Hexarelin modulates lung mechanics, inflammation, and fibrosis in acute lung injury

Abstract

Introduction:: Acute respiratory distress syndrome (ARDS) is an acute form of diffuse lung injury characterized by (i) an intense inflammatory response, (ii) increased pulmonary vascular permeability, and (iii) the loss of respiratory pulmonary tissue. In this article we explore the therapeutic potential of hexarelin, a synthetic hexapeptide growth hormone secretagogue (GHS), in an experimental model of ARDS. Hexarelin has anti-inflammatory properties and demonstrates cardiovascular-protective activities including the inhibition of cardiomyocyte apoptosis and cardiac fibrosis, both of which may involve the angiotensin-converting enzyme (ACE) system.

Methods:: In our experimental model, ARDS was induced by the instillation of 100 mM HCl into the right bronchus; these mice were treated with hexarelin (320 μg/kg, ip) before (Pre) or after (Post) HCl challenge, or with vehicle. Respiratory system compliance, blood gas analysis, and differential cell counts in a selective bronchoalveolar lavage (BAL) were determined 6 or 24 hours after HCl instillation. In an extended study, mice were observed for a subsequent 14 days in order to assess lung fibrosis.

Results:: Hexarelin induced a significant improvement in lung compliance and a reduction of the number of total immune cells in BAL 24 hours after HCl instillation, accompanied with a lower recruitment of neutrophils compared with the vehicle group. At day 14, hexarelin-treated mice presented with less pulmonary collagen deposition compared with vehicle-treated controls.

Conclusions:: Our data suggest that hexarelin can inhibit the early phase of the inflammatory response in a murine model of HCl-induced ARDS, thereby blunting lung remodeling processes and fibrotic development.

Fig. 1 -

Respiratory system static compliance (panel A) and oxygenation (panel B). Respiratory system static compliance derived from the pressure-volume curve construction and oxygenation (PaO₂) was measured by arterial blood from the left ventricle. A) Analysis of variance (ANOVA) in sacrifice 6 h experiment p<0.05, Tukey post hoc test; *p<0.05 vs. Healthy; ANOVA in Sacrifice 24 h experiment p<0.01, Tukey post hoc test; *p<0.05 vs. Healthy, °p<0.05 vs. Post-Hex, # p<0.05 vs. Pre-Hex. B) ANOVA in Sacrifice 6 h experiment p<0.01, Tukey post hoc test; *p<0.01 vs. Vehicle, Post-Hex and Pre-Hex; ANOVA in Sacrifice 24 h experiment p<0.01, Tukey post hoc test; *p<0.01 vs. Vehicle, Post-Hex, and Pre-Hex. Healthy: no surgical interventions or treatment (n = 5); Vehicle: HCl instillation + vehicle treatment (n = 8); Post-Hex: HCl instillation + Hexarelin treatment (n = 8); Pre-Hex: Hexarelin pretreatment + HCl instillation (n = 8).

Fig. 2 -

Peripheral and local inflammation: total white blood cells (A) and cell count in bronchoalveolar lavage (BAL) in right (panel B) and left lung (panel C). White blood cells were collected from the arterial blood and stained with Turk solution. Alveolar cells were collected by performing BAL and stained with Diff-Quik reagent solution. A) Analysis of variance (ANOVA) in Sacrifice 6 h experiment p<0.01, Tukey post hoc test; *p<0.01 vs. Healthy, °p<0.05 vs. Healthy; ANOVA in Sacrifice 24 h experiment p<0.05, Tukey post hoc test; #p<0.05 vs. Pre-Hex. B) Polymorphonuclear (PMN): ANOVA in Sacrifice 6 h experiment p = NS; ANOVA in Sacrifice 24 h experiment p<0.01, Tukey post hoc test; *p = 0.01 vs. Healthy, °p = 0.01 vs. Pre-Hex. Macrophages: ANOVA in Sacrifice 6 h experiment p = 0.02, Tukey post hoc test; *p = 0.01 vs. Healthy; ANOVA in Sacrifice 24 h experiment p<0.01, Tukey post hoc test; **p<0.01 vs. Healthy, °°p<0.05 vs. Vehicle, #p<0.01 vs. Pre-Hex. C) PMN: ANOVA in Sacrifice 6 h experiment p = NS; ANOVA in Sacrifice 24 h experiment p = 0.01, Tukey post hoc test; *p<0.05 vs. Healthy, °p<0.01 vs. Pre-Hex. Macrophages: ANOVA in Sacrifice 6 h experiment p = NS; ANOVA in Sacrifice 24 h experiment p<0.01, Tukey post hoc test; *p = 0.01 vs. Healthy, °p<0.05 vs. Pre-Hex. Healthy: no surgical interventions or treatment (n = 5); Vehicle: HCl instillation + vehicle treatment (n = 8); Post-Hex: HCl instillation + Hexarelin treatment (n = 8); Pre-Hex: Hexarelin pretreatment + HCl instillation (n = 8).

Fig. 3 -

Body weight increment in 48 hours (panel A) and in 14 days (panel B). A) Analysis of variance (ANOVA) p<0.0001, Tukey post hoc test; *p<0.01 vs. Vehicle and Hexarelin. B) ANOVA p = 0.048, Tukey post hoc test; *p<0.05 vs. Healthy. Healthy: no surgical interventions or treatment (n = 15); Vehicle: HCl instillation + Vehicle treatment (n = 18); Hexarelin: HCl instillation + Hexarelin treatment (n = 18).

Fig. 4 -

Lung fibrosis: OH-Proline lung content (A) and effects of hexarelin on lung histology (B) in acid-injured mice 14 days after lung injury induction. The collagen deposition was evaluated by the assessment of OH-proline content in lung-homogenized tissue with chloramine T and Ehrlich’s solutions. In right lung: Analysis of variance (ANOVA) p<0.05, Tukey post hoc; *p<0.05 vs. Hexarelin, °p<0.05 vs. Healthy. In left lung: ANOVA p = NS. Healthy: no surgical interventions or treatment (n = 10); Vehicle: HCl instillation + vehicle treatment (n = 13); Hexarelin: HCl instillation + Hexarelin treatment (n = 13). Histology: Representative images are shown.Tissues were prepared for Masson’s trichrome staining. (B, D, F: 40× magnification; C, E, G: 400× magnification.) (B, C) Healthy: no surgical interventions or treatment (n = 4); (D, E) Vehicle: HCl instillation + vehicle treatment (n = 3); and (F, G) Hexarelin: HCl instillation + Hexarelin treatment (n = 5). Areas of fibrosis (stained in green) are indicated with arrows; boxes on the 40× images indicate the locations of the 400× images.