The effects of Jiawei Duhuo Jisheng mixture on Wnt/β-catenin signaling pathway in the synovium inflamed by knee osteoarthritis: An in vitro and in vivo experiment

Abstract

Ethnopharmacological relevance: Knee osteoarthritis (KOA) is one of the common age-degenerative diseases. Recent studies have demonstrated that the pathogenesis of KOA is closely related to synovial lesions. Jiawei Duhuo Jisheng mixture (JDJM) has shown great potential in the treatment of KOA. However, the effect and mechanism of JDJM on synovial lesions of KOA remain unclear.

Aim of the study: The regulatory effect of JDJM on the Wnt/β-catenin signaling pathway in KOA inflamed synovium was studied via in vitro and in vivo experiments, respectively.

Materials and methods: For the in vitro experiment, fibroblasts were isolated from the rabbit synovium with KOA. The fibroblasts were grouped as follows: the vehicle group was given 0.5% FBS; the inhibitor group was treated with 0.5% volume fraction of XAV939; the normal serum groups and JDJM serum groups were treated with 5%, 10%, and 20% volume fractions of normal serum and JDJM-containing serum. The expression levels of Wnt3a, β-catenin, Cyclin D1, metalloproteinase-7(MMP-7) and cyclooxygenase-2(COX-2) were detected by different assays 48 and 72 h after the intervention. For the in vivo experiment, the rabbit KOA model was prepared using the improved Hulth modeling method, whereby all rabbits were randomly divided into normal control, model control, positive control, and traditional Chinese medicine (TCM) groups. The expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 were detected by different assays in the 2, 4, and 8 weeks of treatment.

Results: In the two test results of in vitro experiments, the normal serum group was compared with the JDJM-containing serum group with the same volume fraction, demonstrating that mRNA transcription and protein expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7, and COX-2 in the latter decreased (P < 0.05), with more pronounced effects observed in the group treated with 20% volume fraction of JDJM serum. Compared with the inhibitor group, there was no significant difference (P > 0.05) in the mRNA transcription and protein expression levels, i.e., Wnt3a, β-catenin, Cyclin D1, and MMP-7 were observed in the JDJM serum groups, except for a significant decrease (P < 0.05) in the level of mRNA transcription and protein expression of COX-2. Based on the in vivo experiment, compared to the model control group, articular cartilage, synovial hyperplasia, and the inflammatory reaction of the TCM group at different treatment times were significantly improved. The mRNA transcription level of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 detected by RT-qPCR and the protein expression level of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 detected by Western blot were significantly reduced (P < 0.05), and the effect was more evident at the eighth week.

Conclusion: JDJM can regulate the synovial Wnt/β-catenin signaling pathway in KOA models, reduce the mRNA transcription and protein expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7, and COX-2 in the synovium, thus inhibiting synovial inflammation and protecting articular cartilage, which could be the key mechanism of action in treating this disease.

Keywords: Fibroblasts; Jiawei Duhuo Jisheng mixture; Knee osteoarthritis; Synovium; Wnt/β-catenin signaling pathway.