Betulinic acid alleviates ankylosing spondylitis and remodels Th17/Treg-associated immune landscapes revealed by single-cell transcriptomics.

To systematically evaluate the therapeutic effects of betulinic acid (BA) in an ankylosing spondylitis (AS) mice model and characterize the molecular changes associated with immune remodeling and Th17/Treg-related transcriptional alterations. Using β-1,3-glucan-induced SKG mice and CD3/CD28-activated Jurkat cells. Mice were divided into four groups: control (Con), AS model, methotrexate (MTX), and BA treatment. Peripheral blood mononuclear cells (PBMCs) were profiled using the 10 × Genomics single-cell transcriptomic platform, followed by T-cell subset identification, pseudotime trajectory analysis, and cell communication network reconstruction. Single-cell changes in Smad2-associated gene regulatory patterns and immune states were evaluated using scTenifoldKnk-based virtual knockout analysis. Histopathology and immunohistochemistry were used to evaluate RANK/RANKL expression in spinal tissues, while flow cytometry, immunofluorescence, ELISA, qPCR, and Western blot were performed to assess immune cell proportions, mitochondrial reactive oxygen species (ROS), and inflammatory signaling pathways. BA markedly alleviated spinal deformity, enthesitis, and bone destruction in AS mice and suppressed RANK/RANKL-mediated osteoclast activation. Single-cell transcriptomic analysis revealed pronounced Th17/Treg imbalance and activation of IL6-STAT3, MAPK/Ras, and TGF-β/SMAD signaling pathways in AS, whereas BA treatment reshaped T-cell developmental trajectories by reducing Rorc-associated differentiation while enhancing Foxp3-related dynamics. BA also disrupted the Th17-macrophage inflammatory amplification loop, reduced Th1 and Th17 populations, increased Th2 and Treg proportions (P < 0.05), and decreased mitochondrial ROS accumulation and aberrant CCR9 and TGFB1 expression. Virtual knockout analysis revealed that Smad2-associated regulatory networks were substantially influenced by the immune microenvironment. BA treatment was associated to decreased expression of inflammatory mediators and changed expression of a number of signaling molecules found through single-cell transcriptome investigations, such as ERK1/2, KRAS, MAPK14, and SMAD2, according to experimental validation (P < 0.05). BA exerts anti-inflammatory and immunomodulatory effects in AS and is associated with modulation of mitochondrial oxidative stress and multiple immune-related signaling signatures, thereby reshaping T cell developmental trajectories and contributing to partial restoration of immune homeostasis.