CPA1 S282P mutation leads to chronic pancreatitis in rabbits

Abstract

Chronic pancreatitis (CP) is a progressive and irreversible fibroinflammatory disease that markedly increases susceptibility to pancreatic cancer and remains without effective targeted therapies. Among the genetic contributors to CP, the carboxypeptidase A1 p.Ser282Pro ( CPA1 ^S282P ) variant has been proposed to promote disease through misfolding-induced endoplasmic reticulum stress (ERS), although the broader pathogenic landscape remains incompletely defined. This study generated a rabbit model mimicking the human CPA1 ^S282P mutation using the SpRY-ABE-8.17 system. Homozygous CPA1 ^S282P rabbits exhibited characteristic human CP phenotypes following alcohol induction, including visceral pain, elevated serum lipase and amylase, inflammatory cell infiltration, and extensive pancreatic fibrosis. Biochemical analyses confirmed that the p.S282P mutation induced CPA1 misfolding and elevated the expression of ERS markers GRP78 and CHOP in both transfected HEK293T cells and homozygous mutant rabbits. Notably, the CPA1 ^S282P mutation markedly disrupted intra-pancreatic lipid homeostasis, contributing to the development of CP in mutant rabbits. This study successfully established the first rabbit model of CP that accurately recapitulates CP caused by a defined human point mutation. Additionally, this study provides insights into a previously unrecognized link between CPA1 and intra-pancreatic lipid metabolism, offering a foundation for identifying novel therapeutic targets for human CP.

Keywords: Carboxypeptidase A1; Chronic pancreatitis; Lipid metabolism; Point mutation; Rabbit.

Figure 1

Generation of CPA1^S282P homozygous rabbits using SpRY-ABE-8.17 system A: Greater CPA1 gene homology between rabbits (87.16%) and humans compared to mice (82.91%) (https://www.ncbi.nlm.nih.gov/gene). B: Target sequence and sgRNA design for CPA1^S282P locus. Target T-to-C base editing site is marked in red. Sanger sequencing chromatograms of genomic DNA from CPA1^S282P homozygous rabbits and controls. Black box indicates substituted nucleotide. C: Schematic illustrating establishment of a cloned CPA1^S282P homozygous rabbit model using standard microinjection procedures. D: CPA1^S282P homozygous rabbits of newly born F0 generation. E: Percent survival of CPA1^S282P rabbits and controls. F: Body weight of CPA1^S282P rabbits and controls at different ages (n=4). G: Pancreatic amylase levels in CPA1^S282P rabbits and controls (n=6). H: Pancreatic lipase levels in CPA1^S282P rabbits and controls (n=6). Survival curves, body weight, pancreatic lipase, and pancreatic amylase tracking showed no difference between CPA1^S282P and control rabbits at different ages. I: Normal histological development in pancreas of controls and CPA1^S282P rabbits at different ages by H&E staining (n=4). Per. Ident: Percentage Identity. ns: Not significant. Error bars indicate mean±SEM. Scale bars: 20 μm.

Figure 2

Alcohol-induced CP in CPA1^S282P rabbits A: Schematic showing alcohol induction experiment. B, C: Reduced exercise capacity of CPA1^S282P rabbits in open field tests, showing reduced number of steps and distance traveled within 1 h (n=4). D: Increased heart rates of CPA1^S282P rabbits after alcohol induction (n=4). E: Pancreatic amylase levels in CPA1^S282P rabbits and controls after alcohol induction (n=6). F: Pancreatic lipase levels in CPA1^S282P rabbits and controls after alcohol induction (n=6). Pancreatic lipase and amylase levels were significantly elevated in alcohol-induced CPA1^S282P rabbits compared to controls. G: Body weight of CPA1^S282P rabbits and controls after alcohol induction (n=4). Body weight was significantly decreased in alcohol-induced CPA1^S282P rabbits compared to controls. H: Pictures of CPA1^S282P rabbits versus controls (yellow arrow) after 1 month of alcohol induction. Pancreatic edema, diffusion, and inflammation were observed in CPA1^S282P rabbits after induction (red arrow). Error bars indicate mean±SEM. ns: Not significant; *: P<0.05; **: P<0.01; ****: P<0.0001. EtOH: Alcohol induction. bpm: Beat per minute.

