Traditional Chinese medicine (TCM) formulas have attracted increasing attention worldwide in the past few years for treating complex disease including rheumatoid arthritis. However, their mechanisms are complex and remain unclear. Guan-Jie-Kang (GJK), a prescription modified from "Wu Tou Decoction," was found to significantly relieve arthritis symptoms in rats with adjuvant-induced arthritis after 30-day treatment, especially in the 24 g/kg/day group. By analyzing 1749 targets related to 358 compounds in the five herbs of GJK, we identified the possible anti-arthritis pathways of GJK, including the calcium signaling and metabolic pathways. Bone damage levels were assessed by micro-computed tomography, and greater bone protective effect was observed with GJK treatment than with methotrexate. Receptor activator of nuclear factor κB ligand (RANKL)-RANK signaling, which is related to calcium signaling, was significantly regulated by GJK. Moreover, a target metabolomics assay of serum was conducted; 17 metabolic biomarkers showed significant correlations with treatment. An integrated pathway analysis revealed that pyruvate metabolism, purine metabolism, and glycolysis metabolism were significantly associated with the effects of GJK in arthritis treatment. Thus, this study establishes a new omics analytical method integrated with bioinformatics analysis for elucidating the multi-pathway mechanisms of TCM.

Objective To investigate the underlying drug enhancement mechanisms of the Chuanwu (Aconiti Radix) and Huangqi (Astragali Radix) combination and toxicity reduction of Chuanwu combined with Gancao (Glycyrrhizae Radix et Rhizoma) in Wutou Decoction (乌头汤, WTD), and to elucidate the compatibility principle. Methods The active compounds and potential effective targets of the selected combinations were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicines Integrated Database (TCMID). The toxicity of Chuanwu (Aconiti Radix) was investigated by selecting all five toxic compounds from the literature and the TCMSP database, and obtaining their targets through SwissTargetPrediction. Targets related to rheumatoid arthritis (RA) were searched using DisGeNET, GenCards, and Online Mendelian Inheritance in Man (OMIM). Mutual targets between the drug pairs and RA were selected as potential RA therapy targets. The medicinally active compound-target network was constructed using Cytoscape 3.9.0. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Results We obtained 191 active compound targets for Gancao (Glycyrrhizae Radix et Rhizoma), 171 for Huangqi (Astragali Radix), and 103 for Chuanwu (Radix Aconiti) (hypoaconitine’s target was obtained through literature and SwissTargetPrediction). A total of 5872 genes were obtained for RA. A drug-active compound-target network involving 13 effect-enhancing and nine toxicity reduction targets was constructed. PGR was the main effect enhancement target, and KCNH2 was the main toxicity reduction target. The effect-enhancing targets were related to 23 GO terms (such as positive regulation of transcription from RNA polymerase II promoter, steroid hormone-mediated signaling pathway, plasma membrane, and protein binding) (P < 0.01), and 13 KEGG pathways related to synergism [such as estrogen signaling pathway, cholinergic synapse, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway]. The toxicity reduction targets were related to 28 GO terms (mainly involes G-protein coupled receptor signaling pathway, plasma membrane, and drug binding) (P < 0.01), and five KEGG pathways related to toxicity reduction (cholinergic synapse, calcium signaling pathway, regulation of actin cytoskeleton, neuroactive ligand-receptor interaction, and serotonergic synapse). Conclusion The combination of Chuanwu (Aconiti Radix) and Huangqi (Astragali Radix) plays an important effect-enhancing role in WTD and involves the estrogen and PI3K/Akt signaling pathways, with PGR as the core. The Chuanwu (Aconiti Radix) and Gancao (Glycyrrhizae Radix et Rhizoma) combination decreases toxicity in WTD and is associated with the cholinergic synapse and calcium signaling pathways, with KCNH2 as the core.