Dysbiosis of intestinal microbiota and decrease in paneth cell antimicrobial peptide level during acute necrotizing pancreatitis in rats

Abstract

Objectives: Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP.

Methods: Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNFα, IL-1β and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and α-defensin5 were measured by real-time PCR, western blot and immunofluoresence.

Results: ANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNFα, IL-1β and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and α-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme.

Conclusion: The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.

Fig 1. Pathological changes in the pancreas and the distal ileum in rats with ANP.

(A) The histopathological changes and the pathological scores of the pancreas of rats (HE, ×200). (B) The histopathological changes and the pathological scores of the distal ileum of rats (HE, ×200). vs.SO*p<0.05, vs.ANP24h^#p<0,05.

Fig 2. Changes in intestinal barrier permeability and the expression of inflammatory cytokines in the plasma and the distal ileum during ANP.

(A) Measurement of plasma diamine oxidase (DAO) activity and D-lactate at 24h and 48h after ANP induction. (B) Plasma inflammatory cytokines TNFα, IL-1β and IL-17A levels in rats at 24h and 48h after ANP induction. (C) Intestinal inflammatory cytokines TNFα, IL-1β and IL-17A at 24h and 48h after ANP induction. vs.SO*p<0.05, vs.ANP24h^#p<0,05.

Fig 3. Changes in intestinal microbiota diversity and structure in ANP.

(A) Rarefaction curve and Shannon diversity curve of single sample in each group. (B) β-diversity comparison via PCA showed a separation between the SO48h and ANP48h groups. Principle components (PCs) 2 and 3 explained 14.7% and 10.97% of the variance respectively. Each symbol represents one sample. (C) The estimators of intestinal bacterial α-diversity in the SO48h and ANP48h groups. (D) Relative abundance of phyla and genera in intestinal microbiota of the two groups.

Fig 4. Relative abundance of significantly different phyla and genera betweenthe SO48h and ANP48h groups.

(A) The significantly different phyla between the two groups. (B) The significantly different genera between the two groups. *p<0.05

Fig 5. Antimicrobial peptides expression decreased in ANP.

(A) Antimicrobial peptides (lysozyme and α-defensin5) mRNA expression by real-time PCR in the distal ileum at 24h and 48h after ANP induction. (B) Lysozyme protein expression by western blot in the distal ileum at 24h and 48h after ANP induction. (C) Lysozyme (red) protein expression produced by Paneth cell in the distal ileum by immunofluorescence. Nuclei were counterstained blue with DAPI. (original magnification, ×200). (D) Quantification of the number of Paneth cells among the SO and ANP groups at 24h and 48h. (E) The relative abundance of Escherichia-Shigella correlated inversely with lysozyme protein expression in the SO48h and ANP48h groups. r = -0.57, p<0.05. vs.SO*p<0.05, vs.ANP24h^#p<0.05.