Mechanistic Insights into Yunpi-Xiefei-Huatan decoction effects on autophagy and MUC5AC secretion in rat tracheal epithelial cells via the TGF-β3/Smad2/Smad3 pathway

Ethnopharmacological relevance: Bronchial asthma (BA) is a common chronic respiratory disease in children. Yunpi-Xiefei-Huatan decoction (YXHD) is known to relieve cough and reduce phlegm, and is widely applied in the clinical treatment of childhood asthma. However, its mechanism of action has not been fully elucidated.

Aim: This study aimed to elucidate the mechanism by which serum containing YXHD influences the secretion of MUC5AC by Rat tracheal epithelial cells (RTE), which is known to be potentially mediated through TGF-β3/SMAD2/SMAD3 pathway-related Autophagy.

Materials and methods: UPLC-QTOF-MS was used to analyze blood components of YXHD. RTE cells were treated with TGF-β3 to induce Autophagy and MUC5AC expression. Cell proliferation was measured by EdU assays. Levels of NADPH Oxidase 4 (NOX4) and Reactive Oxygen Species (ROS) in cell supernatant were quantified by ELISA, with MUC5AC localization determined by immunofluorescence (IF). Transmission electron microscopy (TEM) was used to count autophagosomes and autolysosomes. Autophagic flux was analyzed using mCherry-GFP-LC3B fluorescence labeling. Western blotting (WB) was performed to analyze autophagy-related proteins and key proteins in the TGF-β3/SMAD2/SMAD3 signaling pathway. RT-qPCR was conducted to measure mRNA expressions of TGF-β3, SMAD2, SMAD3, and MUC5AC. The therapeutic effects of different YXHD concentrations on Autophagy and MUC5AC expression were evaluated, with rapamycin and 3-methyladenine as controls. RTE cells were transfected with Ad-rSmad2-shRNA and Ad-rBeclin-shRNA to investigate the interaction between Autophagy and the TGF-β3/SMAD2/SMAD3 signaling pathway, with the aim of elucidating the underlying mechanism.

Results: Using UPLC-QTOF-MS, we identified 18 characteristic chromatographic peaks and analyzed the secondary mass spectra of five bioactive components present in the blood-entering fraction. TGF-β3 (10 ng/mL) significantly induced Autophagy in RTE cells (as shown by upregulated protein levels of ATG5 and Beclin1), increased the LC3-II/LC3-I ratio, suppressed p62 protein levels, and increased the number of autophagosomes and autolysosomes. Furthermore, TGF-β3 (10 ng/mL) markedly enhanced intracellular NOX4 and ROS levels, promoted cellular proliferation, and upregulated MUC5AC mRNA transcription, protein expression, and mean fluorescence intensity. YXHD-containing serum potently inhibited Autophagy in RTE cells, as indicated by downregulated Beclin1 and ATG5 protein levels, a reduced LC3-II/LC3-I ratio, increased p62 levels, decreased autophagosome and autolysosome counts, and suppressed Beclin1 and LC3B mRNA levels. YXHD-containing serum attenuated NOX4 and ROS production, inhibited cell proliferation, and reduced MUC5AC mRNA expression, protein levels, and fluorescence intensity. Transfection with Ad-rBeclin1-shRNA inhibited autophagic flux, reduced Beclin1 protein levels and the LC3-II/LC3-I ratio, increased p62 levels, and diminished MUC5AC fluorescence. Intervention with 5 % YXHD and shBeclin synergistically exacerbated these autophagy-suppressive effects and further reduced MUC5AC fluorescence. Similarly, Ad-rSmad2-shRNA transfection decreased TGF-β3 protein levels, reduced the p-Smad2/SMAD2 and p-Smad3/SMAD3 ratios, downregulated TGF-β3, SMAD2, and SMAD3 mRNA expression, and attenuated Autophagy markers (Beclin1, LC3-II/LC3-I) while increasing p62 levels and reducing MUC5AC fluorescence. Additionally, treatment with 5 % YXHD and shSmad2 produced additive inhibitory effects on these parameters.

Conclusion: YXHD-containing serum inhibited Autophagy activation in RTE cells by suppressing TGF-β3/SMAD2/SMAD3 pathway activity, which downregulated MUC5AC expression.

Autophagy; MUC5AC; RTE; TGF-β3/Smad2/Smad3; YXHD-Containing serum.