ConA e ConBr previnem aumento de [Ca2+]c e necrose de células acinares pancreáticas e melhoram pancreatite aguda alcoólica experimental

Abstract

Ethanol/POA causes calcium elevations, mitochondrial collapse, necrosis of pancreatic acinar cells and acute experimental pancreatitis, It is one of the best models characterized for the study of acute alcoholic pancreatitis, which until now has no cure or effective treatment. In this sense, the ConA and ConBr lectins have important biological activities of cellular and systemic protection in several disease models, and, therefore, present themselves as a potential therapeutic alternative. The objective of this study was to evaluate the effect of ConA and ConBr on in vitro and in vivo pancreatic injury caused by Ethanol/POA. Acinar cells were isolated from pancreas of male Swiss mice and human, incubated with ConA or ConBr (10 μg/ml, 1h), followed by incubation with Ethanol/POA (100 μM, 30 min). In order to analyze the calcium channel participation, tapsigargine (2 μM, Ca2+ pump of the Endoplasmic Reticulum-SERCA) was used. In order to evaluate the lectin domain, the lectins were incubated for 1 h with α-methyl-mannoside (α-MM; 0.1M). The analyzes were carried out using fluorophores and evaluated by confocal microscope or fluorimeter. ConA or ConBr coupled to fluorescein were used to evaluate their interaction with the acinar cell. Data expressed as mean ± standard error of the mean and considered statistical when p <0.05. The necrosis caused by Ethanol/POA (24.9 ± 2.6%) in murine acinar cells, compared to control (9.0 ± 2.6%), was prevented (p<0,05) by ConA (8.1 ± 1.3%) and ConBr (9.4 ± 0.8%). This protective effect was abolished by α-MM (ConA: 19.1 ± 3.5 and ConBr: 26.9 ± 6.4%). In human acinar cells ConA (64.21 ± 7.82%) and ConBr (61.70 ± 4.70%) also prevented necrosis caused by Ethanol/POA (84.37 ± 3.45%). The increase of [Ca2+]c caused by Ethanol/POA (488.3 ± 25.07AUC), compared to control (203.3 ± 1.2 AUC), was prevented by ConA (304.7 ± 12.79 AUC) and ConBr (262.6 ± 15.42 AUC). In the evaluation of stock-operated Ca2+ channels (SOCs), the increase of [Ca2+]c levels (2090 ± 139.5 AUC) promoted by administration of 10 mM Ca2+ in cells pre-incubated with TPG, compared to control (190, 3 ± 2.8 AUC), was decreased by ConA (1395 ± 104.6 AUC) and ConBr (1132 ± 131.2 AUC). The increase of [Ca2+]c caused by interaction of Ethanol/POA with inositol triphosphate (IP3R) receptors (413.4 ± 28.30 vs Control: 193.3 ± 2.8 AUC) was decreased by ConA and ConBr (220.8 ± 08.03 e 232.0 ± 20.52 AUC, respectively). The increase of [Ca2+]mit promoted by Ethanol/POA (1248 ± 105.2 AUC), compared to control (189.4 ± 3.7), was reversed by ConA and ConBr (444.8 ± 22.86 e 440.3 ± 67.82 AUC, respectively). However, lectins did not prevent the entry of calcium into isolated mitochondria in MPTP analysis (POA: 0.32 ± 0.06 vs ConA: 0.31 ± 0.05 and ConBr: 0.32 ± 0.06). In addition, the amylase release caused by Ethanol/POA (18.7 ± 2.2) was prevented by ConA and ConBr (13.41 ± 1.1 and 13.25 ± 1.1, respectively). ConA and ConBr images coupled to fluorescein demonstrated that ConA is internalized and promotes apoptosis, whereas ConBr interacts with the cell membrane and does not exert this effect. In addition, the histopathological, inflammatory, biochemical and nociceptive processes caused by the administration of Ethanol/POA in mice were decreased by both lectins. These data together reinforce the role of these proteins as potential molecules for investigations of treatment in the course of acute alcoholic pancreatitis.