Ghrelin protects against lipopolysaccharide-induced acute respiratory distress syndrome through the PI3K/AKT pathway

Abstract

Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS.

Keywords: PI3K; acute respiratory distress syndrome; endothelial cells; ghrelin.

Figure 1

Ghrelin attenuates LPS-induced lung pathological alterations. Histopathological changes of lungs determined by H&E staining (×200). Male Balb/c mice were randomized into control, ghrelin, LPS, and ghrelin + LPS groups (n = 6, each group analyzed in triplicate). Lung histological changes were assessed 24 h after LPS instillation as described in Experimental procedures. A, representative images of H&E-stained sections of lung tissues for control, ghrelin, LPS, and ghrelin + LPS group. B, pathological scores of lung samples in each group are shown, indicating that treatment with ghrelin significantly ameliorates LPS-induced lung edema, hemorrhage, alveolar collapse, and inflammatory cell infiltration. Three slices of each mouse were used, and the observer randomly observed and photographed six fields of view. The observers were blinded to each experimental group. (n = 6, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group). LPS, lipopolysaccharide.

Figure 2

Effect of LPS and ghrelin on cell viability.A and B, viability of EA.hy 926 cells was estimated using the Cell Counting Kit-8 assay. Cells were cultured in DMEM with different concentration of LPS, with a median inhibitory concentration (IC₅₀) value of 150 μg/ml for 24 h. Later, cells were stimulated with 150 μg/ml LPS alone or in the presence of the indicated concentrations of ghrelin. According to the result, ghrelin at 100 nM plays a significant role on cell viability. Representative of three independent experiments, mean ± SEM, ∗p < 0.05 versus control. DMEM, Dulbecco’s modified Eagle’s medium; LPS, lipopolysaccharide.

Figure 3

Ghrelin plays an antiapoptotic effect after LPS insults in EA.hy 926 cells.A, representative image of TUNEL assay in LPS-treated cells at 24 h. Apoptotic cells are quantified by counting the percentages of TUNEL-positive nuclei. B, detection of early and late apoptosis of cells after 24-h treatment with LPS was analyzed with Annexin V-FITC and PI staining through the flow cytometer. C and D, quantitative analysis of the ratios of apoptotic cells is shown in the bar graphs. Both the TUNEL assay and flow cytometer demonstrated that addition of ghrelin diminished the ratios of TUNEL-positive cells and apoptotic cells under LPS insult condition. E, Western blot analysis shown that ghrelin increased the level of antiapoptotic protein Bcl-2 under LPS exposure, as well as reduces the level of proapoptotic protein Bax. F, quantification of Bcl-2 and Bax expression by densitometric analysis. Data are presented as the mean ± SEM, n = 4, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group. LPS, lipopolysaccharide; PI, propidium iodide.

Figure 4

Ghrelin improved endothelial cell migration and differentiation ability impaired by LPS.A, effect of ghrelin at a concentration of 100 nM on the migration of endothelial cells. Cells were divided into four groups, the control group, ghrelin group, LPS group, and ghrelin + LPS group. Yellow line delineates the margin of the recovery. Images were captured at 5× magnification. B, Matrigel tube formation assay was performed to assess the effect of ghrelin on angiogenesis. C and D, graphical representation summarizing data from four different experiments in endothelial cells. Ghrelin significantly improves the migration and tube formation of endothelial cells under LPS insult condition. N = 6, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group. LPS, lipopolysaccharide.

Figure 5

Ghrelin stabilized endothelial cell–cell adherens junctions after LPS insult in EA.hy 926 cells.A and B, transwell assay was performed to indicate pretreatment with ghrelin before insulting by LPS reduced the permeability of endothelial cell junction. B, the absorbance value of VE-cadherin in tissue. C, Western blot analysis showed that ghrelin enhanced the total protein expression of VE-cadherin under LPS insult conditions. D, the relative abundances of protein bands were quantified by measuring the corresponding band intensities; the relative values are expressed normalized to VE-cadherin signals as shown in the bar graphs. The VE-cadherin level was lower in the LPS-treated group than in the control group. As expected, the ghrelin + LPS group exhibited higher expression than in the LPS group. Values are expressed as the mean ± SEM, n = 6 per group. ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group. LPS, lipopolysaccharide; VE-cadherin, vascular endothelial cadherin.

