cathepsin-g and Pulmonary-Edema

This study aims to evaluate the role of chymostatin in paraquat-induced acute lung injury. Institute of Cancer Research mice were randomly distributed into the NS, DMSO, chymostatin, paraquat or chymostatin treatment groups. Six mice from each group were intraperitoneally injected with chloral hydrate at 0, 1, 2, 4, 8, 12, 24 and 48 h after treatment administration. Blood samples were collected through cardiac puncture. Lung tissues were stained with haematoxylin and eosin for the observation of lung histology. The degree of pulmonary oedema was determined on the basis of lung wet-to-dry ratio (W/D). The serum activity of cathepsin G was determined through substrate fluorescence assay. The serum levels of endothelial cell-specific molecule-1 (endocan), tumour necrosis factor-a (TNF-a), interleukin-1β (IL-1β), IL-6 and high-mobility group box protein 1 (HMGB1) were determined through enzyme-linked immunosorbent assay. The expression levels of endocan and nuclear NF-κBp65 in the lung were quantified through Western blot. Chymostatin alleviated the pathological changes associated with acute alveolitis in mice; decreased the lung W/D ratio, the activity of cathepsin G and the serum concentrations of TNF-a, IL-1β, IL-6 and HMGB1; and increased the serum concentration of endocan. Western blot results revealed that chymostatin up-regulated endocan expression and down-regulated nuclear NF-κBp65 expression in the lung. Chymostatin reversed the inflammatory effects of paraquat-induced lung injury by inhibiting cathepsin G activity to up-regulate endocan expression and indirectly inhibit NF-κBp65 activity.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Cathepsin G; Cytokines; Cytoprotection; Disease Models, Animal; Female; HMGB1 Protein; Inflammation Mediators; Lung; Mice, Inbred ICR; Oligopeptides; Paraquat; Proteoglycans; Pulmonary Edema; Time Factors; Transcription Factor RelA

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

Topics: Amino Acid Sequence; Animals; Caesalpinia; Cathepsin G; Cats; Electrophoresis, Polyacrylamide Gel; Leukocyte Elastase; Myeloblastin; Protease Inhibitors; Pulmonary Edema; Seeds; Serine Endopeptidases; Serine Proteinase Inhibitors