01.17 Apigenin Limits Pancreatic Injury in Recurrent Acute Pancreatitis by Inhibiting MAPK Pathway Activation

A. A. Mrazek1, M. Falzon2, J. Zhou2, M. R. Hellmich1, C. Chao1 1University Of Texas Medical Branch,Surgery,Galveston, TEXAS, USA 2University Of Texas Medical Branch,Pharmacology And Toxicology,Galveston, TEXAS, USA

Introduction: Chronic pancreatitis (CP) is progressive disease involving irreversible histologic damage and pancreatic insufficiency. CP is the result of repeated acute pancreatitis RAP, where damaged pancreatic tissue is replaced with scar. There is no cure for CP, and current treatment options are limited to supportive care and symptom palliation rather than targeting disease pathogenesis. Development of a novel therapeutic that reduces the severity of RAP would thereby prevent or delay progression to CP. We have found that the naturally-occurring flavanoid, apigenin, induced a protective phenotype in a preclinical mouse model of RAP, preserving pancreatic architecture and limiting stromal fibrosis. As a potential molecular target, we investigated the effect of apigenin on MAPK pathway activation during pancreatitis.

Methods: RAP was induced in 6-8 week-old C57BL/6 mice (Harlan Laboratories, Houston, TX) using serial cerulein (CR) injections: 50 μg/kg, 5 hourly intraperitoneal (IP) injections, 3 d/wk, for 4 wks (Bachem, Torrance, CA):. Apigenin treatment was initiated after one week of RAP-induced pancreatitis: 50 μg, oral gavage, once daily, 6 d/wk, for 3 wks (Sigma-Aldrich, St. Louis, MO). Control mice received the vehicles (saline IP, and 0.5% methylcellulose with 0.025% Tween20 by oral gavage) following the same schedule. Mouse pancreata were harvested, formalin-fixed, paraffin-embedded, and sectioned. Immunohistochemistry was performed using a heat-mediated antigen retrieval method, avidin/biotin blocking, phospho-extracellular signal-regulated kinase (pERK) antibody (Cell Signaling, Danvers, MA), VECTASTAIN Elite ABC kit and DAPI-containing mounting medium (Vector Lab, Burlingame, CA). Slides were counter-stained with hematoxylin (Thermo Fisher Scientific, Kalamazoo, MI). Ten non-overlapping 400x images of each pancreas and analyzed using the validated free-ware ImmunoRatio program, which calculated the percentage of positively DAB-stained nuclei. Nuclear translocation of pERK served as conjugate for MAPK pathway activation. Statistical analysis consisted of one-way ANOVA and post-hoc Tukey’s test.

Results: Basal pERK expression was predominately cytoplasmic within the vehicle and apigenin groups. CR-induced RAP resulted in significant MAPK pathway activation, i.e. pERK translocation to the nucleus (nuclear positivity). Daily 50 μg doses of apigenin, while continuing to induce RAP, resulted in a significant reduction of pERK nuclear translocation (p < 0.001) compared to pancreata treated with CR alone.

Conclusion: Apigenin inhibited MAPK pathway activation by limiting pERK nuclear translocation. The down-stream effector of the MAPK pathway, pERK, may serve as a transcription factor for the expression of genes regulating cell proliferation, differentiation, and apoptosis. Thus, down-regulation of the MAPK/ERK pathway is one of apigenin’s molecular mechanism by which pancreatic injury is reduced during RAP.