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. 2023 Apr 23;24(9):7715.
doi: 10.3390/ijms24097715.

Repetitive Cerulein-Induced Chronic Pancreatitis in Growing Pigs-A Pilot Study

Ewa Tomaszewska  1 , Małgorzata Świątkiewicz  2 , Siemowit Muszyński  3 , Janine Donaldson  4 , Katarzyna Ropka-Molik  5 , Marcin B Arciszewski  6 , Maciej Murawski  7 , Tomasz Schwarz  8 , Piotr Dobrowolski  9 , Sylwia Szymańczyk  1 , Sławomir Dresler  10   11 , Joanna Bonior  12
Affiliations

Repetitive Cerulein-Induced Chronic Pancreatitis in Growing Pigs-A Pilot Study

Ewa Tomaszewska et al. Int J Mol Sci. .

Abstract

Chronic pancreatitis (CP) is an irreversible and progressive inflammatory disease. Knowledge on the development and progression of CP is limited. The goal of the study was to define the serum profile of pro-inflammatory cytokines and the cell antioxidant defense system (superoxidase dismutase-SOD, and reduced glutathione-GSH) over time in a cerulein-induced CP model and explore the impact of these changes on selected cytokines in the intestinal mucosa and pancreatic tissue, as well as on selected serum biochemical parameters. The mRNA expression of CLDN1 and CDH1 genes, and levels of Claudin-1 and E-cadherin, proteins of gut barrier, in the intestinal mucosa were determined via western blot analysis. The study showed moderate pathomorphological changes in the pigs' pancreas 43 days after the last cerulein injection. Blood serum levels of interleukin (IL)-1-beta, IL-6, tumor necrosis factor alpha (TNF-alpha), C-reactive protein (CRP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGTP), SOD and GSH were increased following cerulein injections. IL-1-beta, IL-6, TNF-alpha and GSH were also increased in jejunal mucosa and pancreatic tissue. In duodenum, decreased mRNA expression of CDH1 and level of E-cadherin and increased D-lactate, an indicator of leaky gut, indicating an inflammatory state, were observed. Based on the current results, we can conclude that repetitive cerulein injections in growing pigs not only led to CP over time, but also induced inflammation in the intestine. As a result of the inflammation, the intestinal barrier was impaired.

