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

doi:10.12307/2023.522

Abstract

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

CLC Number:

Zhao Zhongsheng, Chen Zhenyuan, Huang Yunmei, Zhang Ling, Wu Guangwen, Chen Jun. Role and mechanism of Rongjin Niantong Fang for extracellular matrix metabolism of chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(28): 4448-4455.

Figures/Tables 8

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