A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (K
Amino Acid Sequence ; Animals ; Leeches/chemistry* ; Pancreatic Elastase/antagonists & inhibitors* ; Protease Inhibitors/pharmacology* ; Proteins

OBJECTIVEPulmonary hypertension is a proliferative vascular disease characterized by pulmonary vascular structural remodeling. Until now, the pathogenesis of pulmonary hypertension is still not fully understood. Although considerable progress has been made, there is, to date, no cure for advanced pulmonary vascular disease. Recently, a number of studies suggest that endogenous vascular elastase (EVE) play a role in the vascular changes associated with pulmonary hypertension. The purpose of the study was to determine whether an elastase inhibitor might reverse advanced pulmonary vascular disease produced in rats by injection of monocrotaline.

METHODSOne hundred and twenty male Sprague-Dawley rats were used in this study. The rats were divided into three groups: control, model and ZD-0892 groups. In the model and ZD-0892 groups, the rats were subjected to a single subcutaneous injection of monocrotaline (60 mg/kg) in the hind flank, while the rats in control group received an equivalent volume of 0.9% saline. From day 21, the rats in the ZD-0892 and model groups received twice-daily gavage tube feedings of either ZD-0892 at a dose of 240 mg/kg per day or its administration vehicle, while the rats in control group were subjected to an equivalent volume of 0.9% saline. On days 21, 28 and 35 post-injection, the elastolytic activity was measured with a fluorescence microplate reader and pulmonary artery pressure was detected via catheterization. Meanwhile, the lungs were evaluated morphologically, using the barium-gelatin perfusion technique.

RESULTSThe injection of monocrotaline led to severe pulmonary hypertension in rats 21 days later and pulmonary artery elastolytic activity increased remarkably. A 1-week treatment with ZD-0892 resulted in declines in elastase activity. This was associated with significant declines in pulmonary artery pressure, decreases in muscularization of peripheral arteries and reductions in medial hypertrophy. After 2 weeks, elastase activity returned to normal level. Pulmonary artery pressure and structure were normalized.

CONCLUSIONIncreased elastase activity is important in the development of vascular changes and progressive pulmonary hypertension. ZD-0892 can suppress the elastase activity and completely reverse the fatal pulmonary hypertension induced by monocrotaline in rats.

Animals ; Hypertension, Pulmonary ; chemically induced ; drug therapy ; Male ; Monocrotaline ; toxicity ; Pancreatic Elastase ; antagonists & inhibitors ; Pulmonary Artery ; drug effects ; pathology ; physiopathology ; Pyrroles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonamides ; pharmacology

Endothelin (ET) receptor antagonists have been developed to produce a reduction of ET related effects in various diseases, as well as in animal models of airway inflammation. We aimed to investigate the anti-inflammatory potential of bosentan on a rat model of emphysema. Thirty Wistar male rats were classified as control group (group 1), intratracheally (i.t.) instilled with saline, treated with vehicle solution; elastase group (group 2), i.t. instilled with porcine pancreatic elastase (PPE), treated with vehicle solution; and PPE+bosentan group (group 3), i.t. instilled with PPE, treated with bosentan. The levels of TNF-alpha, IL-1beta, IL-6, and IL-8 in bronchoalveolar lavage fluid (BALF) and lung tissue, cell counts in BALF, and histologic analysis of all groups were evaluated. Neutrophile granulocytes (NG) and alveolar macrophages (AM) were increased more in group 2 than in group 1 (P<0.001, P=0.04, respectively). Compared with group 2, neutrophil granulocyte (NG) and alveolar macrophages (AM) counts were decreased in group 3 (P< 0.001). Histological examination confirmed a diffuse neutrophilic inflammation and irregular alveolar air space enlargement in group 2. Treatment with bosentan partially reduced the enlarged lung volumes. Compared with group 1, the BALF levels of TNF-alpha and IL-6, and the lung tissue levels of IL-1beta, IL-6, and IL-8 were increased in group 2 (P=0.028, P=0.005, P=0.001, P=0.019, P<0.001, respectively). The TNF-alpha and IL-8 levels of BALF (P=0.007, P=0.001, respectively), and the TNF-alpha, IL-1beta, IL-6, and the IL-8 levels of lung tissue (P=0.031, P=0.017, P=0.007, P<0.001) were decreased in group 3 compared to group 2. In conclusion, bosentan decreased the inflammatory response by reducing numbers of inflammatory cells and proinflammatory cytokines.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Bronchoalveolar Lavage Fluid/cytology/immunology ; Cytokines/*biosynthesis ; Disease Models, Animal ; Emphysema/*drug therapy/etiology/immunology/pathology ; Inflammation Mediators/metabolism ; Lung/drug effects/immunology/pathology ; Male ; Pancreatic Elastase/administration & dosage/toxicity ; Rats ; Rats, Wistar ; Receptors, Endothelin/*antagonists & inhibitors ; Sulfonamides/*pharmacology