Effect and mechanism of apelin on lipopolysaccharide induced acute pulmonary vascular endothelial barrier dysfunction

Figures

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  Figure 1. Dynamic time pulmonary function indexes and inflammatory response in ALI mice. (A) Wet/dry weight ratio of lungs in mice after LPS treatment at 0, 6, 12, 24, 48 and 72 h (n = 5–10). (B) Evans blue assay for lung vascular endothelial permeability in LPS-treated mice at 0, 6, 12, 24, 48 and 72 h (n = 6). (C) HE staining of mice lung tissues at 0, 6, 12, 24, 48 and 72 h after LPS treatment (×200 magnification, scale bar: 100 μm for all images). (D) Lung injury score at 0–72 h (n = 5). Data are presented as the mean ± SEM. *P < 0.05 vs. the control group.

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  Figure 2. Fli-1 is involved in the process of LPS-induced ALI. (A) Fli-1, SRC protein expression levels in lung tissue of mice at 0, 6, 12, 24, 48 and 72 h after LPS treatment (n = 3). Fli-1 original blots are presented in Supplementary Fig. 1. (B) Fli-1 mRNA expression level changes (n = 3). (C) Dual fluorescent protein tracking assay to detect pulmonary vascular endothelial permeability. (D) Pulmonary vascular endothelial permeability was correlated with Fli-1 protein, Fli-1 mRNA and SRC protein, as well as Fli-1 protein—SRC protein. Data are presented as the mean ± SEM. *P < 0.05 vs. the control group.

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  Figure 3. Protective effect of apelin against ALI. (A) Lung wet/dry weight ratio to detect changes in whole lung permeability after apelin control effect. (B) Evans blue assay to detect changes in pulmonary vascular endothelial permeability after apelin prevention and treatment. (C) HE staining demonstrating the inflammatory response of lung tissue after apelin control (×200 magnification, scale bar: 100 μm for all images). (D) Lung injury score on HE staining (n = 5). Data are presented as the mean ± SEM. *P < 0.05 vs. the control group, and #P < 0.05 vs. the LPS group.

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  Figure 4. Effect of apelin prevention and treatment on related proteins and genes. (A) Changes in Fli-1, APJ and adherens junction-related proteins in lung tissue after prevention and treatment of ALI mice by apelin, detected by western blot (n = 3–4). The bar graphs represent the statistical results of the grayscale values of each group of proteins. (B) Effect of apelin prevention and treatment on VE-cadherin internalization (n = 4). (C) qRT-PCR detection of apelin on Fli-1 and adherens junction-related protein mRNA levels in lung tissue of ALI mice after prevention and treatment (n = 4). Data are presented as the mean ± SEM. *P < 0.05 vs. the control group, and #P < 0.05 vs. the LPS group.

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  Figure 5. Functional indexes and morphological changes of pulmonary permeability in ALI mice affected by apelin after Fli-1 inhibition. (A) Western blot of Fli-1 used to evaluate the inhibitory effect of irinotecan on Fli-1. (B) Evans blue assay to evaluate the effect of irinotecan on vascular endothelial permeability. (C) Changes in pulmonary vascular endothelial permeability after inhibition of Fli-1, evans blue assay for apelin effect in ALI mice (n = 8). (D) Effect of apelin on lung wet/dry weight ratio in ALI mice after inhibition of Fli-1 (n = 4). (E) HE staining to observe the inflammatory response of lung tissue treated with apelin in ALI mice, after inhibition of Fli-1 (×200 magnification, scale bar: 100 μm for all images). (F) Lung injury score on HE staining (n = 5). (G) Levels of inflammatory factor IL-6 in BALF (n = 4). (H) Levels of inflammatory factor TNF-α in BALF (n = 4). (I) Tissue immunofluorescence staining of VE-cadherin in pulmonary vascular endothelium (×200 magnification, scale bar: 100 μm for all images). Data are presented as the mean ± SEM. *P < 0.05 vs. the control group, #P < 0.05 vs. the LPS group and $P < 0.05 vs. the LPS + apelin group. (J) Relative…

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  Figure 6. The effects of apelin on pulmonary vascular endothelial adherens junction-related proteins and genes in ALI mice after inhibition of Fli-1. (A) Effect of apelin on lung tissue Fli-1, APJ and adherens junction-related proteins in ALI mice by western blot after inhibition of Fli-1. (B) Inhibition of Fli-1, the degree of internalization of VE-cadherin in each group (n = 3). (C) qRT-PCR to detect the effect of apelin on mRNA levels of Fli-1 and adherens junction-related proteins in lung tissue of ALI mice after Fli-1 inhibition (n = 4). Data are presented as the mean ± SEM. *P < 0.05 vs. the control group, #P < 0.05 vs. the LPS group and $P < 0.05 vs. the LPS + apelin group.