1α,25-Dihydroxyvitamin D3 Ameliorates Seawater Aspiration-Induced Lung Injury By Inhibiting The Translocation Of NF-κB and RhoA

Abstract

Our previous study have reported that 1α,25-Dihydroxyvitamin D3 (calcitriol) suppresses seawater aspiration-induced ALI in vitro and in vivo. We also have confirmed that treatment with calcitriol ameliorates seawater aspiration-induced inflammation and pulmonary edema via the inhibition of NF-κB and RhoA/Rho kinase pathway activation. In our further work, we investigated the effect of calcitriol on nuclear translocation of NF-κB and membrane translocation of RhoA in vitro. A549 cells and rat pulmonary microvascular endothelial cells (RPMVECs) were cultured with calcitriol or not for 48 h and then stimulated with 25% seawater for 40 min. After these treatments, cells were collected and performed with immunofluorescent staining to observe the translocation of NF-κB and RhoA and the cytoskeleton remodeling. In vitro, seawater stimulation activates nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. In addition, seawater administration also induced cytoskeleton remodeling in A549 cells and RPMVECs. However, pretreatment with calcitriol significantly inhibited the activation of NF-κB and RhoA/Rho kinase pathways, as demonstrated by the reduced nuclear translocation of NF-κB and membrane translocation of RhoA in A549 cells. Meanwhile, treatment of calcitriol also regulated the cytoskeleton remodeling in both A549 cells and RPMVECs. These results demonstrated that treatment with calcitriol ameliorates seawater aspiration-induced ALI via inhibition of nuclear translocation of NF-κB and membrane translocation of RhoA and protection of alveolar epithelial and pulmonary microvascular endothelial barrier.

Keywords: 1α,25-Dihydroxyvitamin D3; NF-κB; RhoA; acute lung injury; seawater.

Fig. 1

Effects of calcitriol on nuclear translocation in A549 cells. After treatment, cells were washed, fixed, and stained with anti- NF-κB p65 antibody and DAPI. Cells were analyzed by fluorescence microscope, and individual and merged stainings are shown (magnification ×40). NG: normal group; SG: seawater group; CG: 10⁻⁶M calcitriol group; DG: dexamethasone group.

Fig. 2

Effects of calcitriol on RhoA translocation in A549 cells. After treatment, cells were washed, fixed, and stained with anti-RhoA and anti-pancadherin antibodies. Cells were analyzed by confocal microscopy, and individual and merged stainings are shown (magnification ×60). NG: normal group; SG: seawater group; CG: 10⁻⁶M calcitriol group; DG: dexamethasone group.

Fig. 3

Effects of calcitriol on cytoskeletal remodeling in A549 cells. A549 cells were treated for 40 min with seawater in the presence or absence of calcitriol and dexamethasone. Phenotype of cultured A549 cells were examined by confocal microscopy following staining for F-actin (magnification ×60). NG: normal group; SG: seawater group; CG: 10⁻⁶M calcitriol group; DG: dexamethasone group.

Fig. 4

Effects of calcitriol on cytoskeletal remodeling in RPMVECs. RPMVECs were treated for 40 min with seawater in the presence or absence of calcitriol and dexamethasone. Phenotype of cultured RPMVECs were examined by confocal microscopy following staining for F-actin (magnification ×60). NG: normal group; SG: seawater group; CG: 10⁻⁶M calcitriol group; DG: dexamethasone group.