Keywords: cerulein; chronic pancreatitis; cytokines; junction proteins; pig.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Changes in (a) body weight, (b) and daily weight gain of pigs during the experimental period. Shadowed area shows the 6-day period of daily cerulein injections (1 µg/kg b.w./day) in the CER-pig group. Black: the control group (C-pigs); red: the cerulein-injected group (CER-pigs). Data are presented as mean ± SE. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to day 0). Statistical significance: # p < 0.05; ## p < 0.01 (between groups).
Figure 2
Figure 2
Changes in serum nutritional parameters (a) TCHOL and (b) ALB and other biochemical parameters: (c) TPRT, (d) LDL, (e) HDL, (f) TG, (g) UREA, (h) AST, and (i) ALT of pigs during the experimental period. Shadowed area shows the 6-day period of daily cerulein injections (1 µg/kg b.w./day) in CER-pig group. Black: the control group (C-Pigs); red: the cerulein-injected group (CER-pigs). Data are presented as mean ± SE. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to day 0). Statistical significance: # p < 0.05; ## p < 0.01 (between groups). LDL—low-density lipoprotein, HDL—high-density lipoprotein, AST—aspartate aminotransferase, AL—alanine aminotransferase.
Figure 3
Figure 3
Changes in serum cytokines and inflammatory parameters: (a) IL-1-beta, (b) IL-6, (c) IL-10, (d) TNF-alfa, (e) GSH, (f) CRP, (g) LHD, (h) GGTP, and (i) SOD of pigs during the experimental period. Shadowed area shows the 6-day period of daily cerulein injections (1 µg/kg b.w./day) in CER-pig group. Black: the control group (C-pigs); red: the cerulein-injected group (CER-pigs). Data are presented as mean ± SE. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to day 0). Statistical significance: # p < 0.05; ## p < 0.01; ### p < 0.001 (between groups). IL-1-beta—interleukin 1 beta, IL-6—interleukin 6, IL-10—interleukin 10, TNF alpha—tumor necrosis factor alpha, GSH—reduced glutathione, CRP—C-reactive protein, LDH—lactate dehydrogenase, GGTP—gamma-glutamyl transpeptidase, SOD—total superoxidase dismutase.
Figure 4
Figure 4
Pathomorphological changes observed in pig pancreas sections. Representative hematoxylin and eosin (H&E)-stained sections of pancreas: (A) control group with normal (healthy) pancreas. Acinar cells stained blue at their base due to the high content of RNA and the presence of nuclei, and pink at their apex (lumenal aspect) due to a high content of zymogen granules with proteins (digestive enzymes); arrowheads show acinar cells forming acinar glands of exocrine pancreas; asterisk marks a small pancreatic islet (small cells, pale cytoplasm; islets of Langerhans) in the more abundant and darker acinar tissue where the islet cells are smaller and have paler cytoplasm than the surrounding acinar cells; arrows show an intralobular duct with a modest collagenous wall; the lumen of the small duct contains homogenous pink staining protein-rich pancreatic juice. (B–F) are representative photographs of the pancreas from pigs with cerulein-induced pancreatitis, as the inflammation is not evenly distributed in the pancreatic parenchyma. The images show the characteristics of the pancreatitis observed. Yellow arrowheads show cellular necrosis (B,D) or apoptosis (E,F); white arrowheads show degeneration of acinar cells, pale cytoplasm with no zymogen granules, acinar edema; asterisks mark a darker pancreatic islet (small cells, pale cytoplasm—islets of Langerhans) in the more abundant pale, swollen and degenerative acinar tissue (F); arrows show intralobular ducts with degradation of ductal epithelium (D,E) or with lymphatic infiltration (F). All scale bars represent 100 µm.
Figure 5
Figure 5
USG pancreas examination. (a) USG probe placement allowed visualization of a part of the pancreas, situated behind (underneath in the scan) the spleen, well-visible in the foreground straight under integumentary tissues. (b) Representative USG images on the timeline to show changes in pancreas echotexture after cerulein treatment. Quantitative analysis of USG images: (c) mean pixel intensity (MPI); (d) mean pixel heterogeneity (MPH). Black: the control group (C-pigs); red: the cerulein-injected group (CER-pigs). Data are presented as mean ± SE. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (compared to day 0). Statistical significance: # p < 0.05; ## p < 0.01; ### p < 0.001 (between groups).
Figure 6
Figure 6
Changes in interleukins and reduced GSH in (a) duodenal and (b) jejunal mucosa, and (c) pancreatic tissue. Data are presented as mean ±SE. Statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001 (Tukey’s HSD test). IL-1-beta—interleukin 1 beta, IL-6—interleukin 6, TNF alpha—tumor necrosis factor alpha, GSH—reduced glutathione.
Figure 7
Figure 7
Effect of cerulein-induced chronic pancreatitis on intestinal barrier integrity. Quantitative real-time PCR analysis of mRNA expression of CDH1 and CLDN1 genes, encoding E-cadherin and Claudin-1 protein, respectively, in (a) duodenum and (d) jejunum. Due to the exponential nature of the mRNA expression calculated using the -ddCT method, the geometric means with standard errors are presented. Western blot analysis of E-cadherin and Claudin-1 in (b) duodenum and (e) jejunum, β-actin was used as loading control. (c) Protein expression level of Claudin-1 and E-cadherin in (c) duodenum and (f) jejunum; The protein levels are normalized to the corresponding β-actin levels and expressed as the fold change relative to the amount present in C-pigs; Uncropped original western blot membranes of E-Cadherin and Caludin-1 can be found in Supplementary Figure S1; (g) blood serum concentrations of D-lactate, an indicator of the permeability of the intestinal mucous membrane. Error bars represent standard errors. * p < 0.05. (Tukey’s HSD test).
Figure 8
Figure 8
Experimental layout. The experiment lasted 56 days in total: 7 days of adaptation (−7 to 0), 6 days of cerulein injections were performed with 24 h intervals (between day 0 and day 6). Weighing, blood collection and USG pancreas examination were performed in one-week intervals; * on day 35, due to COVID-19 restrictions, only USG examination was performed. On day 49, after final measurements, the pigs were subjected to pharmacological euthanasia and pancreas and small intestine sections (duodenum, jejunum) were dissected.
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