhenyuan1, Huang Yunmei1, Zhang Ling1, Wu Guangwen3, Chen Jun1, 3   

  1. 1Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; 2Department of Orthopedics, Chongqing Hospital of traditional Chinese Medicine, Chongqing 400021, China; 3Key Laboratory of Orthopedics & Traumatology of Traditional Chinese Medicine and Rehabilitation, Ministry of Education, Fuzhou 350122, Fujian Province, China
  • Received:2022-05-31 Accepted:2022-08-13 Online:2023-10-08 Published:2023-01-29
  • Contact: Chen Jun, Associate professor, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; Key Laboratory of Orthopedics & Traumatology of Traditional Chinese Medicine and Rehabilitation, Ministry of Education, 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; Department of Orthopedics, Chongqing Hospital of traditional Chinese Medicine, Chongqing 400021, China
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 82074465 (to WGW); General Project of Fujian Natural Science Foundation, No. 2020J01750 (to WGW)

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摘要：

文题释义：

细胞外基质：是多细胞有机体细胞周围多种大分子组成的复杂网络，主要由胶原蛋白、非胶原蛋白、弹性蛋白、蛋白聚糖与氨基聚糖5类物质组成。软骨细胞外基质具有保护软骨细胞免受机械应力破坏的作用。

背景：课题组前期动物实验研究表明，荣筋拈痛方可通过LncRNA GAS5/miR-21影响软骨基质分解和合成代谢，以达到防治膝骨关节炎的作用。LncRNA GAS5/miR-21能否作为防治膝骨关节炎的靶点，仍需从细胞水平研究验证。
目的：从细胞水平探讨荣筋拈痛方防治膝骨关节炎的作用机制。
方法：①8周龄SPF级雄性SD大鼠40只，随机分组后给予生理盐水、荣筋拈痛方(0.45 g/mL)、盐酸氨基葡萄糖胶囊(0.015 g/mL)灌胃，制备含药血清。②分离培养SD大鼠膝关节软骨细胞，采用白细胞介素1β干预软骨细胞，诱导退变软骨细胞模型，采用CCK-8法明确含药血清干预软骨细胞的量效时效关系。③将第2代软骨细胞分为空白组(空白血清)、模型组(白细胞介素1β+空白血清)、治疗组(白细胞介素1β+荣筋拈痛方含药血清)、对照组(白细胞介素1β+盐酸氨基葡萄糖胶囊含药血清)诱导干预48 h，Real-time PCR法和Western blot法检测各组软骨细胞血清干预后LncRNA GAS5、miR-21、基质金属蛋白酶抑制物3(tissue inhibitor of metalloproteinases 3，TIMP-3)、基质金属蛋白酶(matrix metalloproteinase，MMP)3、MMP-9、MMP-13和解聚蛋白样金属蛋白酶5(a disintegrin and metalloproteinase with thrombospondin motifs，
ADAMTS-5)基因及蛋白表达。④慢病毒转染软骨细胞过表达LncRNA GAS5，分为LncRNA GAS5空载体+空白血清组、LncRNA GAS5空载体+荣筋拈痛方含药血清组、LncRNA GAS5过表达+空白血清组、LncRNA GAS5过表达+荣筋拈痛方含药血清组，各组干预后Real-time PCR检测LncRNA GAS5、miR-21、TIMP-3、MMP-9、MMP-13、ADAMTS-5基因表达。

结果与结论：①荣筋拈痛方含药血清体积分数为10%、培养48 h；盐酸氨基葡萄糖胶囊含药血清体积分数为15%、培养48 h，能显著提高软骨细胞活性；②干预48 h后，与模型组相比，荣筋拈痛方含药血清和盐酸氨基葡萄糖胶囊含药血清均能抑制LncRNA GAS5、MMP-3、MMP-9、MMP-13、ADAMTS-5基因和蛋白的表达(P < 0.05)，促进miR-21、TIMP-3基因和蛋白表达(P < 0.05)；③与空载体组相比，LncRNA GAS5过表达组LncRNA GAS5、MMP-9、MMP-13、ADAMTS-5基因表达量明显升高(P < 0.05)，miR-21、TIMP-3基因表达量明显降低(P < 0.05)；与LncRNA GAS5过表达+空白血清组相比，LncRNA GAS5过表达+荣筋拈痛方含药血清组MMP-9、MMP-13和ADAMTS-5 mRNA表达量均降低(P < 0.05)，miR-21、TIMP-3基因表达量升高(P < 0.05)；④结果表明，荣筋拈痛方通过调控LncRNA GAS5/miR-21，促进TIMP-3的表达，抑制MMP-3、MMP-9、MMP-13、ADAMTS-5等表达，从而延缓退变软骨细胞外基质降解，起到防治膝骨关节炎的作用。

https://orcid.org/0000-0002-1470-0396(赵忠胜)

