Dysbiosis Of Intestinal Microbiota And Decrease In Paneth Cell Antimicrobial Peptide Level During Acute Necrotizing Pancreatitis In Rats

Background and 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.Hypertriglyceridemia?HTG?aggravates the course of acute pancreatitis?AP?.High-fat diet?HFD?has the ability to affect the structure of intestinal microbiota and the secretory function of Paneth cells.Here,we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP and HTG-related ANP.Methods:Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24 h and 48 h,respectively.Rats fed a high-fat diet for 2 weeks to induce HTG.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 TNF?,IL-1? and IL-17 A concentration by ELISA,respectively.16 S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48 h.Lysozyme and ?-defensin5 were measured by real-time PCR,western blot and immunofluoresence.Results:The normal-lipid ANP?NANP?rats had more severe histopathological injuries in pancreas and distal ileum,injured intestinal barrier and increased expression of TNF?,IL-1? and IL-17 A in plasma and distal ileum compared with those of the normal lipid sham-operated?SO?group.Principal component analysis?PCA?showed structural segregation between the SO and NANP groups.Operational taxonomic unit?OTU?number and ACE index revealed decreased microbiota diversity in the NANP group.Taxonomic analysis showed dysbiosis of intestinal microbiota structure.At phyla level,Candidatus_Saccharibacteria and Tenericutes decreased significantly.At genus level,Escherichia-Shigella and Phascolarctobacterium increased significantly,while Candidatus_Saccharimonas,Prevotellaceae_UCG-001,Lachnospiraceae_UCG-001,Ruminiclostridium₅ and Ruminococcaceae_UCG-008 decreased significantly.Lysozyme and ?-defensin5 mRNA expression levels decreased significantly in NANP group at 48 h.Protein expression of lysozyme decreased in NANP groups at 24 h and 48 h.Meanwhile,the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme.Compared with those of the NANP groups,the HTG-related ANP?HANP?groups had more severe histopathological injuries in pancreas and distal ileum,aggravated intestinal barrier dysfunction and increased TNF?,IL-1? and IL-17 A expression in plasma and distal ileum.PCA showed structural segregation between the HANP and NANP group.?-diversity estimatiors in the HANP group revealed decreased microbiota diversity compared with that in the NANP group.Taxonomic analysis showed dysbiosis of intestinal microbiota structure.In the HANP group,at phyla level,Candidatus_Saccharibacteria and Tenericutes decreased significantly,whereas Actinobacteria increased.At genus level,Allobaculum,Bifidobacterium and Parasutterella increased significantly,while Alloprevotella,Anaerotruncus,Candidatus_Saccharimonas,Christensenellaceae_R-7_group,Rikenellaceae_RC9_gut_group,Ruminiclostridium₅,Ruminococcaceae_UCG-005andRuminococcaceae_UCG-014 decreased.Compared with those in the NANP rats,mRNA expression of lysozyme and ?-defensin5 and protein expression of lysozyme decreased significantly in the HANP rats.Moreover,in the NANP and HANP rats,Allobaculum abundance was inversely correlated with lysozyme expression,while Anaerotruncus abundance was positively correlated with it by Spearman test.Conclusion:The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.HTG might aggravate intestinal barrier dysfunction by influencing intestinal microbiota and Paneth cell secretory function in ANP.