Fig 2: CST1 enhances SERPINB2 expression via AKT signaling pathway in bronchial epithelial cells. (A, B) Overexpression CST1 increased p-AKT and SERPINB2 expression in BEAS-2B cells (each group n = 3). (C) SC-79 has a positive effect on SERPINB2 expression (n = 2). (D) The effect of E64d on SERPINB2 mRNA expression detected by qRT-PCR (each group n = 3). (E, F) E64d enhanced p-AKT and SERPINB2 expression (each group n = 3). (G) Knockdown CST1 using anti-CST1 siRNA significantly decreased the expression of p-AKT and SERPINB2 (n = 2). (H) MK2206 has a negative effect on SERPINB2 expression (n = 2). Data are mean ± SD (Mann-Whitney test and one-way ANOVA were used to compare between or across groups).CST1, cystatin SN; SERPINB2, serpin peptidase inhibitor, clade B, member 2; p-AKT, phosphorylated AKT; BEAS-2B, immortalized human lung epithelial; SC-79, the agonist of AKT; E64d, a chemical compound of CST1; qRT-PCR, quantitative real-time polymerase chain reaction; MK2206, the inhibitor of AKT; SD, standard deviation; ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.*P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: The expression of CST1, AKT signaling pathway, and SERPINB2 in bronchial epithelial cells. (A) The mRNA expression of CST1 in BEAS-2B cells treated with cytokines IL-4, IL-13, and IL-17A, respectively (each group n = 6). (B, C) The protein expression of CST1, p-AKT, AKT, and SERPINB2 in BEAS-2B cells stimulated with cytokine IL-4 (each group n = 3). (D) The mRNA expression of CST1 and SERPINB2 in BEAS-2B cells treated with HDM (concentration 0, 25, 50, 100 μg/mL, respectively (each group n = 3). (E) The protein expression of CST1 in BEAS-2B cells treated with HDM (concentration 0, 25, 50, and 100 μg/mL, respectively). (F, G) The protein expression of CST1, p-AKT, AKT, and SERPINB2 in BEAS-2B cells stimulated with HDM (50 μg/mL, each group n = 3) (one-way ANOVA followed by Tukey’s multiple comparison test was used).CST1, cystatin SN; SERPINB2, serpin peptidase inhibitor, clade B, member 2; BEAS-2B, immortalized human lung epithelial; IL, interleukin; p-AKT, phosphorylated AKT; HDM, house dust mite; ANOVA, analysis of variance.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: The mRNA expression (A-C) and protein levels (D-F) of CST1 were correlated with airway eosinophilic indicator (FeNO), blood EOS%, and serum total IgE level (Spearman’s rank-order correlation was used).CST1, cystatin SN; FeNO, fractional exhaled nitric oxide; EOS, eosinophils; IgE, immunoglobulin E; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig 5: CST1 enhances airway eosinophilic inflammation infiltration in OVA-induced asthma models. (A) Flow charts of the OVA-induced asthma mouse model. (B) Representative images of H&E (original magnification: ×400) and PAS (original magnification: ×400) staining of mouse lung sections. Scale bar, 50 μm. (C) The peri-bronchial inflammation scores were calculated based on H&E staining. (D) Cell counting for eosinophils, neutrophils, lymphocytes, and macrophages in BALF (n = 5–6 mice per group). Data are mean ± SD. (E) The transcript levels of inflammatory cytokines in mouse lung tissues were detected by qRT-PCR (one-way ANOVA followed by Tukey’s multiple comparison test was used).CST1, cystatin SN; OVA, ovalbumin; H&E, hematoxylin and eosin; PAS, periodic acid-Schiff staining; BALF, bronchial alveolar lavage fluid; SD, standard deviation; qRT-PCR, quantitative real-time polymerase chain reaction; ANOVA, analysis of variance.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.