1α,25-dihydroxyvitamin D3 ameliorates seawater aspiration-induced acute lung injury via NF-κB and RhoA/Rho kinase pathways

Abstract

Introduction: Inflammation and pulmonary edema are involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have reported that 1α,25-Dihydroxyvitamin D3 (calcitriol) suppresses inflammation, it has not been confirmed to be effective in seawater aspiration-induced ALI. Thus, we investigated the effect of calcitriol on seawater aspiration-induced ALI and explored the probable mechanism.

Methods: Male SD rats receiving different doses of calcitriol or not, underwent seawater instillation. Then lung samples were collected at 4 h for analysis. In addition, A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not and then stimulated with 25% seawater for 40 min. After these treatments, cells samples were collected for analysis.

Results: Results from real-time PCR showed that seawater stimulation up-regulated the expression of vitamin D receptor in lung tissues, A549 cells and RPMVECs. Seawater stimulation also activates NF-κB and RhoA/Rho kinase pathways. However, we found that pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways. Meanwhile, treatment of calcitriol also improved lung histopathologic changes, reduced inflammation, lung edema and vascular leakage.

Conclusions: These results demonstrated that NF-κB and RhoA/Rho kinase pathways are critical in the development of lung inflammation and pulmonary edema and that treatment with calcitriol could ameliorate seawater aspiration-induced ALI, which was probably through the inhibition of NF-κB and RhoA/Rho kinase pathways.

Figure 1. Effects of calcitriol on seawater aspiration-induced lung histopathologic changes (A), wet/dry weight ratios (B) and Evans blue leakage (C).

Histopathologic, W/D ratio and Evans blue leakage examination of the lung tissues were performed at 4 h after seawater aspiration. (a) normal group; (b) seawater group; (c)–(e) 1 µg/kg, 5 µg/kg and 25 µg/kg calcitriol groups; (f) dexamethasone group. n = 8, ***P<0.001 versus group a, # P<0.05, ## P<0.01, ### P<0.001 versus group b. Histopathologic changes showed pulmonary edema, infiltration of inflammatory cells, hemorrhage and alveolar distortion in seawater group. In calcitriol group, lung injury was significantly alleviated compared with seawater group and dose dependence was observed. Dexamethasone also alleviated lung damage compared with seawater group, but no significant difference compared with 25 µg/kg calcitriol group. (Hematoxylin-eosin stain, 20×).

Figure 2. Effects of calcitriol on the levels of TNF-α, IL-1β and IL-6 (A) in lung tissues.

All data were obtained at 4 h after seawater aspiration. (a) normal group; (b) seawater group; (c)–(e) 1 µg/kg, 5 µg/kg and 25 µg/kg calcitriol groups; (f) dexamethasone group. n = 8, ***P<0.001 versus group a, # P<0.05, ## P<0.01, ### P<0.001 versus group b. Effects of calcitriol on the NF-κB p65 phosphorylation (B) in lung tissues. Proteins were obtained at 4 h after seawater aspiration. The ratios of pNF-κB versus NF-κB were obtained for each group to examine the pNF-κB content. NG: normal group; SG: seawater group; CG: 25 µg/kg calcitriol group; DG: dexamethasone group. n = 6, ***P<0.001 versus group NG, # P<0.05, ## P<0.01 versus group SG.

Figure 3. Effects of calcitriol on the GTP-RhoA and p-MYPT1 in lung tissues.

Proteins were obtained at 4 h after seawater aspiration. The ratios of GTP-RhoA versus total-RhoA and p-MYPT1 versus MYPT1 were obtained for each group to examine the GTP-RhoA (A) and p-MYPT1 (B) contents. NG: normal group; SG: seawater group; CG: 25 µg/kg calcitriol group; DG: dexamethasone group. n = 6, ***P<0.001 versus group NG, ### P<0.001 versus group SG, ° P<0.05 versus group DG.

Figure 4. Effects of calcitriol on NF-κB p65 activation and RhoA/ROCK pathway in A549 cells.

After 4 h seawater treatment, proteins were obtained for western blot. The ratios of pNF-κB versus NF-κB, GTP-RhoA versus total-RhoA and p-MYPT1 versus MYPT1 were obtained for each group to examine the pNF-κB, GTP-RhoA and p-MYPT1 content. NG: normal group; SG: seawater group; CG: 10⁻⁶ M calcitriol group; DG: dexamethasone group. n = 6, ***P<0.001 versus group NG, ## P<0.01, ### P<0.001 versus group SG, ° P<0.05 versus group DG.

Figure 5. Effects of calcitriol on RhoA/ROCK pathway in RPMVECs.

Proteins were obtained at 4 h after seawater administration. The ratios of GTP-RhoA versus total-RhoA and p-MYPT1 versus MYPT1 were obtained for each group to examine the GTP-RhoA (A) and p-MYPT1 (B) contents. NG: normal group; SG: seawater group; CG: 10⁻⁶ M calcitriol group; DG: dexamethasone group. n = 6, ***P<0.001 versus group NG, ### P<0.001 versus group SG.

Figure 6. Effects of calcitriol on monolayer permeability in A549 cells and RPMVECs.

A549 cells and RPMVECs were treated for 40 min with seawater in the presence or absence of calcitriol and dexamethasone. After A549 (A) and RPMVEC (B) monolayer formation, cells were treated for 40 min with seawater. The permeability was shown as fold of NG. NG: normal group; SG: seawater group; CG: 10⁻⁶ M calcitriol group; DG: dexamethasone group. n = 6, ***P<0.001 versus group NG, ## P<0.01, ### P<0.001 versus group SG, ° P<0.05 versus group DG.