Modulation of Hypoxia-Induced Pulmonary Vascular Leakage in Rats by Seabuckthorn (Hippophae rhamnoides L.)

Abstract

Cerebral and pulmonary syndromes may develop in unacclimatized individuals shortly after ascent to high altitude resulting in high altitude illness, which may occur due to extravasation of fluid from intra to extravascular space in the brain, lungs and peripheral tissues. The objective of the present study was to evaluate the potential of seabuckthorn (SBT) (Hippophae rhamnoides L.) leaf extract (LE) in curtailing hypoxia-induced transvascular permeability in the lungs by measuring lung water content, leakage of fluorescein dye into the lungs and further confirmation by quantitation of albumin and protein in the bronchoalveolar lavage fluid (BALF). Exposure of rats to hypoxia caused a significant increase in the transvascular leakage in the lungs. The SBT LE treated animals showed a significant decrease in hypoxia-induced vascular permeability evidenced by decreased water content and fluorescein leakage in the lungs and decreased albumin and protein content in the BALF. The SBT extract was also able to significantly attenuate hypoxia-induced increase in the levels of proinflammatory cytokines and decrease hypoxia-induced oxidative stress by stabilizing the levels of reduced glutathione and antioxidant enzymes. Pretreatment of the extract also resulted in a significant decrease in the circulatory catecholamines and significant increase in the vasorelaxation of the pulmonary arterial rings as compared with the controls. Further, the extract significantly attenuated hypoxia-induced increase in the VEGF levels in the plasma, BALF (ELISA) and lungs (immunohistochemistry). These observations suggest that SBT LE is able to provide significant protection against hypoxia-induced pulmonary vascular leakage.

Figure 1

(a) Experimental design for the dose-response study to evaluate the efficacy of the extract in protection against hypobaric hypoxia-induced transvascular leakage in rats. The animals from group (ii) to (vi) were exposed to hypoxia of 9144 m for 5 h in a decompression chamber. Eight animals were used in each group. (b) Experimental design for effective dose studies. The animals from group (ii) to (vi) were exposed to hypoxia of 9144 m for 5 h in a decompression chamber except VIth batch. Eight animals were used in each group.

Figure 2

Effect of hypobaric hypoxia on lung morphology. Rats were exposed to stimulated altitude of 9144 m at 24°C for 5 h.

Figure 3

Effect of SBT LE (50, 100 and 200 mg/kg bw) on hypobaric hypoxia-induced vascular permeability. Values are mean ± SD (n = 8 per group). Significant test between groups were determined by using one-way ANOVA followed by Tukey test. *P < .05 versus Control; **P < .01 versus Control; ^# P < .05 versus Hypoxia; ^## P < .01 versus Hypoxia.

Figure 4

Prevention of extravasation of total protein and albumin by SBT LE (50, 100 and 200 mg/kg bw) in rats exposed to simulated altitude of 9144 m for 5 h. Values are mean ± SD (n = 8 per group). Significant test between groups were determined by using one-way ANOVA followed by Tukey test. *P < .05 versus Control; **P < .01 versus Control; ^# P < .05 versus Hypoxia; ^## P < .01 versus Hypoxia.

Figure 5

BALF inflammatory cytokines in SBT LE (100 mg/kg bw) treated animals exposed to simulated high altitude of 9144 m for 5 h. Values are mean ± SD (n = 8 per group). Significant test between groups were determined by using one-way ANOVA followed by Tukey test. *P < .05 versus Control; **P < .01 versus Control; ^# P < .05 versus Hypoxia; ^## P < .01 versus Hypoxia. TNF-α, tumor necrosis factor-alpha; IL-6, Interleukin-6; IL-10, Interleukin-10; MCP-1, Macrophage chemoacttractant protein-1.

Figure 6

BALF and plasma VEGF levels in control and hypoxic rats pretreated with SBT LE (100 mg/kg bw). Values are mean ± SD (n = 8 per group). Significant test between groups were determined by using one-way ANOVA followed by Tukey test. *P < .05 versus Control; **P < .01 versus Control.

Figure 7

Reduction of VEGF immunohistochemical staining after pretreatment with DEXA and SBT LE (100 mg/kg bw) in rats exposed to simulated high altitude of 9144 m for 5 h. Minimal VEGF immunostaining was evident in lung parenchyma in control animals in contrast to abundant VEGF immunostaning in the hypoxic group. Pretreatment with DEXA, SBT LE reduced VEGF staining and maintained the capillary integrity. The photographs are representative of three animals examined under each condition. Original magnification: ×200.

Figure 8

Effect of SBT LE on vascular reactivity. The animals were pretreated with DEXA and SBT LE (100 mg/kg bw). Vascular reactivity response in control and treated groups. Significant test between groups were determined by using one-way ANOVA followed by Tukey test. *P < .05 versus control. Ach, acetylcholine.

Figure 9

Mechanism of prevention of SBT LE against hypoxia-indused vascular leakage. The cross indicates that SBT LE is able to inhibit the hypoxia-induced pathophysiological process, thereby inhibiting the capillary leakage.