Effect Of Antimicrobial Peptide Of Paneth Cells On Intestinal Injury During Acute Necrotizing Pancreatitis In Rats And Its Preliminary Mechanism

ObjectivesAcute necrotizing pancreatitis(ANP)is often accompanied by intestinal barrier dysfunction and intestinal microbiota dysbiosis.Paneth cells protect intestinal barrier function and are associated with intestinal microbiota by secreting antimicrobial peptides.Here,we investigated the effect of antimicrobial peptide of Paneth cells on intestinal injury during acute necrotizing pancreatitis in rats and studied its preliminary mechanism.MethodsRats with ANP were established by retrograde injection of 3.5%sodium taurocholate into biliopancreatic duct.The paneth cells in rats were depleted by injection with dithizone(100 mg/kg)through the tail vein.ANP+dithizone combination group rats were induced ANP at 6h after the dithizone injection.All rats were sacrificed respectively at 6h,12h,24h,36h and 48h after modeling.Kaplan-Meier survival analysis was performed.Injuries of pancreas were assessed by histopathological Schmidt score.Intestinal injury is evaluated using the following indicators:Injuries of distal ileum were assessed by histopathological Chiu’s score;Intestinal permeability was evaluated by plasma D-lactate and diamine oxidase activity(DAO);Plasma and distal ileum TNFα,IL-1β and IL-17A concentration were measured to evaluate systemic and intestinal inflammation by ELISA respectively;Intestinal microbiota translocation was observed by Fluorescence in situ hybridization(FISH).The expression of lysozyme were detected by real-time PCR and immunofluoresence.Gas chromatography-mass spectrometry(GC-MS)was used to investigate the concentration of stool short-chain fatty acids.Fecal Ruminococcaceae_UCG-005 and Ruminococcaceae UCG-014 were measured by real-time PCR and Western blot was used to detect the expression of endoplasmic reticulum stress protein BIP and ATF6 in distal ileum mucosa.ResultsSurvival analysis showed that survival rates of dithizone group,ANP group and ANP+dithizone combination group at 48h were 98.8%,78.7%and 50.5%respectively.The pathological lesions of the pancreas in ANP group were more serious than those in sham-operated(SO)group at each time point.Pancreatic injury and histopathological scores in combination group were even more serious than those of ANP group at each time point,especially at 24h and 36h(p<0.05).Compared with those of SO group,the ANP rats had more severe histopathological injuries in distal ileum,injured intestinal permeability,increased expression of TNFα,IL-1β and IL-17A in plasma and distal ileum and intestinal microbiota translocation.Compared with those of ANP group,Combination group had more severe histopathological injuries and scores in distal ileum at each time point(p<0.01),especially at 6h.The plasma DAO and D-lactate levels increased gradually in ANP group and combination group over time,especially in combination group at 6h,12h and 24h(vs ANP group,p<0.05),There was significant upregulation of intestinal IL-17A and IL-1βlevels in combination group compared with those of ANP group at 6h,12h,24h(6h,12h p<0.01,24h p<0.05,respectively),the expression of intestinal TNF-α in combination group were higher than those of ANP group at 6h,12h,24h,36h and 48h(6h p<0.01;12h,24h,36h,48h p<0.05,respectively).The expression of serum inflammatory cytokines increased with a time-dependent manner in ANP group and combination group.The plasma TNFα,IL-1β and IL-17A levels in combination group were higher than those of ANP group at 6h,12h and 24h(TNFα,IL-1β 6h p<0.01,12h,24hp<0.05;IL-17A 6h,12h,24h p<0.05,respectively).FISH showed there are more obvious intestinal microbiota translocation in combination group.PCR showed the expression of lysozyme mRNA in ANP group was lower than those in SO group and decreases of lysozyme gene expression was negatively correlated with upregulation of DAO,D-lactate and inflammatory factors.Immunofluorescence displayed that the lysozyme expression and Paneth cell was the lowest in combination group.Concentration of stool short-chain fatty acids in ANP group such as acetic acid,propionic acid,butyric acid and isobutyric acid decreased over time.The concentration of stool short chain fatty acids in combination group were generally lower than those of ANP group.For combination group,the concentration of acetic acid,propionic acid,isobutyric acid and valeric acid increased slightly with time passing by,while butyric acid and isovaleric acid did not change obviously.The concentration of acetic acid,propionic acid,butyric acid and valeric acid in combination group significantly decreased than those of the ANP group at 6h and 12h(p<0.05).Isobutyric acid and isovaleric acid at 6h in combination group evidently reduced compared with ANP group(p<0.05).The gene expression of rat feces Ruminococcaceae UCG-005 and Ruminococcaceae UCG-014 decreased gradually in ANP group and decreased most significantly in combination group,The expression of Ruminococcaceae UCG-005 and Ruminococcaceae UCG-014 in combination group obviously reduced than those of ANP group at 6h,24h(p<0.01).The expressions of BIP and ATF6 protein in distal ileum mucosa increased gradually in ANP group and combination group over time,especially in combination group at 6h,24h and 48h(vs ANP group,p<0.05).ConclusionDepletion of Paneth cell decreased the survival rate of ANP rats,exacerbated intestinal and pancreas damage,aggravated intestinal permeability,elevated the expression of inflammatory cytokines in plasma and intestinal,promoted intestinal microbiota translocation with the reduction of antimicrobial peptides during ANP.Decreases of Paneth cell antimicrobial peptides may result in reduction of Ruminococcaceae_UCG-005 and Ruminococcaceae_UCG-014 which is associate with Paneth cell,and decrease of corresponding bacterial metabolite acetic acid,reduction of acetic acid may enhance intestinal epithelial endoplasmic reticulum stress(ESR)which participated in intestinal injury during ANP in rats.