Integrated Metabolomics and Proteomics to Decipher Simiao Pill Improving Lipid Homeostasis through PTGES3-mediated Arachidonic Acid Metabolism in AIA Model

BACKGROUND: Simiao Pill (SMP) has been demonstrated to suppress inflammation and modulate immune function, thereby influencing the onset and progression of rheumatoid arthritis (RA). Nonetheless, the specific molecular mechanisms and targets through which SMP mediates metabolic regulation and enhances immune function have yet to be fully elucidated. OBJECTIVE: In this study, we employed an integrated approach combining the analysis of dysregulated metabolites and proteins to identify, screen, and validate the metabolic regulatory targets of SMP in adjuvant-induced arthritis (AIA) rats by using pseudotargeted metabolomics and 4D-DIA quantitative proteomics methodologies. METHODS: An AIA rat model was developed, and SMP was administered to AIA rats. Subsequently, assessments were conducted on paw edema, arthritis scores, histopathological changes and IL-1 β content of inflammatory factors in AIA rats. UHPLC-QTOF-MS/MS was employed to analyze endogenous metabolites in the serum. Metabolic pathway and protein profile were performed on the biomarkers. The protein-lipidphenotype map for the SMP-treated rats was constructed and the primary target closely related to the metabolic regulation of SMP was further screened and verified. RESULTS: Pseudotargeted metabolomics analysis revealed that SMP can mitigate the down-regulation of lipid levels in AIA rats. Pathway enrichment analysis identified arachidonic acid metabolism as the most significantly affected metabolic pathway and SMP was found to substantially ameliorate the dysregulation of this pathway in AIA rats. Subsequent protein profiling led to the identification of five key proteins, with noteworthy obvious corrective effects observed on Ptges3 and Alox15 due to SMP treatment. A comprehensive protein- lipid-phenotypic landscape of SMP-treated rats was analyzed for the specific molecular expressions associated with the arachidonic acid pathway. According to the correlation matrix of dysregulated metabolite/ protein, we found that Ptges3 was ranked as the primary target closely related to the metabolic regulation of SMP, a finding further validated through immunofluorescence staining in rat joint and synovial cells. CONCLUSION: Our study confirmed that SMP exerts an anti-arthritic effect by modulating the arachidonic acid metabolic network via the Ptges3 protein in rat joints and human rheumatoid arthritis synovial fibroblasts. This finding offers a novel mechanistic insight into the pharmacological action of SMP in AIA rats. It informs future research on the therapeutic potential of SMP in RA.