Wang, Oliver H.

Abstract Background: Hemorrhagic necrotizing pancreatitis (HNP) is a highly lethal form of pancreatitis that lacks mechanism-based therapy. Intra-acinar enzyme activation is fundamental to pancreatitis initiation, yet it does not explain why only a subset of episodes progress to catastrophic hemorrhagic necrosis. Objective: To identify determinants of progression to lethal HNP and test a mechanism-based preventive intervention. Design: Using humanized trypsinogen mouse models and secretagogue regimens, we compared high-dose cerulein with low/moderate-dose cerulein and bombesin. We quantified pancreatic protease activity, performed blinded histopathologic scoring, assessed hemorrhage/vascular disruption, and evaluated enzyme localization and ductal injury by PRSS1 immunostaining. De-identified human pancreatic histology from HNP were examined. We tested a repurposed strategy using clinically approved agents for other indications: secretin to stimulate ductal fluid secretion and isosorbide mononitrate (ISMN) to promote ductal outflow via sphincter of Oddi relaxation. Results: High-dose cerulein induced robust intrapancreatic protease activation but predominantly caused edematous pancreatitis. Paradoxically, bombesin and low/moderate-dose cerulein elicited lower total pancreatic protease activity yet produced severe, frequently lethal HNP with vascular disruption and lobular ischemic necrosis. HNP was associated with intraductal enzyme accumulation and ductal injury. Secretin or ISMN alone showed limited protection, whereas combined secretin+ISMN prevented hemorrhagic necrotizing injury. Conclusion: Pancreatitis severity is determined not only by the magnitude of enzyme activation but also by where enzyme activity is exerted, implicating intraductal enzyme accumulation as a mechanistic determinant and therapeutic target in HNP. Coordinated enhancement of ductal secretion and outflow with clinically approved agents prevents HNP in vivo, supporting a translatable prevention strategy.

Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein–coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine- N -oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.