Does Vitamin D Work Synergistically with Anti-Asthmatic Drugs in Airway Remodeling?

Abstract

Vitamin D is commonly known for its properties of airway remodeling inhibition. Due to this, we decided to analyze the action of calcitriol with anti-asthmatic drugs in airway remodeling. The HFL1 cell line was treated with calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β in different combinations. Real-time PCR was used to analyzed the expression of ACTA2, CDH-1, Vimentin, ADAM33, MMP-9 and CysLTR1 on the mRNA level, whereas Western blot was used to analyze gene expression on the protein level. One-way analysis variants, the Kruskal-Wallis test, Student's t-test or Welch's t-test were used for statistical analysis. Concerning the results, pre-treatment with calcitriol increased the inhibitory effect of beclomethasone 17-propionate and montelukast sodium on the expression of ACTA2 (p = 0.0072), Vimentin (p = 0.0002) and CysLTR1 (p = 0.0204), and 1,25(OH)₂D₃ had an influence on the effects of beclomethasone 17-propionate and montelukast sodium and of CDH1 expression (p = 0.0076). On the protein level, pre-treatment with calcitriol with beclomethasone 17-propionate and montelukast sodium treatment decreased ACTA2 expression in comparison to the LT (LTD4 and TGF-β) control group (p = 0.0191). Hence, our study not only confirms that vitamin D may inhibit airway remodeling, but also shows that vitamin D has a synergistic effect with anti-asthmatic drugs.

Keywords: 1,25(OH)2D3; airway remodeling; bronchial asthma; calcitriol; vitamin D.

Figure 1

Calcitriol inhibits the expression of ACTA2 in dose- or time-dependent manner. (A) mRNA expression and (C) protein level of ACTA2 after pre-treatments with three chosen concentrations of 1,25(OH)₂D₃ and treatment with 10 ng/mL of TGF-β. (B) mRNA expression and (D) protein level of ACTA2 after pre-treatments with 1,25(OH)₂D₃ for three chosen incubation times and treated with 10 ng/mL of TGF-β. The results of experiments (n = 3) are presented in relation to GAPDH as a mean ± SEM. Western blots are shown for genes, in which the statistical significance has been observed at the mRNA level. Original Western blots can be found at Figure S1. # p < 0.05, ## p < 0.01, #### p < 0.0001 versus NT group; $ p < 0.05, $$$$ p < 0.0001 versus TGF-β group; & p < 0.05 and &&&& p < 0.0001 versus MT group. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by corresponding post hoc was used to determine differences in several groups, and Student’s t-test or Welch’s t-test was used to determine statistically significant differences between two means. RQ—relative quantification of genes expression normalized to GAPDH, NT—non-treatment cells, TGF-β—cells treated with TGF-β, MT—cells treated with 0.1% of methanol and TGF-β.

Figure 2

Calcitriol, beclomethasone 17-propionate and montelukast sodium effect on the expression of ACTA2, CDH1 and Vimentin. (A–C) mRNA expression and (D,E) protein level of ACTA2, CDH-1 and Vimentin after exposure to calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β. The results of experiments (n = 3) are presented in relation to GAPDH as mean ± SEM. Western blots are shown for genes, in which the statistical significance has been observed at the mRNA level. Original Western blots can be found in Figure S1. @@ p < 0.01 versus NT group; # p < 0.05 versus T group; $ p < 0.05, $$ p < 0.01 versus L group; % p < 0.05, %% p < 0.01 versus LT group; and & p < 0.05 versus DMT group; * p < 0.05, ** p < 0.01, *** p < 0.001. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by corresponding post hoc was used to determine differences in several groups, and Student’s t-test or Welch’s t-test was used to determine statistically significant differences between two means. RQ—relative quantification of genes expression normalized to GAPDH, NT—untreated cells, T—cells treated with TGF-β, L—cells treated with LTD4, LT—cells treated with LTD4 and TGF-β, DMT—cells treated with methanol, DMSO and TGF-β, BT—cells treated with beclomethasone 17-propionate and TGF-β, MLT—cells treated with montelukast sodium, LTD4 and TGF-β, MBLT—cells treated with beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β, CT—cells treated with calcitriol and TGF-β, CBT—cells treated with calcitriol, beclomethasone 17-propionate and TGF-β, CMLT—cells treated with calcitriol, montelukast sodium, LTD4 and TGF-β, CBMLT—cells treated with calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β.

Figure 3

Calcitriol, beclomethasone 17-propionate and montelukast sodium effect on the expression of Vimentin, ADAM33 and MMP-9. (A,B) mRNA expression and (C,D) protein level of ADAM33 and MMP-9 after exposure to calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β. The results of experiments (n = 3) are presented in relation to GAPDH or ACTB as mean ± SEM. Western blots are shown for genes, in which the statistical significance has been observed at the mRNA level. Original Western blots can be found at Figure S1. @ p < 0.05, @@ p < 0.01 versus NT group; # p < 0.05, ## p < 0.01 versus T group; $ p < 0.05 versus L group; % p < 0.05, %% p < 0.01 versus LT group; * p < 0.05, ** p < 0.01. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by corresponding post hoc was used to determine differences in several groups, and Student’s t-test or Welch’s t-test was used to determine statistically significant differences between two means. RQ—relative quantification of genes expression normalized to GAPDH, NT—untreated cells, T—cells treated with TGF-β, L—cells treated with LTD4, LT—cells treated with LTD4 and TGF-β, DMT—cells treated with methanol, DMSO and TGF-β, BT—cells treated with beclomethasone 17-propionate and TGF-β, MLT—cells treated with montelukast sodium, LTD4 and TGF-β, MBLT—cells treated with beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β, CT—cells treated with calcitriol and TGF-β, CBT—cells treated with calcitriol, beclomethasone 17-propionate and TGF-β, CMLT—cells treated with calcitriol, montelukast sodium, LTD4 and TGF-β, CBMLT—cells treated with calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β.

Figure 4

Calcitriol, beclomethasone 17-propionate and montelukast sodium effect on the expression of CysLTR1. mRNA expression CysLTR1 after exposure to calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β. The results of experiments (n = 3) are presented in relation to GAPDH as mean ± SEM. Western blots are shown for genes, in which the statistical significance has been observed at the mRNA level. Original Western blots can be found at Figure S1. % p < 0.05, %% p < 0.01 versus LT group; * p < 0.05. One-way analysis of variance (ANOVA) or Kruskal-Wallis test followed by corresponding post hoc was used to determine differences in several groups, and Student’s t-test or Welch’s t-test was used to determine statistically significant differences between two means. RQ—relative quantification of genes expression normalized to GAPDH, NT—untreated cells, T—cells treated with TGF-β, L—cells treated with LTD4, LT—cells treated with LTD4 and TGF-β, DMT—cells treated with methanol, DMSO and TGF-β, BT—cells treated with beclomethasone 17-propionate and TGF-β, MLT—cells treated with montelukast sodium, LTD4 and TGF-β, MBLT—cells treated with beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β, CT—cells treated with calcitriol and TGF-β, CBT—cells treated with calcitriol, beclomethasone 17-propionate and TGF-β, CMLT—cells treated with calcitriol, montelukast sodium, LTD4 and TGF-β, CBMLT—cells treated with calcitriol, beclomethasone 17-propionate, montelukast sodium, LTD4 and TGF-β.