Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by loss of immune tolerance and persistent inflammation. Imbalance between Th17 and regulatory T (Treg) cells represents a central pathogenic mechanism in SLE. Paeoniflorin (PF), a monoterpene glycoside extracted from Paeonia lactiflora, exhibits immunomodulatory properties, yet its mechanisms in SLE remain unclear. This study investigated whether PF ameliorates SLE by modulating the gut microbiota-lipid metabolite axis and restoring Th17/Treg immune balance.

Network pharmacology was employed to predict PF-related targets and pathways in SLE. For in vivo experiments, 7-8-week-old female MRL/lpr mice were randomly divided into PF-treated group (50 mg/kg by oral gavage) and PBS control group, with C57BL/6 mice serving as normal controls, for 8 weeks of intervention. Disease severity was assessed using semi-quantitative scoring systems for skin lesions, proteinuria, and lymphadenopathy. Serum autoantibodies (anti-dsDNA, ANA, total IgG) and renal function were measured. Th17 and Treg cell populations in spleen, submandibular lymph nodes, intestinal intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs) were analyzed by flow cytometry. Gut microbiota composition was profiled by 16S rRNA gene sequencing. Untargeted LC-MS/MS-based metabolomics was performed on intestinal contents to identify differential lipid metabolites. Correlation analyses were conducted among microbial alterations, metabolic changes, and immune parameters.

Network pharmacology analysis revealed significant enrichment of PF targets in IL-17 signaling pathway and Th17 cell differentiation. PF treatment markedly alleviated lupus-like manifestations in MRL/lpr mice, reduced skin lesion scores, proteinuria, and lymphadenopathy, decreased serum levels of anti-dsDNA antibodies, ANA, total IgG, and IL-17, and improved renal function and histopathological injury. Flow cytometric analysis demonstrated that PF significantly reduced the proportion of CD4⁺IL-17⁺ Th17 cells while increasing CD4⁺Foxp3⁺ Treg cells in spleen, lymph nodes, IELs, and LPLs, thereby restoring Th17/Treg homeostasis. PF also enhanced intestinal motility, reduced intestinal permeability, and upregulated tight junction proteins ZO-1 and occludin. 16S rRNA sequencing showed that PF reshaped gut microbial composition, decreasing the abundance of inflammation-associated taxa including Ligilactobacillus and Mucispirillum, while enriching beneficial bacteria such as Akkermansia and Dubosiella. Metabolomic analysis revealed that PF significantly downregulated glycerophospholipid metabolism, particularly reducing lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC), which have been reported to promote Th17 differentiation. Pearson correlation analysis linked these microbial and metabolic alterations with improvements in disease activity indices and Th17/Treg rebalancing.

Paeoniflorin alleviates SLE by regulating a gut microbiota-lysophospholipid-immune axis, suppressing pro-Th17 lipid metabolism and restoring Th17/Treg homeostasis. These findings provide novel mechanistic insights and a potential therapeutic strategy targeting gut microbiota metabolism for SLE treatment.