Metformin Ameliorates Inflammation and Airway Remodeling of Experimental Allergic Asthma in Mice by Restoring AMPK α Activity

Abstract

Metformin has been involved in modulating inflammatory state and inhibiting cell proliferation and angiogenesis. This study aimed to determine whether metformin alleviates airway inflammation and remodeling of experimental allergic asthma and elucidate the underlying mechanism. We sensitized and challenged mice with ovalbumin (OVA) to induce allergic asthma. During the challenge period, metformin was administered by intraperitoneal injection. By histopathological and immunohistochemical analyses, metformin-treated mice showed a significant alleviation in airway inflammation, and in the parameters of airway remodeling including goblet cell hyperplasia, collagen deposition and airway smooth muscle hypertrophy compared to those in the OVA-challenged mice. We also observed elevated levels of multiple cytokines (IL-4, IL-5, IL-13, TNF-α, TGF-β1 and MMP-9) in the bronchoalveolar lavage fluid, OVA-specific IgE in the serum and angiogenesis-related factors (VEGF, SDF-1 and CXCR4) in the plasma from asthmatic mice, while metformin reduced all these parameters. Additionally, the activity of 5'-adenosine monophosphate-activated protein kinase a (AMPKα) in the lungs from OVA-challenged mice was remarkably lower than control ones, while after metformin treatment, the ratio of p-AMPKα to AMPKα was upregulated and new blood vessels in the sub-epithelial area as evidenced by CD31 staining were effectively suppressed. These results indicate that metformin ameliorates airway inflammation and remodeling in an OVA-induced chronic asthmatic model and its protective role could be associated with the restoration of AMPKα activity and decreased asthma-related angiogenesis.

Keywords: AMPK; airway inflammation; airway remodeling; asthma; metformin.

FIGURE 1

Metformin inhibits inflammatory infiltration in lungs of OVA sensitized and challenged mice. (A). Representative images of HE-stained lung tissue sections from each group. (B). Blinded inflammation score evaluated from HE staining. (C,D). Measurement of airway wall thicknesses and smooth muscle thicknesses from HE-stained lung sections by using Image-Pro Plus software. Data were presented as mean ± SEM (n = 8 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100–200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Metformin attenuates airway remodeling in histology of OVA sensitized and challenged mice. (A). Representative images of PAS-stained and Masson’s trichrome-stained lung sections from each group. (B). Blinded scoring of percentage of PAS positive epithelial cells. (C). Blinded quantitative analyses of area of peribronchial Masson’s trichrome staining by Image-Pro Plus. Data were presented as mean ± SEM (n = 8 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100–200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Metformin has an anti-fibrotic effect in a murine model of chronic asthma. (A). Representative images of a-SMA, TGF-β and MMP-9 immunoreactivity in lung tissues. (B–D). Blinded quantitative analysis of immunohistochemical staining density [(integral optical density)/area; IOD/area] for a-SMA, TGF-β and MMP-9 by Image-Pro Plus. Data were presented as mean ± SEM (n = 8 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100–200 μm of internal diameter). All images are at ×200 magnification. Scale bar = 100 μm *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Metformin reduces multiple cytokine levels in the BALF from the asthmatic mice. (A). Comparison of total cell numbers and differential cell counts in the BALF. (B). Comparison of cytokine concentrations (IL-4, IL-5, IL-13, TNF-α, TGF-β and MMP-9) in the BALF. Data were presented as mean ± SEM. n = 8. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Metformin reduces the levels of OVA-IgE and angiogenesis related cytokines in the blood. (A). Comparison of OVA-IgE concentration in the serum. (B). Comparison of VEGF, CXCR4, and SDF-1 concentrations in the plasma. Data were presented as mean ± SEM. n = 8. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Metformin restores AMPK activity and impairs angiogenesis in the sub-epithelium in lungs of asthmatic mice. (A,B). Representative images and blinded quantitative analyses of p-AMPK immunoreactivity in lung sections from each group. The red arrow points to the vascular endothelium stained with p-AMPKα. (C,D). Representative Western blots and densitometric analyses of p-AMPK and AMPK in lung tissues from each group. (E). Representative images of microvessels with anti-CD31 immunohistochemical and immunofluorescent staining of lung sections. Red arrows point to microvessels stained with CD31. (F). Blinded quantitative analysis of the number and area of microvessels positive for CD31 immunostaining in the sub-epithelial region. (G). Blinded quantitative analysis of fluorescence intensities for CD31 staining in the sub-epithelial region. Data were presented as msean ±SEM (n = 8 animals, 20 bronchioles/3 sections of an animal, each bronchiole with 100–200 μm of internal diameter). All images are at ×400 magnification. Scale bar = 50 μm *p < 0.05, **p < 0.01, ***p < 0.001.