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. 2021 Jan 30;7(1):e05927.
doi: 10.1016/j.heliyon.2021.e05927. eCollection 2021 Jan.

Changes of vitamin D receptors (VDR) and MAPK activation in cytoplasmic and nuclear fractions following exposure to cigarette smoke with or without filter in rats

Fatist Okrit  1 , Poonchavist Chantranuwatana  2 , Duangporn Werawatganon  1 , Maneerat Chayanupatkul  1 , Sompol Sanguanrungsirikul  1
Affiliations

Changes of vitamin D receptors (VDR) and MAPK activation in cytoplasmic and nuclear fractions following exposure to cigarette smoke with or without filter in rats

Fatist Okrit et al. Heliyon. .

Abstract

Cigarette smoke (CS) is a major cause of obstructive lung disease which is associated with significant disability and mortality. Vitamin D receptor (VDR) together with, mitogen activated protein kinases (MAPKs; ERK, JNK and p38) are the cellular transmission signals that mechanistically respond to CS and are recently found to have a role in lung pathogenesis. There are a few in vitro studies on subcellular VDR distribution involved MAPK but in vivo effects of cigarette smoke exposure with and without filter on this complex remain unclear. This study investigated subcellular VDR distribution and MAPK expression at early stages of both types of cigarette smoke exposure (CSE) in a rat model. Male Wistar rats were randomly divided into no-filter, filter and control groups. After 7 and 14 days of CSE, lung tissues were obtained to determine histopathology and protein expression. Cytoplasmic and nuclear VDR distribution significantly decreased on both CSE groups and corresponded with immunohistochemistry detection. The ratio of phosphorylated ERK to total ERK significantly increased in cytoplasm of both CSE on day 7. In particular, nuclear ERK MAPK significantly escalated in the filter group on day 14. In consistent with changes in intracellular markers, histopathological examination in both CSE groups showed significant increases in tracheal and peribronchiolar epithelial proliferation, alveolar macrophages and an increased trend of parenchymal infiltration. In summary, the evidence of lung injuries along with VDR depletion and MAPK activation observed in both CSE types indicated that there was no benefit of using cigarette filter to prevent protein damage or protect cells against cigarette smoke exposure in this model.

Keywords: Cigarette smoke exposure (CSE); Mitogen activated protein kinases (MAPKs); Vitamin D receptors (VDR) distribution.

