Network Pharmacology-Based Approach to Investigate the Active Ingredients and Therapeutic Mechanisms of Jingu Tongxiao Pill against Osteoarthritis

[Image: see text] This study aimed to investigate the active ingredients and therapeutic mechanisms of Jingu Tongxiao Pill (JGTXP), a commonly used Chinese patent medicine, in treating osteoarthritis (OA) via network pharmacology analysis combined with experimental validation. First, we administered JGTXP to rat plasma and identified the candidate active compounds. Next, target prediction, protein–protein interaction, compound-target network construction, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted for JGTXP. Lastly, the network-derived key targets and pathways were validated in vitro and in vivo. Finally, we identified 106 compounds in JGTXP and 24 absorbed compounds in the rat plasma. Network analysis revealed that JGTXP interferes with OA mainly via regulating the inflammatory response, collagen catabolic process, and osteoclast differentiation, and the nuclear factor kappa B (NF-κB) signaling pathway plays a pivotal role in these processes. Experimentally, JGTXP exerted potential protective effects on articular cartilage and inhibited expression of inflammatory mediators and collagen catabolism-related proteins, including interleukin 1 beta (IL-1β), interleukin 6, tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase (MMP) 3 and MMP13, in a papain-induced OA rat model. Consistently, mRNA expression levels of these factors and nitric oxide release were suppressed by JGTXP in an LPS-induced RAW 264.7 inflammation model. The reporter gene assay showed that JGTXP could reduce the transcriptional activity of NF-κB. Consecutive western blot analysis demonstrated that nuclear NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) expression were inhibited while cytoplasmic NF-κB p65 was upregulated by JGTXP. Using a combination of chemical profiling, network pharmacology analysis, and experimental validation, we preliminarily clarified the active ingredients of JGTXP intervention for OA and demonstrated that JGTXP ameliorates OA, at least partially, by regulating the NF-κB signaling pathway.