Pulmonary activation of vitamin D3 and preventive effect against interstitial pneumonia

Abstract

Calcitriol [1,25(OH)2D3] is usually investigated in studies on the preventive effect of activated vitamin D against interstitial pneumonia. Although cholecalciferol (vitamin D3) can be easily obtained in the diet and has a longer half-life than calcitriol, there have been few investigations of its effect on interstitial pneumonia. We used human pulmonary fibroblast cell lines (HPFCs) and a mouse model of bleomycin-induced pulmonary fibrosis to evaluate whether vitamin D3 was activated in the lungs and had a preventive effect against interstitial pneumonia. Expression of the vitamin D receptor gene and genes for enzymes metabolizing vitamin D was evaluated in two HPFCs, and the suppressive effect of vitamin D3 on induction of inflammatory cytokines was also assessed. Gene expression of the vitamin D receptor and vitamin D-metabolizing enzymes was observed in both human pulmonary fibroblast cell lines. Vitamin D3 suppressed bleomycin-induced expression of inflammatory cytokines and fibrosis markers by the HPFCs. In mice, symptoms of bleomycin-induced pulmonary fibrosis were improved and expression of fibrosis markers/fibrosis inducers was decreased by a high vitamin D3 diet. Vitamin D3 is activated locally in lung tissues, suggesting that high dietary intake of vitamin D3 may have a preventive effect against interstitial pneumonia.

Keywords: interstitial pneumonia; prevention; pulmonary fibrosis; vitamin D.

Conflict of interest statement

No potential conflicts of interest were disclosed.

Copyright © 2019 JCBN.

Figures

Fig. 1

Schedule of tests in the mouse model of bleomycin-induced pulmonary fibrosis. Six-week-old C57BL/6JJcl mice were randomized to three groups (n = 5 each): a control (CTL) group (control diet + PBS i.v.), a bleomycin (BLM) group (control diet + bleomycin i.v.), and a high VD3 + bleomycin (VD + BLM) group (high VD3 diet + bleomycin i.v.). The control diet contained 200 IU/100 g of VD3 and the high VD3 diet contained 1,000 IU/100 g. From four days after starting each diet, bleomycin (10 mg/kg) was administered via the tail vein once daily for five days in the BLM and VD + BLM groups, while PBS was administered in the CTL group. Lungs were harvested at four days after the finish of administration.

Fig. 2

Expression of the vitamin D receptor and vitamin D-metabolizing enzymes in human fibroblasts, and VD3-dependent suppression of the induction of IL-1β gene expression in pulmonary fibroblasts by bleomycin. (A) To assess gene expression in MRC-5 and MRC-5 SV1 TG1 cells, RT-PCR was performed using specific primers for the vitamin D receptor (VDR) or vitamin D-metabolizing enzymes, including CYP27A1 (27A1), CYP2R1 (2R1), and CYP27B1 (27B1). (B) MRC-5 SV1 TG1 cells were treated with 25 µg/ml bleomycin for 48 h and RT-PCR was performed using specific primers for αSMA, IL-1β, and GAPDH. (C, D) Band intensities of the PCR products were converted to numerical data by image analysis software, and the data for αSMA and IL-1β were normalized by the GAPDH value.

Fig. 3

Pretreatment of pulmonary fibroblasts with VD3 suppresses bleomycin-induced expression of pro-fibrotic genes, fibrosis markers, and inflammatory markers. (A) MRC-5 SV1 TG1 cells underwent pretreatment with VD3 or 1,25(OH)2D3 overnight and then were incubated with 25 µg/ml bleomycin for 24 h, after which RT-PCR was performed using specific primers for αSMA, COLA2, SPP1, IL-1β, TGF-β1, and β-actin (ACTB). (B–D) Band intensities of the PCR products were converted to numerical data by image analysis software, and the data for SPP1, IL-1β, and TGF-β1 were normalized by the β-actin value.

Fig. 4

High VD3 diet suppresses alveolar wall thickening in mice with bleomycin-induced pulmonary fibrosis and improves the fibrosis scores estimated by the modified Ashcroft method. (A–C) Representative specimens of lung tissue from each group with HE staining (bar = 100 µm; bar in the inset = 30 µm). (A) Control group. (B) Bleomycin group. (C) High VD3 + bleomycin group. (A) Obvious lesions were not observed in the lungs of the control group. (B) Alveolar wall thickening (arrowhead) and multiple fibrotic lesions (arrows) were observed in the bleomycin group. (C) Histological changes were milder in the high VD3 + bleomycin group compared with the bleomycin group. (D) Image analysis (Image J) of the HE-stained area of alveolar walls in the microscopic field shown as a percentage. The value showing maximum deviation from the mean was excluded in each group. CTL: control group; BLM: bleomycin group; VD + BLM: high VD3 + bleomycin group. The alveolar wall area was 51.5 ± 4.7% in the control group, 63.4 ± 2.6% in the bleomycin group, and 51.9 ± 1.2% in the high VD3 + bleomycin group. *p = 0.034 (Mann-Whitney U test), and Cohen’s d = –6.108. (E–G) Representative specimens of lung tissues from each group with Sirius red/fast green staining (bar = 100 µm). (E) Control group (score = 1). (F) Bleomycin group (score = 5). (G) High VD3 + bleomycin group (score = 4). Compared with the control group, the alveolar walls are thicker and there are more fibrotic lesions in the other groups. (H) Comparison of the mean modified Ashcroft score. The value showing maximum deviation from the mean was excluded in each group. The mean score was 0.716 ± 0.583 in the control (CTL) group, 3.818 ± 0.474 in the bleomycin (BLM) group, and 3.794 ± 0.231 in the high VD3 + bleomycin (VD + BLM) group.

Fig. 5

High VD3 diet suppresses expression of TGF-β1 in mice with bleomycin-induced pulmonary fibrosis. Representative result of RT-PCR performed using lung tissues from mice in each group. CTL: control group; BLM: bleomycin group; VD + BLM: high VD3 + bleomycin group. (A) RT-PCR with specific primers for SPP1, IL-1β, and TGFβ1. (B) Band intensities of the PCR products were converted to numerical data by image analysis software and each value was normalized by the β-actin value. Outliers were excluded before the data were compared. *p = 0.025 (t test), Cohen’s d = –1.32.