T. Jain1, S. Iyer1, P. Sahay1, V. Dudeja1 1University Of Alabama at Birmingham, General Surgery, Birmingham, Alabama, USA
Introduction: Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas which leads to significant morbidity in the form of chronic pain and endocrine and exocrine deficiency. The underlying pathophysiology of CP is an area of active investigation. The Hedgehog (Hh) pathway is a developmental signaling pathway that has been implicated in several fibrotic diseases. Here, we evaluate the effects of targeting Hh signaling in CP.
Methods: CP was induced in C57BL/6J male mice by intraperitoneal (IP) injections of L-Arginine (2 doses 1hr apart weekly for 4 weeks). After induction of CP, daily IP injections of Vismodegib, an FDA approved inhibitor of Smoothened, were used for inhibiting Hh pathway. DMSO was used as vehicle control. Some mice were sacrificed after conclusion of L-arginine injections (pre-treatment group) while the rest were sacrificed after 4 weeks of Vismodegib or Vehicle control treatment. Pancreata were examined by IHC, flow cytometry and qPCR. To determine early changes induced by Hh signaling inhibition, CP was induced as described above, and mice were sacrificed after 3 days of Vismodegib treatment. RNA sequencing was conducted on Day 3 pancreas to evaluate transcriptional changes induced by Hh signaling inhibition.
Results: IHC and qPCR confirmed upregulation of Hh signaling with L-arginine induced CP in the pre-treatment group. Flow cytometry revealed increased M2 macrophage (CD45+F4/80+CD206+) infiltration while PCR demonstrated increased expression of fibrotic markers including Col1A2 and TGFβ as compared to uninjured pancreas. After 4 weeks of treatment, Vismodegib group demonstrated significant decrease in M2 macrophage infiltration on flow cytometry and fibrotic marker expression on qPCR, when compared to vehicle control. Histological analysis revealed improved pancreatic atrophy upon Vismodegib treatment. Early sacrifice study (Day 3) revealed no differences in pancreatic atrophy or macrophage infiltration, however, global transcriptional changes were seen on RNA sequencing analysis with downregulation of ER stress pathway genes as well as metabolic reprogramming with decreased adipogenesis and oxidative phosphorylation related gene expression.
Conclusion: Hedgehog pathway is activated in CP and its inhibition can abrogate fibroinflammatory transformation of the pancreas with improvement in pancreatic atrophy. Transcriptional changes in metabolic signaling and ER stress pathways precede morphological changes upon Hh inhibition. Further studies are required to elucidate the mechanism underlying improvement in CP upon Hh inhibition. Hh inhibition can emerge as a novel therapeutic strategy against CP.