scRNA-sequencing Reveals Bu-Shen-Yi-Qi formula (BSYQF) ameliorates airway remodeling via regulating imbalanced M1/M2 macrophage subtypes in chronic asthmatic mice.

Airway remodeling is an end result of excessive tissue repair in chronic asthma, a heterogeneous disease with complex pathogenic features. Traditional Chinese Medicine (TCM) is known for its substantial history for asthma treatment and Bu-Shen-Yi-Qi Formula (BSYQF) has strong clinically and laboratory proven evidences as effective asthma controller. However, its underlying therapeutic mechanism remains unknown. In present study, we aim to investigate how BSYQF exerts its therapeutic function in chronic asthmatic mice using single-cell RNA-sequencing. We established a chronic asthma mouse model to evaluate the therapeutic effects of BSYQF, assessing airway inflammation, hyperresponsiveness, and remodeling through functional, histological, and molecular analyses. Single-cell RNA sequencing (scRNA-seq) and bulk transcriptomics were employed to investigate immune cell dynamics, significant modulation of macrophage polarization and key inflammatory pathways. Immunohistochemistry was applied to assess the transforming growth factor-β1 (TGF-β1) expression in lung tissues. Immunofluorescence analyses were conducted to assess airway remodeling, particularly myofibroblast expansion and the expression of key fibrosis-related markers, including Col1a1+α-SMA+ co-expression and osteopontin (OPN) protein. Our result showed that BSYQF treatment, particularly 2.6 g/kg group, significantly improve airway hyperresponsiveness (AHR), mirrored by significantly reduced lung resistance and increased dynamic lung compliance in asthmatic mice (P < 0.05). Histopathological assessment reveals that BSYQF mitigated the airway inflammation and airway remodeling, reflected by markedly reduced goblet cell metaplasia and collagen deposition on subepithelial layer of the airway(P < 0.05). By single cell RNA sequencing technique on eight samples, we profiled over 60 000 mouse lung tissue cells, 21 distinct cell clusters and discovered a unique pattern of BSYQF-targeted M1 and M2 macrophage subtypes in chronic asthmatic mice. Significantly disturbed M1/M2 macrophage ratio was restored by BSYQF treatment and highly resemble to healthy mice status, compared to asthmatic condition (P < 0.05). Our qRT-PCR analysis validated the total of 23 asthmatic genes significantly regulated by BSYQF treatment in M1 and M2 macrophages (P < 0.05), which were highly associated with M2 Mediated airway remodeling. Immunofluorescence analyses showed BSYQF treatment markedly reduced the OPN protein expression on the airway and significantly suppressed the Col1a1+α-SMA+ co-expressed myofibroblast expansion on subepithelial airway layer, compared to asthmatic group (P < 0.05). BSYQF significantly improved airway hyperresponsiveness, reduced airway inflammation and remodeling, and restored the disturbed M1/M2 macrophage balance in asthmatic mice. Mechanistically, BSYQF treatment regulated 23 asthma-related genes, suppressed OPN expression, and inhibited Col1a1+α-SMA+ myofibroblast expansion, highlighting its potential as a therapeutic intervention for chronic asthma.