The broad long-term objective of the work described in this application is to determine the molecular mechanisms responsible for the exocrine pancreatic disorders caused by ethanol. The work proposed is designed to determine the effect of an ethanol-containing diet on the factors that mediate pancreatitis. Our hypothesis states that a ethanol diet alone or in combination with a low dose of cholecystokinin-octapeptide (CCK-8) infusion results in activation of transcription factors involved in regulating the expression of pro-inflammatory cytokines and chemokines. These cytokines/chemokines, in turn, mediate the inflammatory and cell death responses that are the hallmarks of pancreatitis. Using a rat model of continuous infusion of an ethanol-containing diet, we proposed the following specific aims: 1. Determine the effect of the ethanol diet with and without the CCK-8 infusion on activation of transcription factors (i.e., NF- kappaB and P-1) that regulate cytokine/chemokine production in the pancreas and inflammatory and cell death responses. 2. Determine the mechanism(s) of NF-kappaB and AP-1 activation in the pancreas caused by the ethanol diet in the presence and absence of the CCK-8 infusion. 3. Determine the effect of the ethanol diet with and without the CCK-8 infusion on the expression and regulation of specific cytokines/chemokines and their role in mediating inflammatory and cell death responses in pancreatitis. 4. Determine the effect of the ethanol diet with and without the CCK-8 infusion on intrapancreatic trypsin activation and the mechanism(s) of these effects on intrapancreatic trypsin activation. Measurements. to accomplish these goals will include serum amylase and lipase, pancreatic weight, pancreatic necrosis, apoptosis, vacuolization, neutrophils and macrophages using histologic techniques: pancreatic trypsin activation; transcription factor activation using gel shift assays; and expression of cytokines/chemokines using RT-PCR. Western blot analysis, immunocytochemistry and bioassay. The results of the studies described in this application will be elucidation of the mechanism of ethanol's effects in pancreatitis that can be used to design strategies for therapeutic interventions and clinical trials.
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