Robinin protects chondrocytes injury via TLR2/TLR4/NF-κB signaling in osteoarthritis.

Abstract

Osteoarthritis (OA) is a joint disease closely related to aging and characterized by degeneration of articular cartilage. Robinin is a natural agent with various pharmacological properties. Recently, Robinin has been found to have the potential to improve the bone-related diseases. However, its effect on OA development remained unknown. Here, we discuss the specific role and underlying mechanisms of Robinin in interleukin-1beta (IL-1β)-treated chondrocytes and OA mouse model. Chondrocytes were isolated from the mouse to conduct in vitro assays. We evaluated cell viability and apoptosis using Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Western blotting assessed the levels of proteins related to apoptosis, extracellular matrix (ECM), and signaling pathways. Immunofluorescence staining was used to detect the expression of ECM and signaling markers. ELISA was conducted to assess the levels of inflammatory markers. The OA mice model was established using surgical destabilization of the medial meniscus (DMM), and then H&E staining and Safranin O staining were conducted to observe the histopathological changes in synovial tissues. TUNEL assay was used to detect cell apoptosis in vivo. Real-time RT-PCR was operated to measure mRNA level in vitro and in vivo. We discovered that Robinin reversed the IL-1β-induced decrease in chondrocyte viability. Robinin suppressed IL-1β-induced apoptosis of chondrocytes. The ECM destruction and inflammatory response induced by IL-1β were markedly reversed by Robinin incubation in the mouse chondrocytes. Besides, the upregulated cytokine mRNA levels in IL-1β-treated chondrocytes were reduced by Robinin treatment. The downregulation of COL2A1 level and upregulation of MMP13 and ADAMTS5 levels were counteracted by Robinin treatment. Robinin reduced the protein levels of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) but enhanced the level of phosphorylated p65 (p-p65) in IL-1β-stimulated chondrocytes and OA mice. Robinin mitigated inflammation, cell apoptosis and cartilage destruction in synovial tissues from the OA mice. In conclusion, Robinin alleviated OA development in vitro and in vivo via TLR2/TLR4/NF-κB signaling pathway.