Pulmonary vascular remodeling and distribution of ET-1 in HPS rat lung.

<p>(A.B) Representative images of pulmonary vascular remodeling sections from HPS and SHAM group rats stained with HE. (C) The arteriole wall thickness/vascular extemal diameter (WT%) is displayed. (D.E) Immunohistochemical localization of preproET-1 in lung sections from normal and HPS rats. (F) The relative level of preproET-1 protein expression compared with SHAM group is displayed. (G) Western blotting and graphical summaries of preproET-1 protein levels in lung tissues of HPS rats. β-actin was used as an internal control. (H) Expression of ET-1 was measured in the lung tissue from HPS and SHAM group rats by ELISA. All datas were representative of four independent experiments. Values were expressed as means±SEM. *P<0.05 compared with SHAM group.</p

ANXA1 decreased p-ERK1/2 accumulation in the nucleus and decreased cyclin D1 expression in PASMCs.

<p>(A)PASMCs were transfected with Ad-ANXA1 48 h before ET-1-stimulation. Western blot analysis was used to measure the protein expression of total ERK1/2 and nuclear p-ERK1/2 expression in PASMCs 24 h after ET-1 treatment. (B) PASMCs were transfected with Ad-ANXA1 48 h before ET-1-stimulation. Western blot analysis was used to measure the protein expression of cyclinD1 expression in PASMCs 24 h after ET-1 treatment. β-actin was used as an internal control. Each data point represents the mean ± S.E.M. of four independent experiments. *p< 0.05 vs. control group. ^#P< 0.05 vs. ET-1 treated group.</p

ANXA1 overexpression inhibited ET-1-induced inflammatory phenotype and proliferation of PASMCs.

<p>(A.B.C) PASMCs were treated with up to 50 nM of ET-1. Expression of IL-1β, IL-6 and TNF-a was measured in the supernatants of PASMCs by ELISAs at 24 h. (D.E) The change in the value of the incorporation of ³H-TdR and the absorbance of CCK-8 assay was displayed. The inhibitory effect was investigated at 24 h and 48 h. Each data point represents the mean ± S.E.M. of four independent experiments.*p< 0.05 vs. control group. ^#P< 0.05 vs. ET-1 treated group.</p

Carbonylation of ANXA1 induced by the ET-1-mediated ROS decreases ANXA1 protein stability.

<p>(A) PASMCs were treated with ET-1 (10 nM) in the presence or absence of Apo (100 μmol/l)for 60 min. PASMCs were stained by dihydroethidium (DHE), DHE fluorescence intensity was measured by flowcytometry analysis and expressed as fold induction compared with control group. (B) Cells were treated with 10 nM of ET-1 for 6 h, and whole cell extracts were immunoprecipitated with an anti-ANXA1 antibody. ANXA1 carbonylation was detected by Western blot using an anti-DNP antibody. (C) Whole cell extracts were assayed by Western blot analysis to measure ANXA1 levels. (D) PASMCs were pre-treated with MG132 (10 μM) for 1 h and then exposed to the ET-1 for 24 h; Western blot analysis to measure ANXA1 levels. Each data point represents the means ±SEMs for parametric tests; n = 4. *p< 0.05 vs. control group. ^#P< 0.05 vs. ET-1 treated group.</p

ET-1 down regulated ANXA1 by posttranslational mechanisms.

<p>(A)PASMCs were treated with different concentrations of ET-1 for 24 h, and then cells were harvested for ANXA1 mRNA level analysis by qPCR. Data were normalized to GADPH (P > 0.05, n = 5). (B)PASMCs cultures were pre-incubated with Act-D (2.5 μg/mL) or CHX (10 μg/mL) for 1 h, then treated with 10 nM ET-1 for an additional 24 h and ANXA1 protein was analyzed by using Western blot. Each data point represents the mean ± S.E.M. of four independent experiments.*p< 0.05 vs. ET-1 untreated control group. ^#P< 0.05 vs. ET-1 treated control group.</p

The effects of NaHS or DL- propargylglycine (PAG) treatment on myeloperoxidase (MPO) in plasma and lungs after blast limb trauma.

<p>Plasma MPO (A), Lung MPO (B). Data are mean ± SEM (n = 8);*P<0.0.5, ** p<0.01 <i>vs</i> Sham;+p<0.05,++p<0.01 <i>vs</i> control.</p

Sodium Hydrosulphide (NaHS) attenuates the pulmonary micro-vessel endothelial cells injury induced by tumour necrosis factor alpha (TNF-α).

<p>The cells viability is measured with 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5- diphenyl-2H-tetrazolium bromide assay (MTT) assay with TNF-α with or without NaHS (A). The injured cells indicated with lactate dehydrogenase release in the culture media with TNF-α treatment in the presence or absence of NaHS (B). Data are mean ± SEM (n = 12); ** p<0.01 <i>vs</i> Naïve,++p<0.01 <i>vs</i> vehicle.</p

NaHS suppresses the productions of oxidative and inflammatory mediators in pulmonary micro-vessel endothelial cells induced by TNF-α.

<p>Nitric oxide concentration (A), Inductive nitric oxide synthase (B), Intercellular adhesion molecule (ICAM) 1 (C) and interleukin (IL) 6 (D). Data are mean ± SEM (n = 12); ** p<0.01 <i>vs</i> Naïve,++p<0.01 <i>vs</i> vehicle.</p

The effects of NaHS or DL- propargylglycine (PAG) treatment on cytokines in plasma and lungs after blast limb trauma

<p>. Plasma TNF-α (A), interleukin (IL)-6 (B), Lung TNF-α (C), IL- 6 (D). Data are mean ± SEM (n = 8).*P<0.0.5, ** p<0.01 <i>vs</i> Sham;+p<0.05,++p<0.01 <i>vs</i> control.</p

Attenuation of lung injury following blast limb trauma by NaHS treatment.

<p>Blast limb trauma induces systemic inflammatory response, oxidative stress and suppression of CSE/H₂S pathway. All those induce to the disruption of endothelium and the increase of permeability of gas blood barrier, and ultimately lead to lung oedema formation and remote lung injury following blast limb trauma. Exogenous NaHS treatment attenuates lung injury following blast limb trauma via preventing the decrease of plasma H₂S, suppressing the production of cytokines and adhesion molecular, such as TNF-α, IL-6, IL-10, ICAM1, CXCL-2 and CD11b <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059100#pone.0059100-Francis1" target="_blank">[31]</a> and ameliorating oxidative stress though Nrf2, K⁺_ATP and Cl⁻ channel and inhibition of iNOS/NO pathway and L-type Ca2+ channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059100#pone.0059100-Mikami1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059100#pone.0059100-Kimura2" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059100#pone.0059100-Kimura3" target="_blank">[46]</a>.</p