A.B. Kannan1, S. Iyer1, T. Jain1, P. Sahay1, S. Bhandari1, V. Dudeja1 1University Of Alabama at Birmingham, General Surgery, Birmingham, Alabama, USA
Introduction: Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas with no known cure. Notch pathway, evolutionarily conserved developmental pathway is known to be activated in various fibrotic conditions, but it's role in CP remains unclear. Here, we evaluate the role of Notch signaling in CP. Furthermore, we delineate the role of acinar-cell specific Notch signaling in CP progression.
Methods: CP was induced in C57BL6 mice by repeated injections of L-arginine (Schematic Fig A). Mice were subsequently randomized to receive Notch-pathway inhibitor (DAPT, 50mg/kg, oral gavage) or Vehicle control treatment for 4 weeks. Mice were euthanized and the pancreata were evaluated by H&E staining, RNA-Fluorescent in situ hybridization (RNA-FISH), qPCR, immunofluorescence (IF) and flow cytometry. In order to evaluate the role of acinar-cell specific Notch signaling, we developed a double transgenic (DTG) Ptf1aCre-ERT;RBPJ flox/flox mouse, where tamoxifen administration leads to conditional loss of Notch signaling in acini under the control of Ptf1a-cre. L-arginine CP was induced in DTG mice and two weeks later, tamoxifen (i.p., 5 days) was administered. Littermate Ptf1aCre-ve RBPJ flox/flox mice were used as controls. Mice were euthanized two weeks after the last tamoxifen injection and pancreata were harvested and analyzed.
Results: IF studies demonstrated upregulation of Notch signaling upon induction of CP. Further, RNA-FISH indicated that this was a multi-compartment phenomena with upregulation in acini, acinar-to-ductal metaplasia cells(ADM) and pancreatic stellate cells (PSCs). DAPT treatment significantly improved pancreatic histology (Fig B) and atrophy (Fig C), with attenuated pancreatic inflammation and fibrosis. In the DTG mice, acinar-specific loss of Notch signaling was confirmed in Cre-positive mice with RNA-FISH. However, this had no effect on CP progression as the pancreas-to-body weight ratio showed no significant differences between the Cre-positive and the Cre-negative groups. Similar acinar cell content and fibrosis was seen on histological evaluation. Immunohistochemistry and qPCR revealed no significant differences in immune infiltration and pro-fibrotic gene expression respectively.
Conclusion: Notch pathway is activated in CP and its inhibition leads to improvement in pancreatic atrophy, fibrosis and inflammation. However, Notch signaling in acini does not seem to play a role in CP progression. Further studies involving compartment-specific deletion of Notch are required to comprehensively understand its role in CP pathogenesis. Notch inhibition can emerge as a novel therapeutic strategy against CP.