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: 荣筋拈痛方, 软骨细胞, LncRNA GAS5, miR-21, 基质代谢

Abstract: BACKGROUND: Our previous animal experiments have shown that Rongjin Niantong Fang can affect the decomposition and anabolism of cartilage matrix through long non-coding RNA (LncRNA) growth arrest-specific transcript 5 (GAS5)/miR-21, thereby preventing and treating knee osteoarthritis. Whether LncRNA GAS5/miR-21 can be used as a target for the prevention and treatment of knee osteoarthritis still needs to be verified at the cellular level.
OBJECTIVE: To explore the mechanism of Rongjin Niantong Fang in the treatment of knee osteoarthritis at the cellular level. 
METHODS: (1) Forty 8-week-old SPF male Sprague-Dawley rats were randomly divided into blank serum group, Rongjin Niantong Fang-containing serum group, and glucosamine hydrochloride capsule-containing serum group. 0.9% normal saline, Rongjin Niantong Fang (0.45 g/mL), and glucosamine hydrochloride capsule (0.015 g/mL) were intragastrically given to the rats in the corresponding groups to collect drug containing serum. (2) Cells were treated with interleukin-1β to induce degenerative chondrocyte model. (3) Chondrocytes from the knee joint of Sprague-Dawley rats were isolated and cultured. Passage 2 chondrocytes were obtained and divided into blank group (blank serum), and model group (interleukin-1β+blank serum), treatment group (interleukin-1β+Rongjin Niantong Fang-containing serum), and control group (interleukin-1β+glucosamine hydrochloride capsule-containing serum). After 48 hours of intervention, the gene and protein expressions of LncRNA GAS5, miR-21, tissue inhibitor of metalloproteinases 3 (TIMP-3), matrix metalloproteinase (MMP)-3, MMP-9, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) were detected by real-time PCR and western blot, respectively. (4) Chondrocytes were transfected with LncRNA GAS5 overexpression lentiviral vector. Then, the transfected cells were divided into LncRNA GAS5 empty vector + blank serum group, LncRNA GAS5 empty vector + Rongjin Niantong Fang-containing serum group, LncRNA GAS5 overexpression + blank serum group, and LncRNA GAS5 overexpression + Rongjin Niantong Fang-containing serum group. The gene expressions of LncRNA GAS5, miR-21, TIMP-3, MMP-9, MMP-13, and ADAMTS-5 were detected using real-time PCR. 
RESULTS AND CONCLUSION: Culture with the serum containing 10% Rongjin Niantong Fang and 15% glucosamine hydrochloride capsule for 48 hours significantly increased the activity of chondrocytes. Compared with the model group, Rongjin Niantong Fang-containing serum and glucosamine hydrochloride capsule-containing serum could inhibit the gene expression of LncRNA GAS5, MMP-3, MMP-9, MMP-13, and ADAMTS-5 (P < 0.05) and promote the gene and protein expression of miR-21 and TIMP-3 (P < 0.05). Compared with the blank group, the gene expression of LncRNA GAS5, MMP-3, MMP-9, MMP-13, and ADAMTS-5 in the LncRNA GAS5 overexpression group increased significantly (P < 0.05), while the gene expression of miR-21 and TIMP-3 decreased significantly (P < 0.05). Compared with the LncRNA GAS5 overexpression+blank serum group, the gene expression of MMP-9, MMP-13, and ADAMTS-5 was significantly decreased in the LncRNA GAS5 overexpression+Rongjin Niantong Fang-containing serum group (P < 0.05), while the gene expression of miR-21 and TIMP-3 were significantly increased (P < 0.05). To conclude, Rongjin Niantong Fang could delay the degradation of cartilage extracellular matrix by promoting TIMP-3 expression and inhibiting the expression of MMP-3, MMP-9, MMP-13 and ADAMTS-5 through regulating LncRNA GAS5/miR-21, thereby preventing and treating knee osteoarthritis.

Key words: Rongjin Niantong Fang, chondrocyte, LncRNA GAS5, miRNA-21, matrix metabolism

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