Protease Inhibitors Extracted from Caesalpinia echinata Lam. Affect Kinin Release during Lung Inflammation

Abstract

Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases.

Figure 1

PMN quantification in BALF. Rats were pretreated intravenously with buffer (negative or positive controls), CeKI (7.8 or 2.6 mg), or rCeEI (2.6 or 0.84 mg). After 20 min, they received 75 μg LPS/animal (positive control and CeKI and rCeEI groups) or buffer (negative control) injected via the trachea directly into their lungs. Six hours later, lungs were collected and washed with buffer (BALF). BALF (20 µL) was mixed with 0.4% Trypan Blue. Total and differential leukocyte cells were counted manually with a Neubauer chamber using optical microscopy. ^∗Significant difference compared to negative control (ρ < 0.05). ^#Significant difference compared to positive control (ρ < 0.05).

Figure 2

Kinin release in BALF, plasma, and lungs. Rats were pretreated intravenously with buffer (negative or positive controls), CeKI (7.8 or 2.6 mg), or rCeEI (2.6 or 0.84 mg). After 20 min, they received 75 μg of LPS/animal (positive control and CeKI and rCeEI groups) or buffer (negative control) injected via the trachea directly into their lungs. Six hours later, blood was collected, BALF was obtained, and lungs were extracted. Kinin was extracted from BALF (a), plasma (b), or homogenized lung (c) using a treatment with ethanol and then water, acetone, and petroleum ether. For kinin quantification, a radioimmunoassay was performed according to Shimamoto et al., 1978, with some modifications [21]. The experiment was performed twice and radiation values were converted into kinin (pg) using a standard curve. ^∗Significant difference compared to negative control (ρ<0.05). ^#Significant difference compared to positive control (ρ < 0.05). ^&Significant difference compared to CeKI-treated groups (ρ < 0.05).

Figure 3

ACE activity in lungs. Rats were pretreated intravenously with buffer (negative or positive controls), CeKI (7.8 or 2.6 mg), or rCeEI (2.6 or 0.84 mg). After 20 min, they received 75 μg of LPS/animal (positive control and CeKI and rCeEI groups) or buffer (negative control) injected via the trachea directly into their lungs. Six hours later, lungs were extracted and homogenized. Samples of lung (5 µL) were maintained in 50 mM Tris buffer at pH 7.4 containing 50 mM NaCl for 5 min at 37°C before the addition of the substrate Abz-F-R-K(Dnp)-P-OH (10 µM) in a final volume of 200 µL. Fluorescence changes were monitored continuously for 30 min at λ _ex = 320 nm and λ _em = 420 nm. The slope of the generated fluorescence signal was converted into micromoles of substrate hydrolyzed per minute based on a calibration curve obtained from the complete hydrolysis of peptide and adjusted for total protein quantity. ^∗Significant difference compared to negative control (ρ < 0.05). ^&Significant difference compared to CeKI-treated groups (ρ < 0.05).

Figure 4

Potential sources of kinin in LPS-induced lung inflammation model. Kinin may be generated in lung via kininogen hydrolysis and is rapidly degraded by kininases. This peptide can be released into the alveolar space through distinct pathways: (a) in neutrophil-bound kininogen cleavage by plasma kallikrein, (b) kininogen hydrolysis by NE and PR3, or (c) diffusion from the lung interstitium to the alveolar space. In the plasma, pK, PR3, and NE all release kinin. Additionally, kinin might be exchanged between plasma, lung, and alveolar space. K: kininogen (high-molecular-weight, low-molecular-weight, and/or T-kininogen); pK: plasma kallikrein; NE: neutrophil elastase; PR3: proteinase 3; BALF: bronchoalveolar lavage fluid; LKT: leukotrienes; PAF: platelet activator factor.