Melatonin Attenuates Pulmonary Hypertension in Chronically Hypoxic Rats

Abstract

Chronic hypoxia induces pulmonary hypertension and vascular remodeling, which are clinically relevant to patients with chronic obstructive pulmonary disease (COPD) associated with a decreased level of nitric oxide (NO). Oxidative stress and inflammation play important roles in the pathophysiological processes in COPD. We examined the hypothesis that daily administration of melatonin (10 mg/kg) mitigates the pulmonary hypertension and vascular remodeling in chronically hypoxic rats. The right ventricular systolic pressure (RVSP) and the thickness of pulmonary arteriolar wall were measured from normoxic control, vehicle- and melatonin-treated hypoxic rats exposed to 10% O₂ for 14 days. Levels of markers for oxidative stress (malondialdhyde) and inflammation (tumor necrosis factor-α (TNFα), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2)) and the expressions of total endothelial NO synthase (eNOS) and phosphorylated eNOS at serine1177 (ser1177) were determined in the lung tissue. We found that the RVSP and the thickness of the arteriolar wall were significantly increased in the vehicle-treated hypoxic animals with elevated levels of malondialdhyde and mRNA expressions of the inflammatory mediators, when compared with the normoxic control. In addition, the phosphorylated eNOS (ser1177) level was significantly decreased, despite an increased eNOS expression in the vehicle-treated hypoxic group. Melatonin treatment significantly attenuated the levels of RVSP, thickness of the arteriolar wall, oxidative and inflammatory markers in the hypoxic animals with a marked increase in the eNOS phosphorylation in the lung. These results suggest that melatonin attenuates pulmonary hypertension by antagonizing the oxidative injury and restoration of NO production.

Keywords: anti-oxidant; chronic hypoxia; inflammation; lung injury; nitric oxide.

Figure 1

Effect of melatonin on the right ventricular systolic pressure of normoxic control and chronically hypoxic rats treated with vehicle or melatonin. *, p < 0.01, n = 6, versus normoxic group. #, p < 0.01, n = 6, versus vehicle group.

Figure 2

Effect of melatonin on the thickness of smooth muscle layers of pulmonary small resistance vessels in normoxic control and chronically hypoxic rats treated with vehicle or melatonin: (A) photomicrographs of pulmonary small resistance vessels of diameters smaller than 50 μm, scale bar = 25 μm; (B) photomicrographs of pulmonary small resistance vessels of diameters between 50 and 100 μm, scale bar = 25 μm and (C) summary of the wall thickness in vessels of diameters: (i) smaller than 50 μm; and (ii) between 50 and 100 μm. *, p < 0.01, n = 6, versus normoxic group. #, p < 0.01, n = 6, versus vehicle group.

Figure 3

Effect of melatonin on lipid peroxidation on lung tissues of normoxic control and chronically hypoxic rats treated with vehicle or melatonin. *, p < 0.01, n = 6, versus normoxic group. #, p < 0.01, n = 6, versus vehicle group.

Figure 4

Effect of melatonin on gene expression of pro-inflammatory cytokine and inflammatory mediators: (A) tumor necrosis factor-α (TNFα); (B) inducible NO synthase (iNOS); and (C) cyclooxygenase-2 (COX-2), in the lung of normoxic control and chronically hypoxic rats treated with vehicle or melatonin. *, p < 0.01, n = 6, versus normoxic group. #, p < 0.01, n = 6, versus vehicle group.

Figure 5

Effect of melatonin on: (A) gene expression of endothelial NO synthase (eNOS); (B) protein level of total; and (C) phosphorylated eNOS (ser1177) in the lung of normoxic control and chronically hypoxic rats treated with vehicle or melatonin. *, p < 0.01, n = 6, versus normoxic group. #, p < 0.01, n = 6, versus vehicle group.