Chonggu Granules Improve Cartilage Matrix Metabolism in Knee Osteoarthritis via the miR-148a-3p/Wnt/β-Catenin Pathway

Abstract

Purpose

This study aims to explore the effect and underlying mechanism of Chonggu Granules (CGG) in knee osteoarthritis (KOA) in rats.

Methods

A papain-induced KOA model was established in rats. The pathological alterations of extracellular matrix in rat cartilage tissues were observed through hematoxylin and eosin (H&E) staining, followed by Mankin score for quantitative scoring. The ultrastructure of cartilage extracellular matrix was examined under a transmission electron microscopy (TEM). ELISA was used to measure the levels of IL-6, TNF-α, and IL-1β in rat serum. Immunofluorescence was performed for assessing the levels of MMP-3, MMP-13, and Col2al in rat cartilage. Western blot was used to identify the protein expressions of wnt1, GSK-3β, β-catenin, and Aggrecan in rat cartilage. The mRNA relative expressions of miR-148a-3p, wnt1, β-catenin, and GSK-3β in rat cartilage were detected by RT-PCR. Luciferase reporter gene was used to detect the target genes of miR-148a-3p.

Results

CGG significantly improved articular cartilage tissue and extracellular matrix metabolism compared to the model group as indicated by H&E, Mankin score, and TEM data. Moreover, low, medium, and high doses of CGG reduced the levels of IL-6, TNF-α, IL-1β, MMP-3, and MMP-13 in serum to varying degrees but increased the levels of Col2al and Aggrecan. Mechanistically, CGG targeted wnt1 by increasing the expression of miR-148a-3p in a dose-dependent manner, thereby downregulating the mRNA and protein expressions of β-catenin in cartilage tissue and upregulating the mRNA and protein expressions of GSK-3β.

Conclusion

CGG may control the miR-148a-3p/wnt/β-catenin signaling pathway to decrease the levels of its downstream target genes MMP-13 and MMP-3, increase the expressions of Col2al and Aggrecan, and downregulate the contents of inflammatory cytokines IL-6, TNF-α, and IL-1β, thereby improving the metabolism of cartilage extracellular matrix and alleviating the degeneration of articular cartilage in KOA.

Keywords:

• knee osteoarthritis
• cartilage extracellular matrix metabolism
• chonggu granules
• miR-148a-3p
• Wnt/β-catenin signaling pathway

Ethical Statement

This study was approved by the Ethics Committee of Anhui University of Traditional Chinese Medicine (approval number: 2022123). All experiments were conducted in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This study was awarded the Key project of Natural Science Research Foundation for Universities in Anhui Province (KJ2020A0395), Collaborative Innovation Program of Universities in Anhui Province (No. GXXT-2021-085), Traditional Chinese Medicine Inheritance and Innovation Development Research Project of Anhui Red Cross Foundation (No. 2021ZYZD3), University-level Exploratory Research Project of Anhui University of Traditional Chinese Medicine in 2021 (2021zxts49), and Key Project of Health Research of Anhui Province in 2022 (AHWJ2022a005).