Oral Presentation The Pancreas Summit 2025

RAGE Inhibitor Azeliragon Reduces Pyroptosis in Severe Acute Pancreatitis by Blocking the Extracellular Histones/LPS-RAGE Axis (125900)

Qiqi Wang 1 2 , Shiyu Liu 1 , Lijia Huang 1 , Wenjuan Luo 1 , Yuying Li 1 , Diane Latawiec 2 , Hao Yang 3 , David Fernig 4 , Qing Xia 1 , Robert Sutton 2 5 , Peter Szatmary 2 5 , Tingting Liu 1 , Wei Huang 1
  1. West China Centre of Excellence for Pancreatitis, Institute of Integrated Traditional Chinese and Western Medicine, West China hospital, Sichuan university, Chengdu, China
  2. Liverpool Pancreatitis Research Group, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
  3. Division of Liver Surgery and NHC Key Lab of Transplant Engineering and Immunology, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu, China
  4. Department of Biochemistry, Cell and Systems Biology, Institute of Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
  5. Liverpool University Hospitals NHS Foundation Trust, University of Liverpool, Liverpool, UK

Background: Extracellular histones (eHIS) act as damage-associated molecular patterns (DAMPs); the receptor for advanced glycation end-products (RAGE) binds diverse ligands including DAMPs and lipopolysaccharide (LPS), triggering inflammatory cascades. Intestinal barrier injury inevitably occurs in severe acute pancreatitis (SAP) which facilitates LPS translocation and further exacerbates severity. In this study, we investigated: (1) whether eHIS binds to RAGE and LPS to synergistically provoke pyroptosis and inflammation; (2) whether Azeliragon, a small-molecule RAGE inhibitor, could inhibit eHIS/LPS-RAGE axis to ameliorate severity of SAP.

Methods: Binding interactions between core histones, soluble RAGE (sRAGE), and LPS were measured using bio-layer interferometry. Intracellular colocalisation was assessed via confocal microscopy in mScarlet-RAGE-transfected HEK293T cells incubated with FITC-labelled histones. The effects of histones, LPS, and/or Azeliragon on cell death in freshly isolated mouse pancreatic acinar cells (PACs) were analysed. GSDMD expression and IL-1β release in THP-1 cells incubated with histones and/or LPS were determined. Mice received 10 intraperitoneal injections of 100 μg/kg caerulein at hourly intervals, immediately followed by 5 mg/kg LPS to establish a SAP model (CER/LPS-SAP). Azeliragon (4 or 8 mg/kg) was intraperitoneally injected 1 hour before inducing CER/LPS-SAP. Disease severity was evaluated 12 hours after initial model induction.

Results: The dissociation constants of histone subtypes with sRAGE or LPS ranged from 7.97 × 10⁻⁹ to 6.84 × 10⁻⁸ M and 4.7 × 10⁻⁸ to 1.02 × 10⁻⁷ M, respectively. Pretreatment with Azeliragon significantly reduced histone-induced cell death in PACs. Co-culture of histones with LPS exacerbated cell death in all tested in vitro models and synergistically increased N-terminal GSDMD expression and IL-1β release compared to either alone. In the CER/LPS-SAP, serum levels of eHIS and sRAGE were elevated, correlating positively with pancreatic histopathology scores and multi-organ injury indices. RAGE expression was also upregulated in pancreatic, lung, and intestinal tissues. Azeliragon at both doses reduced pancreatic injury indices and aspartate aminotransferase levels with better effect achieved at high dose which additionally decreased serum sRAGE and RAGE expression in multi-organs.

Conclusion: Histones bind with high affinity to RAGE and LPS, indicative of three-way interaction activating pyroptosis with major impact in SAP, which is counteracted by Azeliragon.