Integrated network pharmacology and metabolomics reveal the mechanisms of Compound Nanxing Zhitong Plaster in anti-rheumatoid arthritis.

OBJECTIVE: This study aimed to evaluate the therapeutic efficacy of Compound Nanxing Zhitong Plaster (CNZP) against rheumatoid arthritis (RA) and to investigate its underlying mechanism through an integrated approach combining network pharmacology and metabolomics. METHODS: A collagen-induced arthritis (CIA) rat model was established to assess the anti-RA effects of CNZP. A comprehensive strategy, incorporating network pharmacology (based on previously reported chemical constituents), molecular docking, serum biochemical assays, histopathological examination, immunohistochemistry, and Western blotting, was employed to elucidate its therapeutic actions, associated pathways, and molecular targets. RESULTS: Network pharmacology identified 53 common targets between CNZP and RA, with IL1β, TLR4, and STAT3 as the top three hub genes. GO and KEGG enrichment analyses revealed that the therapeutic effects of CNZP are primarily mediated through inflammatory response, endopeptidase activity, and signaling pathways such as TNF, IL-17, and Toll-like receptor signaling. Quercetin, imperatorin, and isoimperatorin were identified as the core components. Molecular docking confirmed favorable binding affinities of these three components to IL1β, TLR4, and STAT3. Metabolomics analysis revealed eight potential biomarkers, among which six were significantly modulated by CNZP. These metabolites were primarily involved in arginine and proline metabolism, histidine metabolism, and glycerophospholipid metabolism pathways. Integrated analysis of metabolomics and network pharmacology highlighted the arginine and proline metabolism and arginine biosynthesis pathways as the common mechanisms. L-Arginine was pinpointed as the key differential metabolite, with nitric oxide synthase 1 (NOS1) as its associated regulatory enzyme. CNZP treatment is associated with reduced activation of the JAK1/STAT1 pathway and downregulation of NOS1, suggesting a potential involvement of this axis in the therapeutic effect of CNZP against RA. CONCLUSION: CNZP exhibits significant therapeutic effects on RA, potentially by downregulating NOS1 expression and subsequently correcting the metabolic imbalance of L-arginine. Key Points • CNZP demonstrated significant therapeutic efficacy in a CIA rat model; serum metabolomics revealed the associated differential metabolites and key metabolic pathways involved in its action. • Network pharmacology and molecular docking revealed that CNZP exerts multi-target effects on RA by modulating TNF, IL-17, and Toll-like receptor signaling pathways via its core components quercetin, imperatorin, and isoimperatorin. • Integrated analysis suggests that CNZP treatment is associated with downregulation of NOS1 and correction of l-arginine metabolic imbalance, implicating the JAK1/STAT1 pathway in its anti-RA effect. • This preclinical study provides mechanistic evidence for the anti-RA effect of CNZP.