Figure 6

PI3K/AKT activation was involved in ghrelin-mediated lung endothelial cell barrier protection in vitro. The PI3K inhibitor, LY294002 (20 μM), was added 30 min before ghrelin (100 nM) treatment. LPS was added 30 min after ghrelin treatment. A and B, Western blot analysis showed that ghrelin participated in the activation of the PI3K/AKT pathway, which was diminished under LPS insulting, and LY294002 reversed this effect. C and D, Western blot analysis showed that LY294002 inhibited the antiapoptotic protein Bcl-2 and proapoptotic protein Bax. E, the scratch-wound test indicated that the motility of endothelial cells was inhibited by adding LY294002. F, tube formation assay suggest that ghrelin promotes the vascular tube formation under LPS exposure but is reversed by the PI3K inhibitor LY294002. G and H, quantitative analysis of the wound confluence rates and numbers of tube formation are shown in the bar graphs. The data are presented as the mean ± SEM, n = 6, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group, ∗∗p < 0.05 compared with the ghrelin + LPS group. LPS, lipopolysaccharide.

Figure 7

PI3K/AKT activation was involved in ghrelin-mediated protection of lung injury in vivo.A, immunofluorescence staining of p-AKT showed that increased pAkt in the ghrelin treated but not the LPS-only treated lungs. Red (TRICT) represents p-AKT, and blue represents nuclei. Mice divided into four groups were pretreated with or without ghrelin and subjected to intranasal injection with LPS (3 mg/kg) or dimethyl sulfoxide (DMSO), including the control group, LPS group, ghrelin group, and LPS + ghrelin group. B, the absorbance value of p-AKT in cells. Three slices of each mouse were used, and the observer randomly observed and photographed four fields of view. The observers were blinded to each experimental group. Data are expressed as the mean ± SEM, n = 4, ^##p < 0.05 compared with the LPS group, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group. C, H&E staining (magnification, ×200) showed that LY294002 aggravated histological injury in LPS-induced ARDS mice (n = 6 independent mice from each group analyzed in triplicate). Mice divided into four groups were pretreated with or without ghrelin and subjected to intranasal injection with LPS (3 mg/kg) or DMSO, including the control group, LPS group, ghrelin + LPS group, and ghrelin + LY294002 + LPS group. PI3K inhibitor, LY294002, dissolved in DMSO was injected into mice 1 h before LPS or PBS instillation through the tail vein. D, lung injury scores were utilized for quantitative analysis of lung histopathologic damage. E, immunofluorescence staining of VE-cadherin showed that ghrelin reversed the LPS-induced decrease in the expression of VE-cadherin, but this effect disappeared after adding LY294002. Green (TRICT) represents VE-cadherin, and blue represents nuclei. Mice divided into five groups were pretreated with or without ghrelin and subjected to intranasal injection with LPS (3 mg/kg) or DMSO, including the control group, ghrelin group, LPS group, ghrelin + LPS group, and ghrelin + LY294002 + LPS group. F, the absorbance value of VE-cadherin in cells. Three slices of each mouse were used, and the observer randomly observed and photographed six fields of view. The observers were blinded to each experimental group. Data are expressed as the mean ± SEM, n = 6, ∗p < 0.05 compared with the control group, ^#p < 0.05 compared with the LPS group, ∗∗p < 0.05 compared with the ghrelin + LPS group. ARDS, acute respiratory distress syndrome; LPS, lipopolysaccharide; VE-cadherin, vascular endothelial cadherin.