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Figures

Figure 1
Figure 1
Components of cigarette smoke exposure system. The cigarette smoke exposure system was composed of cigarette, ash and smoke storage box, cigarette filter articulation, air compressor and inhalation chamber (Right to left).
Figure 2
Figure 2
Histological features of tracheal epithelial cell changes in rat lungs using H&E staining (100X, n = 5 per group). (A,D) Tracheal epithelial cells of control rats after being exposed to room air for 7 and 14 days showed normal epithelial layers. (B,E) Non-filtered CSE groups at 7 and 14 days, and (C,F) filtered CSE groups at 7 and 14 days showed mild to moderate squamous cell metaplasia. Red arrows indicate mild to moderate squamous cell metaplasia.
Figure 3
Figure 3
Histological features of peribronchiolar epithelial cell proliferation in rat lungs using H&E staining (100X, n = 5 per group). (A,D) Peribronchiolar epithelial cells of control rats after being exposed to room air for 7 and 14 days showed normal epithelial layers. (B,E) Non-filtered CSE groups at 7 and 14 days, and (C,F) filtered CSE groups at 7 and 14 days showed mild to moderate epithelial cell dysplasia. Red arrows indicate mild to moderate epithelial cell dysplasia.
Figure 4
Figure 4
Histological features of lung parenchymal infiltration in rat lungs using H&E staining (100X, n = 5 per group). (A,D) Lung parenchymal infiltration of control rats after being exposed to room air for 7 and 14 days showed few inflammatory cells. (B,E) Non-filtered CSE groups and (C,F) filtered CSE groups at 7 and 14 days showed increased inflammatory cell infiltration. Red arrows indicate inflammatory cell infiltration in lung parenchyma.
Figure 5
Figure 5
Representative images of alveolar macrophage count in rat lungs using H&E staining (100X, n = 5 per group). (A,D) Alveolar macrophage count of control rats after being exposed to room air for 7 and 14 days. (B,E) Non-filtered CSE groups and (C,F) filtered CSE groups at 7 and 14 days showed small clusters of macrophages in the alveolar space. Red arrows indicate alveolar macrophages in the alveolar space of rat lung tissues and solid line arrows indicate particulate matter uptake by alveolar macrophages.
Figure 6
Figure 6
Western blot analyses of VDR distribution from cytoplasmic and nuclear fractions in rat lungs. (A) Cytoplasmic (cVDR) and (B) nuclear VDR (nVDR) protein expression in rat lungs were shown in bar charts and band densities (n = 5 for each group). β-actin and lamin B1 were used as loading control. ∗, p < 0.05 compared with the control group at 7 days. #, p < 0.05 compared with control group at 14 days. a, p < 0.05 comparison between no-filter and filter groups at 7 days. b, p < 0.05 comparison between no-filter and filter groups at 14 days. c, p < 0.05 comparison between no-filter groups at 7 and 14 days. f, p < 0.05 comparison between filter groups at 7 days and 14 days. Data were expressed as mean ± standard error of mean. Full versions of all images are provided in Supplementary figure 1 and 2.
Figure 7
Figure 7
Western blot analyses of ERK distribution from cytoplasmic and nuclear fractions in rat lungs. Extracellular regulated kinase (ERK) protein expression of (A)cytoplasmic and (B) nuclear parts were shown in bar charts and band densities (n = 5 for each group). β-actin and lamin B1 were used as loading control. ∗, p < 0.05 compared with the control group at 7 days. #, p < 0.05 compared with the control group at 14 days. b, p < 0.05 comparison between no-filter and filter groups at 14 days. d, p < 0.05 comparison between no-filter group at 7 days and filter group at 14 days. f, p < 0.05 comparison between filter groups at 7 and 14 days. Data were expressed as mean ± standard error of mean. Full versions of all images are provided in Supplementary figure 3 and 4.
Figure 8
Figure 8
Western blot analyses of JNK distribution from cytoplasmic and nuclear fractions in rat lungs. C-Jun N-terminal kinase (JNK) protein expression of (A) cytoplasmic and (B) nuclear parts were shown in bar charts and band densities (n = 5 for each group). β-actin and lamin B1 were used as loading control. ∗, p < 0.05 compared with the control group at 7 days. #, p < 0.05 compared with the control group at 14 days. Data were expressed as mean ± standard error of mean. Full versions of all images are provided in Supplementary figure 5 and 6.
Figure 9
Figure 9
Western blot analyses of p38 distribution from cytoplasmic and nuclear fractions in rat lungs. p38 protein expression of (A) cytoplasmic and (B) nuclear parts were shown in bar charts and band densities (n = 5 for each group). β-actin and lamin B1 were used as loading control. ∗, p < 0.05 compared with the control group at 7 days. #, p < 0.05 compared with the control group at 14 days. c, p < 0.05 comparison between no-filter groups at 7 and 14 days. Data were expressed as mean ± standard error of mean. Full versions of all images are provided in Supplementary figure 7 and 8.
Figure 10
Figure 10
Immunohistochemistry of VDR distribution in rat lungs (40X, n = 5 per group). (A,D) Control groups at 7 and 14 days, (B,E) No-filter groups at 7 and 14 days, and (C,F) filter groups at 7 and 14 days, respectively. VDR expression were shown in histological images and bar charts. Arrows indicated VDR positive pneumocytes type II cells with dark brown stained nuclei. p < 0.05 when compared the filter group at 14 days with the control group of the same time point. Data were expressed as mean ± standard error of mean.
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