ASK1-p38 cascaded signal mediates pulmonary microvascular endothelial barrier injury induced by the return of PHSML in rats

Abstract

The return of post-hemorrhagic shock mesenteric lymph (PHSML) induces pulmonary vascular endothelial barrier dysfunction, which results in acute lung injury. Activation of the apoptosis signal-regulated kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38 MAPK) pathway has been shown to trigger inflammatory responses. However, whether the ASK1-p38 MAPK pathway is involved in the PHSML-induced pulmonary endothelial barrier dysfunction remains unclear. In the present study, permeability changes of pulmonary capillaries were found in vivo, and activation of the ASK1-p38 MAPK pathway was determined in vitro. PMVEC barrier dysfunction was determined by measuring TEER. Furthermore, junctional and cytoskeletal protein expressions were analyzed. The results showed that hemorrhagic shock led to a marked increase in the permeability of pulmonary capillaries in vivo, which was markedly alleviated by PHSML drainage. In cultured pulmonary microvascular endothelial cells (PMVECs), PHSML reduced the endothelial barrier function accompanied by upregulated p-ASK1 and p-p38 MAPK protein expression, impaired the cytoskeletal protein structure, and down-regulated junction protein expression. These adverse effects were eliminated by applying either Trx1 (ASK1 inhibitor) or SB203580 (p38 MAPK inhibitor). These results indicated that the ASK1-p38 MAPK pathway mediates PHSML-induced pulmonary vascular endothelial barrier dysfunction during hemorrhagic shock.

Fig. 1. Permeability changes of pulmonary capillaries following hemorrhagic shock in rats. (A–C) Images of FITC-albumin leakage from pulmonary capillaries under fluorescence microscopy among the three groups, bar = 50 μm. (D) Ratio of FITC-albumin leakage in different groups. Results are presented as means ± SD (n = 3). *P < 0.05 vs. sham group, ^#P < 0.05 vs. shock group.

Fig. 2. Effects of TRX1 and SB203580 on the p-ASK1 and p-p38 MAPK expressions in PMVECs treated with PHSML. (A) Images of western blotting for ASK1 and p38 MAPK. (B) Relative density analysis of concentration. Results are presented as the mean ± SD (n = 3). *P < 0.05 vs. control group, ^#P < 0.05 vs. PHSML group, ^▲P < 0.05 vs. LPS group.

Fig. 3. Effects of TRX1 and SB203580 on the morphology changes of PMVECs treated with PHSML. (A–J) Images of control, PHSML, lipopolysaccharide (LPS), PHSML + TRX1 (ASK1 inhibitor) or PHSML + SB203580 (p38 MAPK inhibitor) incubated for 6 h (inverted microscope, ×50 and ×200).

Fig. 4. Effects of TRX1 and SB203580 on the permeability of PMVECs treated with PHSML. (A) Permeability coefficient of the PMVEC barrier. (B) TEER of the PMVEC barrier. Results are presented as means ± SD (n = 3). *P < 0.05 vs. control group, ^#P < 0.05 vs. PHSML group, ^▲P < 0.05 vs. LPS group.

Fig. 5. Effects of TRX1 and SB203580 on junction protein expression in PMVECs treated with PHSML. (A) Images of western blotting for VE-cadherin, ZO-1, and claudin-1. (B) Relative density analysis of concentration. Results are presented as means ± SD (n = 3). *P < 0.05 vs. control group, ^#P < 0.05 vs. PHSML group, ^▲P < 0.05 vs. LPS group.

Fig. 6. Effects of TRX1 and SB203580 on F-actin expression in PMVECs treated with PHSML. F-actin was stained with phalloidin FITC (green); nuclei were stained with DAPI (blue); cells co-expressed F-actin and DAPI in the merged image.