Vitamin D enhances the effect of Soufeng sanjie formula in alleviating joint inflammation in CIA mice through VDR-NOTCH3/DLL4 signaling in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA), defined as Bi Zheng syndrome in traditional Chinese medicine (TCM), is a chronic inflammatory and autoimmune disease that can cause substantial articular degradation and impairment. In RA, the polarization of macrophages to a proinflammatoy phenotype contributes to chronic inflammation and joint injury. Soufeng sanjie formula (SF), a traditional Chinese formula used to treat RA, consists of Scolopendra subspinipes mutilans L. Koch, Buthus martensii Karsch, Astragalus membranaceus (Fisch), and Glycine max (L.) Merr seed coats and plays a role in the regulation of macrophage polarization. Vitamin D (VD), a specific activator of the vitamin D receptor (VDR), promotes the differentiation of M2 macrophages and significantly enhances the therapeutic effects of TCM on arthritis. However, whether SF can be combined with VD to regulate macrophage polarization and alleviate RA remains unclear.

AIM OF THE STUDY: In this study, we examined the regulatory mechanisms and therapeutic effects of the combination of SF and VD on macrophages in RA.

MATERIALS AND METHODS: We used network pharmacology to analyze the targets and pathways of SF in RA. Clinical data were analyzed to confirm the expression of key targets. The RA immune landscape for cell-gene correlations was built using bioinformatics. A collagen-induced arthritis (CIA) model was established to evaluate the combined therapeutic effects of SF and VD on joint injury and inflammation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting (WB), and immunofluorescence were used to assess the expression of key molecules in macrophages. Macrophage polarization was analyzed using flow cytometry.

RESULTS: SF acts on RA through multiple pathways, including hub genes, such as VDR, ABL1, DNMT1, and CXCR4. Immune infiltration analysis of RA showed that a reduction in the number of M2 macrophages significantly affected RA. Clinical data analysis and prognostic assessments corroborated the pivotal roles played by hub genes, such as VDR, ABL1, DNMT1, and CXCR4. Crucially, this study demonstrated a strong association between the key hub gene VDR and M2 macrophages. In vivo and in vitro models of RA showed that VD significantly improved the efficacy of SF against arthritis in CIA mice and stimulated macrophage polarization toward the M2 phenotype via modulation of the NOTCH3/DLL4 pathway. Furthermore, siRNA-mediated interference with the expression of VDR confirmed that the downregulation of VDR significantly activated the NOTCH3/DLL4 signaling pathway and blocked the regulation of SF combined with VD on the polarization of macrophages toward the M2 phenotype.

CONCLUSIONS: Our results demonstrate that the combination of SF and VD markedly improves the therapeutic effect of SF on RA via M2 macrophage differentiation through the VDR-NOTCH3/DLL4 signaling pathway. The VD-enhancing effect of SF in improving RA symptoms offers a new strategy and theoretical foundation for the clinical treatment of the condition.