Su1903 MicroRNA-196a As a Prognostic Marker in Pancreatic Neuroendocrine Tumor

Abstract

Background and aim: Alcohol has been associated with activation and survival of activated pancreatic stellate cells (PSC). The effect of ethanol is partly mediated by the generation of oxidative stress within the cells, but other factors are required to induce pancreatic fibrogenesis. Tobacco is recognized as an etiologic factor of chronic pancreatitis (CP) and it is related with fibrogenesis in other tissues such as heart, liver and kidney. However its effect on pancreatic fibrogenesis is still unknown. We hypothesized that tobacco in combination with alcohol stimulates pancreatic fibrogenesis by PSC activation through the generation of oxidative stress within the cells. Our aim was to evaluate the effect of tobacco alone and in combination with alcohol on the activation of PSC and production of extracellular matrix (ECM) proteins and the generation of oxidative stress within the cells. Methods: PSC were isolated from rat pancreas Sprague-Dawley and exposed to tobacco (cigarette smoke condensate) alone (0.01mg/ml) or in combination of increasing concentrations of ethanol (5 to 50mM). PSC activation (α-SMA expression) was measured in both early and primary cell culture by Western blot. Fibronectin-1 (FNT-1) was evaluated by western blot and immunochemistry. Collagen-I was measured by western blot and Masson trichrome. Oxidative stress was examined using 2'-7'-dichlorofluorescin diacetate (DCF-DA) and was detected by flow cytometry. Results are expressed as mean±SEM. Statistical analysis was carried out one-way ANOVA followed by Fisher's LSD post hoc test. Results: Tobacco alone (α-SMA 1.83±0.3; p=0.003 versus negative control) or in combination with 50 mM ethanol (α-SMA 1.53±0.2; p=0.04 versus negative control) induced PSC activation in early culture. Tobacco in combination with 50 mM ethanol increased the expression of collagen-I (3.9±1.2, p<0.001 versus negative control) and FNT-1 (3.6±0.3, p<0.001 versus negative control and ethanol alone). Tobacco also increased the expression of FNT-1 in combination of 10mM alcohol (2.23±0.1, p=0.007 versus negative control). Ethanol (50mM) alone (3.06±0.77, p=0.008 versus negative control) or in combination with tobacco (0.01mg/mL) (2.68±0.49 E50, p=0.027 versus negative control) increased the reactive oxygen species fluorescence emission. Conclusion: Tobacco alone or in combination with alcohol induces the activation of PSC. Tobacco associated with alcohol increases the expression of extracellular matrix proteins and is a potential inducer of oxidative stress. These results support for the first time the synergistic effect of alcohol and tobacco in the pathogenesis of chronic pancreatitis.