Shema Zhichuan Liquid alleviates airway inflammation in Allergic Asthma by regulating autophagy pathways through DNA...

Shema Zhichuan Liquid (SMZCL) is a traditional Chinese medicine formulation used clinically as an adjunct therapy for asthma. Its therapeutic mechanism in allergic asthma (AA), however, remains poorly understood.

This study aimed to evaluate the efficacy of SMZCL in treating AA and to elucidate its molecular mechanisms, with particular focus on the regulation of autophagy via DNMT1-mediated promoter demethylation of autophagy-related genes.

The AA mouse model was established by ovalbumin (OVA)/Al(OH)₃ sensitization and challenge. Lung histopathological, inflammatory cytokine levels, Th2 cell proportion, methylation status of autophagy-related genes, and the expression of autophagy-associated markers were assessed. Additionally, an in vitro inflammatory model was generated using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages to further investigate the mechanism by which SMZCL modulates autophagy through modulating DNA methylation.

In vivo, SMZCL attenuated airway inflammation in AA mice and reduced the proportion of Th2 cells in BALF. It significantly decreased serum levels of IgE, Th2 cytokines IL-4 and IL-13, and pro-inflammatory cytokines TNF-α and IL-6, while increasing the Th1 cytokine IFN-γ. Mechanistically, SMZCL inhibited DNMT1 mRNA and protein expression, decreased the relative methylation levels of the ATG5 and Beclin-1 promoters, upregulated mRNA and protein levels of ATG5, Beclin-1, and LC3, and downregulated p62 mRNA expression. In vitro, SMZCL treatment reduced pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), suppressed DNMT1 expression, induced demethylation of ATG5 and Beclin-1 promoters, and upregulated their expression. SMZCL restored autophagic flux, as evidenced by elevated absolute levels of LC3-II, enhanced p62 degradation, an upward trend in the LC3-II/I ratio, a concurrent increase in both autophagosome and autolysosome puncta in mCherry-GFP-LC3 dual-fluorescence analysis, and further LC3-II accumulation in the presence of chloroquine. Moreover, SMZCL inhibited the expression of mTOR and phosphorylated mTOR (p-mTOR).

SMZCL inhibited DNMT1, leading to demethylation of the ATG5 and Beclin-1 promoters, and concurrently suppressed the mTOR signaling pathway. Through these mechanisms, SMZCL restored autophagic flux, exerted anti-inflammatory effects, and thereby provided protection against AA.