Xinfeng capsule attenuates ankylosing spondylitis by downregulating YTHDC1-mediated m6A modification of LINC01579 and suppressing IL-17/NF-κB signaling

Background Ankylosing spondylitis (AS) is a chronic inflammatory disorder predominantly affecting the sacroiliac and spinal joints. Emerging evidence indicates that N 6 -methyladenosine (m 6 A) RNA modification plays a critical role in inflammatory regulation. Xinfeng Capsule (XFC), a Traditional Chinese Medicine formula (Xin’an medicine), has demonstrated clinical efficacy in alleviating AS-associated inflammation. However, whether XFC modulates AS pathology through m 6 A-dependent epigenetic mechanisms remains unclear. Objective This study aimed to investigate whether XFC mitigates AS inflammation by regulating the IL-17/NF-κB pathway via m 6 A modification. Methods Core therapeutic targets were identified by integrating network pharmacology with RNA-sequencing data. The direct interaction between YTHDC1 and LINC01579 was validated using RNA pull-down and dual-luciferase reporter assays. To identify the functional m6A sites, site-directed mutagenesis of two putative m6A motifs (MUT1 and MUT2) in LINC01579 was performed, and the effects of YTHDC1 knockdown were assessed. An in vitro co-culture model comprising AS patient-derived fibroblast-like synoviocytes (FLS) and peripheral blood mononuclear cells (PBMC) was established, alongside an in vivo proteoglycan-induced arthritis (PGIA) mouse model. To verify the underlying mechanism, gain- and loss-of-function experiments were conducted using overexpression plasmids and small interfering RNAs (siRNAs) targeting LINC01579 and YTHDC1. m 6 A levels were quantified by MeRIP-qPCR. Inflammation and pathway activation were assessed via immunofluorescence, Western blot, and enzyme-linked immunosorbent assay (ELISA). RNA stability was evaluated using actinomycin D assays, while cell migration was measured by scratch assays. Bone destruction was analyzed using micro-CT and histological staining. Additionally, an IL-17 pathway inhibitor (AIN457) and agonist (SR0987) were employed to validate pathway involvement. Results Bioinformatics and network pharmacology analysis identified LINC01579 as a core gene in AS inflammation and YTHDC1 as an upstream regulator of LINC01579 m 6 A modification. RNA pull-down assays confirmed the direct binding of YTHDC1 to LINC01579.Functional studies revealed that mutation of the MUT1 site abolished the regulatory effects of YTHDC1 knockdown on inflammatory cytokines (IL-17, IL-6, TNF-α) and LINC01579 expression. XFC was predicted to inhibit inflammation via the IL-17/NF-κB pathway. In vitro , LINC01579 knockdown significantly enhanced the release of inflammatory mediators and activated IL-17/NF-κB signaling. Conversely, YTHDC1 overexpression increased LINC01579 m 6 A levels, leading to its downregulation. XFC treatment effectively reduced YTHDC1 expression and LINC01579 m 6 A modification, thereby restoring LINC01579 levels and suppressing IL-17/NF-κB activation. In vivo , XFC ameliorated joint inflammation, bone erosion, and joint space narrowing in PGIA mice. Conclusion XFC alleviates AS progression by inhibiting YTHDC1-mediated m6A modification of LINC01579, which prevents its degradation and subsequently dampens IL-17/NF-κB pathway activation. These findings highlight a potential epigenetic mechanism underlying the therapeutic effects of XFC in AS.