Glucocorticoid Alleviates Mechanical Stress-Induced Airway Inflammation and Remodeling in COPD via Transient Receptor Potential Canonical 1 Channel

Abstract

Background: Increased airway resistance and hyperinflation in chronic obstructive pulmonary disease (COPD) are associated with increased mechanical stress that modulate many essential pathophysiological functions including airway remodeling and inflammation. Our present study aimed to investigate the role of transient receptor potential canonical 1 (TRPC1), a mechanosensitive cation channel in airway remodeling and inflammation in COPD and the effect of glucocorticoid on this process.

Methods: In patients, we investigated the effect of pathological high mechanical stress on the expression of airway remodeling-related cytokines transforming growth factor β1 (TGF-β1), matrix metalloproteinase-9 (MMP9) and the count of inflammatory cells in endotracheal aspirates (ETAs) by means of different levels of peak inspiratory pressure (PIP) under mechanical ventilation, and analyzed their correlation with TRPC1. Based on whether patients regularly used inhaled corticosteroid (ICS), COPD patients were further divided into ICS group (n = 12) and non-ICS group (n=15). The ICS effect on the expression of TRPC1 was detected by Western blot. In vitro, we imitated the mechanical stress using cyclic stretch and examined the levels of TGF-β1 and MMP-9. The role of TRPC1 was further explored by siRNA transfection and dexamethasone administration.

Results: Our results revealed that the TRPC1 level and the inflammatory cells counts were significantly higher in COPD group. After mechanical ventilation, the expression of TGF-β1 and MMP-9 in all COPD subgroups was significantly increased, while in the control group, only high PIP subgroup increased. Meanwhile, TRPC1 expression was positively correlated with the counts of inflammatory cells and the levels of TGF-β₁ and MMP-9. In vitro, mechanical stretch significantly increased TGF-β1 and MMP-9 levels and such increase was greatly attenuated by TRPC1 siRNA transfection and dexamethasone administration.

Conclusion: Our results suggest that the increased TRPC1 may play a role in the airway inflammation and airway remodeling in COPD under high airway pressure. Glucocorticoid could in some degree alleviate airway remodeling via inhibition of TRPC1.

Keywords: COPD; TRPC1; airway inflammation; airway remodeling; chronic obstructive pulmonary disease; mechanical stress; transient receptor potential canonical 1.

Figure 1

TRPC1 expression levels in patients. RT-qPCR analysis (A) and Western blotting (C and D) were used to detect the TRPC1 levels in bronchial epithelial cells in control group and COPD group. All values were represented as means ± SD. (N = 33 for patients in control group, N =15 for patients in Non-ICS group, N =12 for patients in ICS group). TRPC1 mRNA and proteins were detected by RT-PCR analysis (B) and Western blotting (E and F) in subgroup of control group and COPD group. LPIP, low peak inspiratory pressure; MPIP, moderate peak inspiratory pressure; HPIP, high peak inspiratory pressure. All values were represented as means ± SD. (N = 11 for patients in each subgroup of control group, N =9 for patients in each subgroup of COPD group). There was no difference in subgroups respectively in control group and COPD group. *P<0.01 compared with control group; ^#P<0.01 compared with Non-ICS group.

Figure 2

The effects of three levels of peak inspiratory pressure on airway remodeling-related cytokines release in ETAs. TGF-β1 in supernatants of ETAs were collected and processed by ELISA (A). MMP-9 in supernatants of ETAs were collected and measured by ELISA (B).*P<0.05 VS the subgroup before mechanical stretch. All data were represented as means ± SD. (N = 11 for patients in each subgroup of control group, N =9 for patients in each subgroup of COPD group). ^ΔP<0.05 VS low PIP subgroup and ^# P<0.05 VS moderate PIP subgroup after ventilation in COPD group.

Figure 3

Correlation between TRPC1 levels in bronchial epithelial cells and FEV1%/Pre in COPD group before mechanical ventilation. Correlation between TRPC1 protein and FEV1%/Pre in COPD patients (A). Correlation between TRPC1 mRNA and FEV1%/Pre in COPD patients (B). Statistical analysis by Spearman’s rank correlation test.

Figure 4

Correlation between TRPC1 levels and inflammation cell numbers in ETAs in COPD group. Correlations between TRPC1 protein and macrophages (A), lymphocytes (B), neutrophils numbers (C) in ETAs in COPD patients. Correlations between TRPC1 mRNA levels and macrophages (D), lymphocytes (E), neutrophils numbers (F) in ETAs in COPD patients. Statistical analysis by Spearman’s rank correlation test.

Figure 5

Correlation between TRPC1 protein levels by Western blotting and the levels of TGF-β₁(×ng/L), MMP-9 (×μg/L) in ETAs in LPIP group (A), MPIP group (B), and HPIP group (C). Statistical analysis by Spearman’s rank correlation test.

Figure 6

The efficiency of knockdown in TRPC1 expression by TRPC1 siRNA was determined by Western blot analysis. Western blotting of TRPC1 in each group in 16HBE cells (A). Relative ptrotein expression of TRPC1 measured by Western blotting (B). All values were represented as means ± SD. (N=3). * P<0.01 vs the control group.

Figure 7

Effect of mechanical stretch on TGF-β₁ and MMP-9 expression in 16HBE cells. The mRNA levels of TGF-β1 and MMP-9 in cell lysates after mechanical stretch was observed using RT-qPCR (A and B), the protein expression of TGF-β₁ and MMP-9 in the supernatant of cultured cells was measured using ELISA (C and D). Data are presented as the means ±SD. (N=6). *P<0.01 compared with the control group; ^#P<0.05 compared with the stretch group.

Figure 8

Detection of cell viability of 16HBE cells by CCK-8 assay. Viabilities of 16HBE cells after dexamethasone treatment (1, 5, 25, 50, 100, 200μM) for 24 h were detected using Cell Counting Kit-8 (CCK-8) assay. Values are presented as the means ±SD.*P<0.01 compared with the control group.

Figure 9

Dex reduced calcium levels and airway remodeling-related cytokins induced by mechanical stress in 16HBE cells. Fluorescence images of intracellular Ca²⁺ in 16HBE cells after stress and dexamethasone treatment. Scale bar: 20µm (A). Ca²⁺ fluorescence relative intensity in control group, stretch group and stretch group treatment with dexamethasone (B). Western blotting was used to analyze the expression levels of TRPC1, TGF-β₁ and MMP-9 in 16HBE cells after stress and dexamethasone treatment (C and D). Data are presented as the means ±SD. (N=3). *P<0.05 compared with the control group; ^#P<0.05 compared with the stretch group.