Figure 3

Alcohol induction causes pancreatic injury in CPA1^S282P rabbits A–C: Cell shrinkage, nuclear condensation (green arrow), vacuolization (black arrow), fibers (yellow arrow), and apoptosis (red arrow) were observed in the pancreas of alcohol-induced CPA1^S282P rabbits compared to WT controls based on H&E (A), Masson (B) and TUNEL staining (C) (n=4). Scale bars: 20 μm. D–F: Significantly elevated pancreatic hydroxyproline (D), serum IL-6 (E), and serum TNF-α (F) levels in alcohol-induced CPA1^S282P rabbits compared to controls (n=6). G–I: Significantly elevated IL-6 (G), TNF-α (H), and F4/80 (I) levels in the pancreas of CPA1^S282P rabbits compared to controls after alcohol induction, as verified by IHC (n=4). Scale bars: 20 μm. J–L: Significantly elevated GRP78 (J), CHOP (K), and XBP1 (L) levels in the pancreas of CPA1^S282P rabbits compared to controls after alcohol induction, as verified by RT-qPCR (n=3). Error bars indicate mean±SEM. ns: Not significant; ^****: P<0.0001. EtOH: Alcohol induction.

Figure 4

Abnormalities of lipid metabolism in the pancreas of CPA1^S282P rabbits A: Lipid metabolism-related pathways were identified in the pancreas of CPA1^S282P rabbits by KEGG pathway enrichment analysis followed by RNA-seq. B: Heatmap of DEGs in CPA1^S282P rabbits. C: PPI network of lipid metabolism-related DEGs between CPA1^S282P rabbits and controls based on RNA-seq. Interaction score=0.4. Data are mean±SEM from four independent experiments. D: Circular plot of GO and KEGG enrichment analyses for lipid metabolism-related DEGs in CPA1^S282P rabbits compared to controls. P<0.05. E: Significant changes in lipid metabolism-associated genes in the pancreas of CPA1^S282P rabbits compared to controls based on RT-qPCR analysis (n=3). F: TEM images showing reduced ribosome density along ER, increased tubular ER, and increased smooth-surface ER in the pancreas of CPA1^S282P rabbits at 1 year of age (red arrow). Error bars indicate mean±SEM. Scale bars: 10 μm. ^****: P<0.0001.

Figure 5

Abnormalities in pancreatic lipid metabolism in CPA1^S282P rabbits A, B: TG and HDL-C levels in pancreatic tissues of CPA1^S282P rabbits at 1–6 months of age were comparable to those of WT rabbits; after 12 months of age, TG levels increased and HDL-C levels decreased in pancreatic tissues of CPA1^S282P rabbits (n=4). C: Lipidomic profiling showed increased levels of glycerol-phosphatidylcholine and glycerol-phosphatidylethanolamine, as well as reduced sphingomyelin levels, in the pancreas of CPA1^S282P rabbits (n=3). D: Heatmap comparing Affymetrix microarray data for human clinical pancreas-related diseases to human CPA1-associated genes based on the GEO database. E: Parametric (top) and nonparametric (bottom) analyses identified significant enrichment of lipid metabolism-related genes among DEGs between human clinical pancreas datasets and CPA1-associated genes (via PMID: 33050938). Error bars indicate mean±SEM. ns: Not significant; ^*: P<0.05; ^**: P<0.01; ^***: P<0.001.

Figure 6

Alcohol induction exacerbates lipid metabolism disorders and ERS in CPA1^S282P rabbits A, B: Oil Red O staining revealed higher lipid droplet accumulation (yellow arrows) in the pancreas of CPA1^S282P rabbits compared to controls after alcohol induction (n=4). C: HDL-C levels in pancreatic tissues were significantly reduced in CPA1^S282P rabbits compared to controls after alcohol induction (n=4). D: TG levels in pancreatic tissues were significantly elevated in CPA1^S282P rabbits compared to controls after alcohol induction (n=4). E: Lipidomic profiling showed that alcohol induction exacerbated lipid metabolism disturbances in the pancreases of CPA1^S282P rabbits (n=3). F: TEM revealed ERS, apoptotic features, structural disorganization, and lipid droplets (yellow arrows) in the pancreas of alcohol-induced CPA1^S282P rabbits (red arrows) compared to controls (white arrows). Scale bars: 10 μm. Error bars indicate mean±SEM. ^**: P<0.01; ^****: P<0.0001. EtOH: Alcohol induction.