3.1 NA mouse model was established
A mouse model with NA was induced by OVA plus LPS sensitization followed by seven sequential daily OVA challenges (Fig. 1a). sRAGE and inhibitors were administered to NA mice as illustrated in Fig. 1b and 1c. As shown in HE staining, the control group manifested a normal bronchial lumen and alveolar structure, while NA mice exhibited a disordered structure with epithelial cell shedding, thicken bronchial wall, and basement membrane with multi-layer inflammatory cell infiltration around (Fig. 2a). Compared with the NC group and PBS group, airway resistance increased significantly in the NA group after methacholine challenge at doses of 12.5, 25, and 50 mg/mL (P < 0.0001) (Fig. 2b). The inflammatory cells in BALF significantly elevated (including total cells, neutrophils, and lymphocytes) in NA mice when compared with the levels in control mice and the PBS group (Fig. 2c).
Figure 1. Protocols for NA mouse model and inhibitor intervention. (a) Sensitization and challenge for mouse model with NA. (b) Protocols for sRAGE administration. (c) Protocols for HMGB1 inhibitor, RAGE inhibitor, and PI3K inhibitor intervention. i.v.: tail-vein injection, i.p.: intraperitoneally injection.
3.2 sRAGE level decreased in NA mouse
Compared with the control group, the sRAGE level was significantly decreased in NA mouse BALF determined by ELISA (Fig. 2d). Given it was negatively correlated with lung function in COPD [6, 7], we evaluated the relation of sRAGE and airway resistance in NA. It showed that there was an opposite trend between sRAGE levels in BALF and airway resistance, however, with no statistical differences (Fig. 2e). A study revealed that sRAGE may be a predictor of neutrophilic airway inflammation of asthma and COPD . Our data verified that sRAGE was negatively related to BALF neutrophils, especially in the NA group with r=-0.9217 and P = 0.0078 by Spearman correlation analysis (Fig. 2f).
Figure 2. sRAGE level decreased in NA mouse. (a) Representative images of HE stained lung tissue (magnification, × 100). The increased number of inflammatory cells infiltrating around the bronchial wall in the NA group. (b) Airway resistance increased significantly in the NA mouse model analyzed by two-way ANOVA. (c) Inflammatory cells, especially total cells and neutrophils were significantly increased in the NA mouse BALF analyzed by one-way ANOVA. (d) sRAGE levels were assessed by ELISA. Kruskal-Wallis test was performed for analysis. (e) The opposite trend of BALF sRAGE expression and airway resistance in the NA group. (f) Negative correlations between BALF sRAGE and neutrophils using Spearman correlation analysis. n = 6 in each group. Results were depicted as means ± SD. ns: no significant difference. **P < 0.01, ****P < 0.0001, compared with the NC and NA group. NC: normal control group; NA: neutrophilic asthma group; BALF, bronchial alveolar lavage fluid. Mch: methacholine. Mac: macrophages. Neu: neutrophils. Lym: lymphocytes.
3.3 Overexpressed sRAGE decreased neutrophilic airway inflammation in NA mice
AAV-9 was used to overexpress sRAGE and then the transfection efficiency was assessed by the green fluorescent protein (GFP) expression. Compared to the NC and NA groups, the NA + sRAGE group exhibited a distinct expression of GFP (Fig. 3a). Further, the sRAGE administration inhibited the number of neutrophils in BALF cytology smears (Fig. 3b). More importantly, sRAGE sharply reduced the influx of total cells and neutrophils in NA mouse BALF (Fig. 3c). Compared with the airway showing increased inflammatory cell infiltration and thick basement membrane in NA mice, sRAGE overexpression significantly inhibited the airway inflammation and the thicknesses of the basement membrane (Fig. 3d). sRAGE intervention significantly inhibited the inflammation score in NA mice (P < 0.05) (Fig. 3e). Interleukin (IL) -17 and IL-6 were reported as the inflammatory mediator of NA, in this study, sRAGE decreased IL-17 and IL-6 levels in BALF (Fig. 3f).
Figure 3. sRAGE reduced neutrophilic airway inflammation of NA mice. (a) Representative immunofluorescence images of lung tissue. (b) Representative images of cytology smears (magnification, × 100). (c) Classified cell count of BALF in the mouse model. (d) Representative images of HE-stained lung tissue (magnification, × 100). sRAGE inhibited the airway inflammation and the thicknesses of the basement membrane. (e) Inflammation score in HE-stained lung tissue. sRAGE decreased the score of the NA mouse model. (f) sRAGE administration inhibited IL-17 and IL-6 levels in BALF. n = 6 in each group. Results were depicted as means ± SD. *P < 0.05 and ****P < 0.0001 using one-way ANOVA for analysis.
3.4 sRAGE reduced the airway remodeling of NA mice
Masson trichrome staining was widely used to identify collagen deposits which were proved to be the typical phenotype of airway remodeling. Compared to the NC group, NA mice exhibited more blue-stained collagen deposits around the bronchus and blood vessels (Fig. 4a). sRAGE administration highly decreased the collagen deposition, whereas the NA + sRAGE control group showed no reduction. Airway remodeling markers were detected by RT-qPCR (Fig. 4b) and Western Blotting (Fig. 4c). The result revealed that TGF-β1 mRNA was increased in NA mice lung (P < 0.05), while VEGF, MMP-9, and α-SMA expression showed no elevation. Consistently, TGF-β1 protein expression was increased in the NA group with VEGF, MMP-9, and α-SMA protein expression no change. Overexpression of sRAGE significantly decreased the mRNA and protein expression of TGF-β1 (P < 0.05), suggesting sRAGE may inhibit airway remodeling in the NA mouse model.
