Abstract
Tongbi Lingding (TBLD) has demonstrated therapeutic effects in early- and mid-stage osteoarthritis (OA), but its active components and underlying mechanisms remain unclear. This study aimed to identify the bioactive compounds of TBLD and explore the potential molecular mechanisms underlying its therapeutic effects against OA. A destabilization of the medial meniscus (DMM)-induced OA mouse model and IL-1β-stimulated chondrocytes were used to evaluate the therapeutic effects of TBLD. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was performed to identify TBLD-associated compounds in blood and knee joints. Network pharmacology, transcriptomic analysis, molecular docking, and in vitro experiments were integrated to investigate the potential mechanisms underlying the effects of TBLD. TBLD markedly alleviated cartilage degeneration and subchondral bone sclerosis in OA mice. UPLC-MS/MS identified 2295 compounds, among which 178 and 102 TBLD-associated compounds were detected in blood and knee joints, respectively. Seventy-six compounds with predicted targets and 153 shared targets were identified. Functional enrichment analysis indicated that these targets were mainly associated with the NOD-like receptor (NLR) and NF-κB signaling pathways. Molecular docking predicted potential interactions between key compounds and NLRP3 and p65. Furthermore, hinokiflavone and dehydroabietic acid suppressed inflammatory responses, restored extracellular matrix metabolism, and inhibited IL-1β-induced MMP3 and MMP13 expression in chondrocytes. Hinokiflavone treatment also reduced the expression of NLRP3 inflammasome-related proteins and attenuated the phosphorylation of NF-κB signaling-related proteins in TNF-α-stimulated chondrocytes. TBLD exerts protective effects against OA that may be associated with changes in NLRP3/NF-κB-related inflammatory signaling, further supporting its potential therapeutic application in OA.