Duhuo Jisheng decoction alleviates knee osteoarthritis via synovial mesenchymal stem cell-derived exosomes mediating the microRNA-194-5p/wnt signaling pathway

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Ethnopharmacological relevance Knee osteoarthritis (KOA) is a prevalent degenerative joint disease with limited effective treatments. Synovial mesenchymal stem cells (SMSCs) are recognized for their superior potential in cartilage regeneration compared to other stem cells. Duhuo Jisheng Decoction (DHJSD), is a traditional Chinese formulation used for centuries to treat KOA. While most studies on DHJSD have focused on chondrocytes, its effects on SMSCs or exosome derived from SMSCs have not been explored. Aim of the study This study aims to investigates the therapeutic effects of DHJSD on repairing cartilage via SMSCs-derived exosomes mediating the microRNA (miR)-194-5p/Wnt Signaling Pathway. Materials and methods Extracts of DHJSD were prepared from single batch of formulations, and LC-MS was used to analyze the components. KOA rat model with bilateral anterior cruciate ligament transection, primary SMSCs and H 2 O 2 -induced chondrocytes were established and treated with DHJSD. H&E, toluidine blue, and safranin O/fast green staining was used to assess the cartilage injury and repair, TUNEL staining was used to evaluate the level of apoptosis. Exosomes derived from DHJSD-pretreated SMSCs (DHJSD-Exo) were collected and identified by transmission electron microscopy and nanoparticle tracking analysis. The exosomal miRNA profile was analyzed through RNA sequencing and quantitative polymerase chain reaction (qPCR), and chondrocytes were intervened with 100 nM miRNA mimic/inhibitor and different doses of DHJSD-Exo. Single-cell RNA sequencing analysis was utilized to classify chondrocyte subpopulations. Additionally, chondrocyte differentiation and apoptosis following treatment with DHJSD-Exo (10 μg/mL) or the Wnt agonist BML-284 (10 μM) were investigated using Western blotting, qPCR toluidine blue staining, crystal violet staining and flow cytometry. Results DHJSD significantly mitigates cartilage destruction and reduces the extracellular matrix degradation and inhibits apoptosis in KOA rats. Also, DHJSD promotes SMSCs proliferation, and enhances chondrogenic differentiation. Additionally, DHJSD modulated the miRNA profile in exosomes derived from serum and DHJSD-pretreated SMSCs (DHJSD-Exo), with DHJSD-Exo further promoting chondrogenic differentiation in H 2 O 2 -induced chondrocytes. Single-cell RNA sequencing results indicated that the Proc sub-cluster of chondrocytes is responsible for cartilage repair and regeneration. Gene Set Enrichment Analysis (GSEA) results indicated that endocytosis and the Wnt signaling pathway are involved in the process of cartilage remodeling. Notably, inhibition of miR-194-5p targets the Wnt3a/β-catenin signaling pathway, suppressing chondrogenic differentiation and promoting apoptosis in H 2 O 2 -induced chondrocytes, while DHJSD-Exo reverses there effect. Overexpression of miR-194-5p exhibited the opposite effects, and the pro-chondrogenic effects of DHJSD-Exo via the Wnt signaling pathway could be blocked by BML-284, a Wnt signaling inhibitor. Conclusion DHJSD not only enhances SMSCs proliferation and chondrogenic differentiation, but also stimulates chondrocyte activity via DHJSD-Exo mediated activation of the miR-194-5p/Wnt signaling pathway, offering promising insights into KOA treatment. Future research should focus on advancing these promising insights toward clinical application, through further mechanistic exploration, translational studies, and ensuring the consistency of the multi-herbal formula.

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