Figure 4. Effects of sRAGE on airway remodeling of NA mice. (a) Representative images of Masson trichrome stained lung tissue (magnification, × 40). sRAGE administration decreased the peribronchial collagen deposition. (b) mRNA expressions of TGF-β1, VEGF, MMP-9, and α-SMA were examined using RT-qPCR. (c) TGF-β1, VEGF, MMP-9, and α-SMA protein were detected by Western Blotting. n = 6 in each group. Results were depicted as means ± SD. ns: no significant difference. *P < 0.05, **P < 0.01. One-way ANOVA was performed for analysis.
3.5 Effects of sRAGE on EMT and signaling pathway in NA mice
EMT is one of the mechanisms of airway remodeling. The expression of E-cadherin and vimentin were evaluated using RT-qPCR (Fig. 5a) and Western Blotting (Fig. 5b). Compared with the control mice, E-cadherin expression was decreased, while vimentin level was increased in the NA group. sRAGE administration markedly upregulated the level of E-cadherin and reduced the vimentin expression, indicating sRAGE regulated the EMT of NA mouse.
As reported that RAGE and PI3K participated in the EMT process of human airway epithelial cells. sRAGE as a decoy receptor competitively bound the ligands of RAGE (HMGB1, S100, etc) to inhibit the inflammatory effect. In this study, RAGE, PI3K, and HMGB1 expressions were elevated in the lung of NA mice measured by RT-qPCR, while the sRAGE administration inhibited the expressions (Fig. 5c, d). To determine whether sRAGE modulated EMT via RAGE/PI3K, we assessed E-cadherin and vimentin expressions after HMGB1 inhibitor, RAGE inhibitor, and PI3K inhibitor intervention (Fig. 5e, f). It showed that the mRNA and protein levels of E-cadherin were improved in the NA + HMGB1 inhibitor group, NA + RAGE inhibitor group, and NA + PI3K inhibitor group, whereas the administration of the inhibitor could not affect the vimentin expression. The data suggesting that RAGE/PI3K may partly participate in the EMT process of NA.
Figure 5. Effects of sRAGE on EMT and signaling pathway in NA mice. (a) The mRNA expression of E-cadherin and vimentin were examined using RT-qPCR. (b) E-cadherin and vimentin protein expressions were detected by Western Blotting. (c, d) The mRNA expression levels of RAGE, PI3K, and HMGB1. (e, f) mRNA and protein expressions of E-cadherin and vimentin were measured after inhibitor intervention. ns: no significant difference. n = 6 in each group. Results were depicted as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA was performed for analysis.
3.8 The effect of sRAGE on EMT in 16HBE cells
HMGB1 was reported to induce EMT of airway epithelial cells. We assessed the function of sRAGE on HMGB1-induced EMT in 16HBE cells. The results showed that the migration ability of 16HBE cells was enhanced in the HMGB1 group (P < 0.0001), while the sRAGE administration inhibited this effect (P < 0.001) (Fig. 6a, b). The mRNA expression of E-cadherin decreased and vimentin elevated in HMGB1 group (1000 ng/mL) (P < 0.05) (Fig. 6c). Compared to the HMGB1 group, E-cadherin mRNA was significantly elevated (P < 0.0001), while the vimentin level was decreased in the HMGB1 + sRAGE group (P < 0.05) (Fig. 6d).
Figure 6. The effect of sRAGE on EMT in 16HBE cells. (a, b) Representative images (magnification, × 40) of wound-healing assay of 16HBE cells treated with sRAGE and HMGB1 after 24 hours post scratch. Results are expressed as the percentage of the recovered wound area. Migration index = migration distance of test group/migrating distance of the control group. (c) E-cadherin and vimentin mRNA was examined in HMGB1-induced 16HBE cells. (d) E-cadherin and Vimentin expressions were detected after sRAGE administration. n = 6 in each group. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA was performed for analysis.
3.9 The signaling pathway involved in EMT of 16HBE cells
RAGE and PI3K mRNA expressions improved in 16HBE cells treated with HMGB1 (P < 0.01) (Fig. 7a), while the sRAGE administration inhibited the improvement. RAGE inhibitor and PI3K inhibitor were used to intervene in the HMGB1-induced 16HBE cells. It showed that E-cadherin mRNA was increased, while vimentin mRNA was declined in the HMGB1 + RAGE inhibitor group and HMGB1 + PI3K inhibitor group (Fig. 7b). Western Blotting demonstrated that HMGB1 down-regulated the protein expression of E-cadherin (P < 0.001) and up-regulated vimentin expression (P < 0.05) in 16HBE cells (Fig. 7c). However, the E-cadherin level was elevated (P < 0.01) with vimentin protein decreasing (P < 0.01) in HMGB1 + RAGE inhibitor group and HMGB1 + PI3K inhibitor group, suggesting that RAGE/PI3K may be involved in EMT of 16HBE cells.
Figure 7. The signaling pathway involved in EMT of 16HBE cells. (a) The mRNA expression of RAGE and PI3K was examined by RT-qPCR. (b) E-cadherin and Vimentin expressions were detected using RT-qPCR after RAGE inhibitor and PI3K inhibitor intervention. (c) The protein levels of E-cadherin and Vimentin were detected by Western Blotting. n = 6 in each group. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA or Mann-Whitney test was performed for analysis.