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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.

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