Targeting TLR4/non-canonical NF-κB pathway by Bushen Bitong recipe enhances BMSCs-based cartilage repair in osteoarthritis

Bushen Bitong Recipe (BSBT) is a traditional Chinese medicine used clinically for osteoarthritis, while bone marrow mesenchymal stromal cell (BMSCs) therapy is increasingly applied for its immunomodulatory and anti-inflammatory properties. However, their combined effects on osteoarthritis have not been investigated. In this study, BSBT containing serum reversed IL-1β induced apoptosis and improved the viability of BMSCs and chondrocytes. Co-treatment with BSBT and BMSCs further enhanced chondrocyte viability and migration, as shown by CCK-8 and Transwell assays. In vivo, X-ray imaging and histological staining (H&E and toluidine blue) demonstrated that the combined therapy preserved joint space, reduced osteophyte formation, and maintained cartilage structure with a more organized arrangement of chondrocytes. At the mechanistic level, TUNEL staining revealed that the combined treatment markedly reduced chondrocyte apoptosis in articular cartilage compared with osteoarthritic controls. These findings were consistent with Western blot analysis, which showed decreased expression of pro-apoptotic proteins Bax and cleaved caspase-3 after co-treatment. Immunofluorescence staining further demonstrated reduced tissue level expression of TLR4, MyD88, and phosphorylated RelB ( p -RelB), indicating attenuation of TLR4/non-canonical NF-κB signaling. Western blot and ELISA analyses supported these observations, showing suppression of TLR4/p-RelB and a concomitant decrease in pro-inflammatory cytokines and oxidative stress. Overall, these results suggest that BSBT and BMSCs exert synergistic protective effects on cartilage by reducing chondrocyte apoptosis and dampening inflammatory signaling, representing a promising therapeutic strategy for knee osteoarthritis. • BSBT enhances BMSC therapy by reducing chondrocyte apoptosis. • BSBT + BMSCs combination improve cartilage structure and cell alignment in KOA rats. • Combination therapy promotes chondrocyte viability and migration. • Protection involves inhibition of TLR4/non-canonical NF-κB signaling.