Ghrelin therapy improves lung and cardiovascular function in experimental emphysema

Abstract

Background: Emphysema is a progressive disease characterized by irreversible airspace enlargement followed by a decline in lung function. It also causes extrapulmonary effects, such as loss of body mass and cor pulmonale, which are associated with shorter survival and worse clinical outcomes. Ghrelin, a growth-hormone secretagogue, stimulates muscle anabolism, has anti-inflammatory effects, promotes vasodilation, and improves cardiac performance. Therefore, we hypothesized that ghrelin might reduce lung inflammation and remodelling as well as improve lung mechanics and cardiac function in experimental emphysema.

Methods: Forty female C57BL/6 mice were randomly assigned into two main groups: control (C) and emphysema (ELA). In the ELA group (n=20), animals received four intratracheal instillations of pancreatic porcine elastase (PPE) at 1-week intervals. C animals (n=20) received saline alone (50 μL) using the same protocol. Two weeks after the last instillation of saline or PPE, C and ELA animals received ghrelin or saline (n=10/group) intraperitoneally (i.p.) daily, during 3 weeks. Dual-energy X-ray absorptiometry (DEXA), echocardiography, lung mechanics, histology, and molecular biology were analysed.

Results: In elastase-induced emphysema, ghrelin treatment decreased alveolar hyperinflation and mean linear intercept, neutrophil infiltration, and collagen fibre content in the alveolar septa and pulmonary vessel wall; increased elastic fibre content; reduced M1-macrophage populations and increased M2 polarization; decreased levels of keratinocyte-derived chemokine (KC, a mouse analogue of interleukin-8), tumour necrosis factor-α, and transforming growth factor-β, but increased interleukin-10 in lung tissue; augmented static lung elastance; reduced arterial pulmonary hypertension and right ventricular hypertrophy on echocardiography; and increased lean mass.

Conclusion: In the elastase-induced emphysema model used herein, ghrelin not only reduced lung damage but also improved cardiac function and increased lean mass. These findings should prompt further studies to evaluate ghrelin as a potential therapy for emphysema.

Keywords: Cardiac function; Experimental emphysema; Ghrelin therapy; Lung function; Lung inflammation; Lung remodelling.

Fig. 1

Schematic flow chart (a) and timeline of study design (b). C group: intratracheal instillation of 50 μL of saline, ELA: intratracheal instillation of 0.2 UI of pancreatic porcine elastase (PPE) once a week during 4 weeks, Sal: i.p. injection of saline (50 μL), Ghr: i.p. injection of ghrelin (200 μg kg^-1 per day)

Fig. 2

Representative photomicrographs of lung parenchyma stained with haematoxylin–eosin. C: control; ELA: elastase-induced emphysema; Sal: i.p. injection of saline; Ghr: i.p. injection of ghrelin. Original magnification ×200. Bars = 100 μm

Fig. 3

Elastic fibre content in alveolar septa (a) and representative photomicrographs of lung parenchyma stained with Weigert's resorcin fuchsin method with oxidation (elastic fibres) (b). Arrows: Elastic fibres (stained black). C: control; ELA: elastase induced emphysema; Sal: i.p. injection of saline; Ghr: i.p. injection of ghrelin. Boxes show the interquartile range (25^th-75^th percentile), whiskers encompass the range (minimum–maximum), and horizontal lines represent the median in 10 animals/group. * vs. C-Sal. # vs. ELA-Sal

Fig. 4

Collagen fibre content in alveolar septa (a) and pulmonary vessel wall (b). Representative photomicrographs of alveolar septa (c) and pulmonary vessels (d), stained with the Picrosirius-polarization method. Note the enlargement of alveolar space in the ELA-Sal group. C: control; ELA: elastase-induced emphysema; Sal: i.p. injection of saline; Ghr: i.p. injection of ghrelin. Boxes show the interquartile range (25^th–75^th percentile), whiskers encompass the range (minimum–maximum), and horizontal lines represent the median in 10 animals/group. * vs. C-Sal. # vs. ELA-Sal

Fig. 5

Transmission electron microscopy of lung parenchyma in control (C) animals treated with i.p. saline (Sal, a, b, and c) or ghrelin (Ghr, d, e, and f), as well as elastase-induced emphysema (ELA) mice treated with i.p. saline (g, h, and i) or ghrelin (j, k, and l). Note normal alveolar epithelium (type 2 epithelial cell, P2) and intact alveolar septa (AS) and capillary membrane (Cap) in C-Sal (a-c) and C-Ghr (d-f). Activated macrophages (Mac*) with lysosomes and glycogen accumulation can be visualized in the alveolar space in ELA-Sal (i). ELA-Sal animals show rupture of alveolar septa with loss of capillaries and increased collagen fibre content (in AS) (g and h). After ghrelin therapy, there is visible repair of the capillary (j), proliferation of type 2 epithelial cells (P2) (k), suggesting epithelial repair, as well as activated macrophages with lysosomes and glycogen accumulation (l)

Fig. 6

Immunohistochemistry for surfactant protein (SP)-D. Boxes show the interquartile range (25^th–75^th percentile), whiskers encompass the range (minimum–maximum), and horizontal lines represent the median in 10 animals/group. * vs. C-Sal. # vs. ELA-Sal

Fig. 7

Immunohistochemistry for F4/80 (total macrophages (a), iNOS (M1 macrophages (b) and arginase-1 (M2 macrophages (c). C: control; ELA: elastase-induced emphysema; Sal: i.p. injection of saline; Ghr: i.p. injection of ghrelin. Boxes show the interquartile range (25^th–75^th percentile), whiskers encompass the range (minimum–maximum), and horizontal lines represent the median in 10 animals/group. * vs. C-Sal. # vs. ELA-Sal

Fig. 8

Levels of keratinocyte-derived chemokine (KC, a mouse analogue of interleukin-8), tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β, and interleukin-10. Levels in lung tissue corrected by Bradford’s method. The non-parametric Mann–Whitney test was used to evaluate between-group differences. Boxes show the interquartile range (25^th-75^th percentile), whiskers encompass the range (minimum–maximum), and horizontal lines represent the median in 10 animals/group. * vs. C-Sal. # vs. ELA-Sal

Fig. 9

Right ventricular (RV) end-diastolic area (a) and pulmonary artery acceleration time/pulmonary artery ejection time (PAT/PET) ratio (b). Representative images of RV area on short-axis B-dimensional views of both ventricles (c). LV: left ventricle. Representative images of pulmonary blood flow (d). A.U.: arbitrary units; C: control; ELA: elastase-induced emphysema; Sal: i.p. injection of saline; Ghr: i.p. injection of ghrelin. Bars are means ± SD of 10 animals per group. * vs. C-Sal. # vs ELA-Sal