Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC196
The natural antioxidant cinnamtannin B-1 reduces the effects of H2O2 on CCK-8-evoked responses in mouse pancreatic acinar cells
G. M. Salido1, P. Santofimia-Castaño1, R. Rivera-Barreno1, A. Gonzalez1
1. Department of Physiology (Cell Physiology Reseacr Group), University of Extremadura, Caceres, Spain.
Intracellular Ca2+ overload and oxidant production play a key role in the intracellular activation of digestive enzymes leading to the development of pancreatitis. Cinnamtannin B-1 is a naturally occurring A-type proanthocyanidin, which belong to a class of polyphenols that is widely distributed throughout the plant kingdom. This compound shows antioxidant properties. The aim of our study was to examine the antioxidant properties of cinnamtannin B-1 against hydrogen peroxide (H2O2) effects in mouse pancreatic acinar cells. Cells were prepared by collagenase digestion of the pancreas, following previously described methods (Gonzalez et al., 1997). We have analyzed cytosolic free Ca2+ concentration ([Ca2+]c), cell oxidative state, amylase secretion and viability of pancreatic acinar cells treated with cinnamtannin B-1 in the presence of various concentrations of H2O2. Cells were loaded with the fluorescent ratiometric Ca2+ indicator fura-2 to monitor [Ca2+]c by single cell analysis of fluorescence. Cell oxidative state was measured in a spectrofluorimeter, following changes of fluorescence of CM-H2DCFDA-loaded mouse pancreatic acinar cells. Amylase release was determined using the Phadebas blue starch colorimetric method, employing a spectrophotometer. Cellular viability was determined following reduction of alamarBlue® and employing an ELISA spectrofluorimeter. Our results show that H2O2 (0.1 - 100 µM) lead to an increase in fluorescence of CM-H2DCFDA, reflecting an increase in oxidation. Cinnamtannin B-1 (10 µM) reduced H2O2-induced oxidation of CM-H2DCFDA. CCK-8 induced oxidation of CM-H2DCFDA similarly to the low micromolar concentrations of H2O2, and cinnamtannin B-1 reduced the oxidant effect of CCK-8. In addition, H2O2 induced a slow a progressive increase in [Ca2+]c. Cinnamtannin B-1 reduced the effect of H2O2 on [Ca2+]c, but only at the lower concentrations of the oxidant. CCK-8 induced a biphasic effect on amylase secretion, resulting in a maximum at 0.1 nM, and a reduction of secretion at higher concentrations. H2O2 inhibited amylase secretion in response to CCK-8. Preincubation of cells with cinnamtannin B-1 reduced the inhibitory action of H2O2 on CCK-8-evoked enzyme secretion. Finally, H2O2 reduced cell viability and the antioxidant protected acinar cells against H2O2. In conclusion, the beneficial effects of cinnamtannin B-1 appear to be mediated by reducing the intracellular Ca2+ overload, ROS production and intracellular accumulation of digestive enzymes evoked by ROS, which is a common pathological precursor that mediates acute pancreatitis. Our results support the use of natural antioxidants in the therapy against oxidative stress-derived deleterious effects on cellular physiology.
Where applicable, experiments conform with Society ethical requirements