D2.109 - Single-Cell Sequencing Reveals the Immunological Mechanism of Astragaloside IV in the Treatment of Allergic Rhinitis Complicated with Asthma

Astragaloside IV (AST), a natural active component of Astragalus membranaceus (Fisch.) Bunge, has potent anti-allergic and immunomodulatory properties. However, the molecular mechanism by which AST treats allergic rhinitis complicated with asthma (ARCA) remains unclear, hindering its clinical translation. This study aimed to elucidate the immunological mechanism of AST in ARCA.

The ARCA model was established with ovalbumin plus aluminum hydroxide. After AST treatment, airway resistance and allergic symptom scores were measured. Pathological staining assessed eosinophil infiltration, goblet cell hyperplasia, and collagen proliferation in nasal and pulmonary tissues. ELISA, Western blot (WB), and immunohistochemistry (IHC) detected allergy-related inflammatory factors. Single-cell sequencing combined with bulk RNA sequencing analyzed cell distribution and signaling pathway changes in nasal and pulmonary tissues. Flow cytometry and immunofluorescence (IF) determined expression and co-expression of professional antigen-presenting cell markers (dendritic cells: CD11c; macrophages: F4/80; B cells: CD19) and MHC II. WB and IHC evaluated JAK2/STAT1 pathway activation. Molecular docking and Drug Affinity Responsive Target Stability (DARTS) assays tested AST’s binding to JAK2 and STAT1.

AST reduced airway resistance and airway allergic symptom scores in ARCA model mice. Meanwhile, AST diminished eosinophil infiltration, goblet cell hyperplasia, and collagen fiber proliferation in nasal and pulmonary tissues. Additionally, AST downregulated the expression levels of OVA-sIgE, IL-4, IL-5, IL-13, and ECP. Single-cell sequencing revealed a marked increase in the number of professional antigen-presenting cells (dendritic cells, macrophages, B cells) in nasal and pulmonary tissues of ARCA mice, whereas AST treatment decreased the abundance of these cells. Bulk RNA sequencing showed that the JAK2/STAT1 signaling pathway was activated in ARCA mice, which was notably suppressed by AST administration. Flow cytometry and IF assays confirmed that the expression levels of CD11c, F4/80, CD19, and MHC II were elevated in nasal and pulmonary tissues of ARCA mice, with only CD19 and MHC II showing significant co-expression; AST treatment  downregulated the expression of CD11c, F4/80, CD19, and MHC II, and reduced the number of CD19/MHC II double-positive cells. WB and IHC results indicated that the expression levels of p-JAK2, STAT1, and p-STAT1 proteins were increased in ARCA mice, which were markedly reduced by AST treatment. Molecular docking analysis showed that the binding energies of AST to JAK2 and STAT1 proteins were -10.3 kcal/mol and -7.5 kcal/mol, respectively. DARTS assays further verified that AST could directly bind to JAK2 and STAT1 proteins.

AST effectively alleviates airway allergic symptoms, inhibits nasal and pulmonary tissue remodeling, and suppresses Th2-type inflammatory responses in ARCA. The underlying mechanism is closely associated with the regulation of the JAK2/STAT1/MHC II signaling pathway and the inhibition of B cell-mediated antigen presentation during the secondary immune response. This study provides novel modern pharmacological evidence for the clinical application of AST in the treatment of ARCA.