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Serine proteinase inhibitors in pulmonary cells and in emphysema

Grant number: 18/11874-5
Support type:Regular Research Grants
Duration: April 01, 2019 - March 31, 2021
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Sergio Daishi Sasaki
Grantee:Sergio Daishi Sasaki
Home Institution: Centro de Ciências Naturais e Humanas (CCNH). Universidade Federal do ABC (UFABC). Ministério da Educação (Brasil). Santo André , SP, Brazil

Abstract

COPD (Chronic obstructive pulmonary disease) is one of the major causes of morbidity and mortality in the world, there is not an effective treatment that revert the physio pathological damages observed in the patients. One of the causes to the development of the COPD is the smoke; however, genetic and environmental factors are involved with the disease emergence in the human. The pulmonary emphysema (pulmonary alveolar destruction) is one of the COPD manifestations. Some proteases are related with emphysema development, MMP-9, MMP-12 and serine proteases like human neutrophil elastase. In previous project founded by FAPESP (2011/07001-7), the serine protease inhibitors rBmTI-A and rBmTI-6 were tested in an emphysema model in mice. These inhibitors presented a protector effect against development of pulmonary emphysema in the mice and an anti-inflammatory effect too. rBmTI-A and rBmTI-6 are Konitz-BPTI like inhibitors and originally they were purified from Rhipicephalus microplus tick. rBmTI-A presents two Kunitz-BPTI type inhibitory domains and inhibit neutrophil elastase, bovine trypsin, plasmin and human plasma Kallikrein. rBmTI-6 presents three Kunitz-BPTI type inhibitory domains and inhibits bovine trypsin and plasmin. As in the previous project we use the entire rBmTI-A (expressed with the two domains), in the present work we propose to clone, express and purify each rBmTI-A domain (d1 and d2) separated, after these the domains will be tested in pulmonary emphysema model in mice to attempt evaluate the potential of each domain to avoid emphysema development. This evaluation will be performed by mean linear intercept (Lm) measurement of pulmonary alveoli, leucocyte quantification form broncoalveloar lavages (BALs), proteolytic activities and cytokines quantification in BALs, and, elastic and collagen fibers quantification in the lungs of experimental mice. The second part of this project consists in investigation of the anti-inflammatory effect of rBmTI-A and rBmTI-6 toward human pulmonary epithelial cells. We will use two cellular lineages, A549 that will be induced to inflammation process using porcine pancreatic elastase and, BEAS-2B that will be induced to inflammation process using LPS. In the both cellular lineages will be quantified the production of prop-inflammatory cytokines from the cells by qPCR and ELISA. Additionally we will investigate the possible involvement of PAR-1 and PAR-2(protease activated receptors 1 or 2) in the response of the inhibitors (anti-inflammatory effect) toward A549 and BEAS-2B cells. (AU)