Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung

Abstract

Aim: To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling.

Methods: Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review.

Results: MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38.

Conclusion: Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Figure 1

The signaling pathway of PI3K/Akt/eNOS in response to MV. C57BL/6J mice were randomly exposed to spontaneous breathing (Control, Cont) or HV_T (20 mL/kg) for 2 h. (A) Immunoblotting of WT mouse lung homogenates shows a decrease in PI3K, Akt, and eNOS phosphorylation after initiation of HV_T MV detected by their active forms of antibody. (B), (C), and (D) show the densitometry of phospho-PI3K, phopho-AKT, and phospho-eNOS normalized to total Akt. n=3–4 mice per condition, ^bP<0.05 vs control. Note: the Figure 1A P-Akt bands represented a combination of the results came from several experiments.

Figure 2

Down-regulation of phospho-Akt in response to mechanical stress in a time-dependent fashion. (A) In a separate experiment of 4 h HV_T MV, we observed that phospho-Akt at its serine residue 473 decreased most noticeably at 30 min and then gradually returned to control level at 2 h. (B) shows the densitometry of phopho-Akt normalized to total Akt. n=3–4 mice per condition, ^bP <0.05.

Figure 3

eNOS defective mice are less tolerant than WT mice in response to high amplitude mechanical stress featured with severe lung edema. Pulmonary vascular leakage was assessed at 4 h by the extravasation of evans blue dye(EBD) albumin in the lung parenchyma. MV with LV_T caused no change in EBD influx in C57BL/6J mouse lungs, but caused a marked increase of EBD extravasation in eNOS^−/− mouse lungs. In addition, HV_T MV caused a 100% increase of EBD in eNOS^−/− mouse lung in comparison with a 50% increase of EBA in WT mice. n=4 mice/experimental condition. ^bP<0.05 vs Control; ^eP<0.05 vs LV_T; ^hP<0.05 vs WT vent-exposed mice.

Figure 4

Effects of eNOS deficiency on pulmonary vascular leakage during mechanical ventilation. As a separate measure of pulmonary edema, wet/dry lung weight ratios were determined at the end of 4 h HV_T MV. HV_T MV caused an increase of the lung wet-dry weight ratio especially in eNOS-deficient mice. n=4 mice/experimental condition. ^bP<0.05 vs LV_T exposed animals; ^eP<0.05 compared with WT mice exposed to HV_T MV.

Figure 5

Lower levels of phosph-Akt expression in eNOS^−/− mice compared with WT mice after exposure to mechanical stress. We specifically evaluated the level of phosphor-Akt, a survivor signal in response to a variety of stresses, in our 4-h VALI model by immunoblotting. (A) Consistent with our data above, decreased phopho-Akt was blunted in WT mice at the end of 4 h HV_T MV, while eNOS^−/− mice showed a significant further decrease of phosphorylated Akt in response to 4 h HV_T over the attenuated expression of phospho-Akt when exposed to LV_T. (B) Densitometry of phopho-Akt normalized to total Akt. n=3–4 mice per condition. ^bP<0.05 vs LV_T; ^eP<0.05 vs eNOS^−/− mice.

Figure 6

Wortmannin (Wort) pretreatment aggravates the pulmonary vascular leakage in WT mice induced by 4 h HV_T MV. C57BL/6J mice (WT) were pretreated with Wort (100 nmol/L, given intraperitoneally), a selective PI3Kase inhibitor, 1 h prior to exposure to MV. Wort itself dose not have effect on EBD assay (data not shown). Exposure of animals to LV_T on the other hand caused no changes in these values as compared to controls (data not shown). However, Wort pretreatment significantly enhanced EBD lung accumulation in C57BL/6J mice caused by 4 h HV_T MV. n=4–6 mice/experimental condition. ^bP<0.05 vs LV_T; ^eP<0.05 vs HV_T MV.

Figure 7

The morphological review of the lungs after pretreatment with Wort and mechanical ventilation. (A) Neutrophil immunostaining: the presence of parenchymal neutrophil was assessed using specific anti-neutrophil antibody. Exposure to 4 h HV_T MV produced an increased staining of neutrophils in the interstitium and alveolus in WT mice (c, arrow) compared to LV_T and Wort only (a and b). However, Wort — a specific inhibitor of PI3K--pretreatment exacerbated this effect (d, arrow). (B) Hematoxyline and eosin stain of lung tissue (×40): the presence of neutrophil infiltration in WT mice lungs when exposed to HV_T for 4 h is similar as seen on the neutrophil immuno-staining but with occasional alveolar hemorrhage (g, arrow) compared to LV_T and Wort only (e and f); Wort worsened the lung injury compared to that in animals ventilated alone (h, arrow). There were no striking histological changes in lungs of C57BL/6J mice exposed to LV_T as compared with control animals (results not shown).

Figure 8

Inhibition of p38 MAP kinase prevents protein leakage in response to HV_T MV. Mice were treated with the p38 MAP kinase inhibitor SB203580 (2 mg/kg, ip) 1 h prior to exposure to 1–2 h HV_T MV. The data showed that the lung capillary leakage was attenuated by using the index of BAL protein at the end of 2 h HV_T MV. n=4. ^bP<0.05 vs LV_T; ^eP<0.05 vs 2 h HV_T MV.

Figure 9

The effect of SB203580(SB) on the activation of Akt in WT mice exposed to 4 h mechanical ventilation. Western blotting on the mice lung exposed to HV_T MV for 4 h demonstrated that the inhibition of p38 MAPK with SB203580 increased the activation of Akt (serine 473 phosphorylation), indicating interplay between these two signal pathways. n=3–4 mice per condition. ^bP<0.05.

Figure 10

The effect of PI3K inhibitor, 20 nmol/L Wort, on the expression of phospho-Akt and phospho-p38 MAPK. Pharmacologic antagonism of PI3K with Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38 in C10, type II alveolar epithelial cells when exposed to 18% cyclic stretch (CS) from 1–2 h. A) the representative blotting; B and C) the densitometric ratio of phospho-p38/actin and phospho-Akt/actin. n=3–4. ^bP<0.05 vs Vehicle; ^eP<0.05 vs static. Note: the P-Akt bands represented a combination of the results came from several experiments.