Roflumilast improves airway inflammationin mice with bronchial asthma through mTOR/ULK1/Atg13 pathway

To explore the regulation of roflumilast on the mammalian target of rapamycin (mTOR)/unc-51 like autophagy activating kinase 1 (ULK1)/autophagy-related gene 13 (Atg13) pathway and its influence on airway inflammation in mice with bronchial asthma (BA).

Sixty mice were randomly divided into normal control group, model group, roflumilast group (1.0 mg/kg), autophagy inhibitor (3-methyladenine, 3MA) group (10.0 mg/kg), autophagy activator[mTOR inhibitor (rapamycin), 1.0 mg/kg]group, roflumilast+rapamycin group (1.0 mg/kg+1.0 mg/kg), with ten mice in each group. The ovalbumin sensitization method was used to induce the establishment of a mouse BA model, and the mice in each group were administered for 4 weeks. After the last administration, the animal lung function meter was used to detect the lung function of the mouse; the bronchoalveolar lavage fluid (BALF) was used to detect the number of inflammatory cells; enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of inflammatory factor interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) in BALF; hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining were used to detect changes in lung tissue structure and airway goblet cell metaplasia; transmission electron microscope was used to observe the formation of autophagic vesicles; TUNEL method was used to detect airway epithelial cell apoptosis; western blot was used to detect the expression level of Atg13, phosphorylated Atg13 (p-Atg13), mTOR, phosphorylated mTOR (p-mTOR), ULK1 specific site Ser757 protein (ULK1 Ser757) and its phosphorylation protein (p-ULKl Ser757), autophagy marker protein microtubule-related protein light chain 3B (LC3B), autophagy-related protein (Beclin1), B-cell lymphoma-2 (Bcl-2), interleukin-33 (IL-33), mucus hypersecretion marker protein mucin (MUC5AC).

Compared with the normal control group, mice in the model group had decreased lung function, aggregated lung tissue inflammatory cell and goblet cell metaplasia, the expression of phosphorylated protein in mTOR/ULK1/Atg13 pathway statistically decreased, and the expression levels of autophagy-related protein, inflammation and apoptosis were statistically increased (P < 0.05). The autophagy and apoptosis of airway epithelial cells were decreased in the roflumilast and 3MA groups, and the phosphorylation protein of mTOR/ULK1/Atg13 pathway was increased (P < 0.05). Rapamycin could reverse the above-mentioned alleviating effects of roflumilast (P < 0.05).

Roflumilast can promote the phosphorylation and activation of mTOR/ULK1/Atg13 pathway, block autophagy, inhibit airway inflammation and apoptosis of epithelial cells, thereby relieving BA pathological symptoms.