Objective:To explore the effect of probiotics Lactiplantibacillus plantarum(LP) WCFS1 by gavage on acute necrotizing pancreatitis (ANP) and associated ileum injury in mice. Methods:Twenty-four healthy male mice were gavaged with broad-spectrum antibiotics for 3 weeks to establish microbiota-depleted mice, and then randomly divided into control group (CON), ANP model group (ANP), LP gavage group (LP) and LP gavage and ANP induced group (LP+ ANP) , with 6 mice in each group. Mice in LP and LP+ ANP group were treated by gavage of LP (1×10 9 CFU/ml, 0.2 ml/day per mouse) for 1 week, while CON and ANP were gavaged with sterile phosphate buffered saline for 1 week instead. The ANP model was induced by intraperitoneal injection with caerulein (100 μg/kg) for 10 times with 1-hour interval between two injections and the 10th injection with lipopolysaccharide(LPS) 5 mg/kg intraperitoneally, and the mice were sacrificed 2 h later. Levels of LP in stool and ileal mucosa were detected by real-time PCR; the pancreas and ileum were collected for pathological examination to observe the extent of tissue inflammation and to score the pathology. Serum amylase activities were determined by enzymatic kinetic chemistry; serum inflammators levels and intestinal permeability were detected by ELISA; levels of inflammators in pancreatic and ileal tissues were detected by real-time PCR; ileal tight-junction proteins (occludin, claudin-1 and ZO-1) were measured by immunofluorescence staining. Results:LP levels in the stool and ileal mucosa of mice were significantly increased after LP gavage, and the differences were statistically significant (913.30±39.12 vs 2.39±1.39, 23.11±0.50 vs 1.38±0.28, all P value <0.05). The pathological scores of pancreatic tissue of CON, LP, ANP and LP+ ANP group were (0.26±0.41), (0.17±0.26), (8.55±0.46) and (6.30±0.45); the serum amylase activities were (219.70±19.73), (217.60±11.30), (2896.24±98.32) and (1837.13±131.60)U/L, IL-1β were (0.87±0.28), (1.4±0.85), (67.41±6.45) and (36.33±5.65)pg/ml, IL-6 were (0.74±0.27), (0.16±0.16), (280586.12±39163.92) and (107912.75±31283.47)pg/ml, IL-10 were (35.52±5.27), (50.99±15.34), (2008.45±184.83) and (3070.35±403.71)pg/ml; the expression level of pancreatic IL-1β mRNA was 1.42±0.39, 0.95±25, 20.53±0.50 and 10.69±1.01, IL-6 mRNA was 1.31±0.44, 0.93±0.023, 21.97±1.71 and 11.54±1.75, IL-10 mRNA was 0.93±0.14, 0.75±0.15, 0.99±0.21 and 1.76±0.19; there was no significant difference between LP and CON group, and pancreatic pathological scores, serum amylase、IL-1β and IL-6 levels, and the expression level of pancreatic IL-1β and IL-6 mRNA were significantly decreased in LP+ ANP group compared with those in ANP group, while serum IL-10 levels and the expression level of pancreatic IL-10 mRNA were significantly increased compared with ANP group, and all the differences were statistically significant (all P values <0.05). The pathological scores of ileal tissue of CON, LP, ANP and LP+ ANP group were 0, 0, (3.17±0.41) and (1.67±0.52); the levels of serum DAO of CON, LP, ANP and LP+ ANP group were (0.03±0.03), (0.02±0.02), (0.50±0.05) and (0.49±0.06)ng/ml; LPS levels were (2.75±0.35), (3.74±0.28), (7.19±0.92) and (5.88±0.38)ng/ml; the expression level of ileal IL-1β mRNA was 1.21±0.20, 1.17±0.09, 1.81±0.25 and 1.63±0.21; IL-6 mRNA was 1.01±0.29, 2.83±0.42, 54.45±8.50 and 16.87±4.42; IL-10 mRNA was 1.12±0.41, 6.09±2.51, 11.65±1.47 and 29.86±2.93. There was no significant difference between LP and CON group, except that the ileal IL-10 mRNA expression was significantly higher than that of CON group. Ileal pathological scores, serum LPS levels and the expression level of ileal IL-6 mRNA were significantly lower in LP+ ANP group than those in ANP group, while the expression level of ileal IL-10 mRNA was significantly higher than that of ANP group; the expression of ileal tight junction proteins (ocludin, claudin-1, ZO-1) was significantly higher than those in ANP group, and all the differences were statistically significant (all P values <0.05). Conclusions:LP WCFS1 gavage could ameliorate the injury of pancreatic and ileal barrier in caerulein-induced ANP mice.

Objective:To establish culture system for mouse pancreatic ductal organoids and investigate the morphology and physiological functions of the organoids.Methods:Pancreatic tissues were taken from C57BL/6 mice (6-8 weeks) and digested by collagenase Ⅳ. The pancreatic ducts were separated and collected and then the pancreatic organoids were cultured in the complete medium after Matrix gel embedding. Morphological evaluation of the organoids was performed after hematoxylin-eosin staining. The expression and localization of markers for organoids were identified by Western Blot and immunofluorescence staining; and the expression and localization of ion channels and antimicrobial peptides of the organoids were detected by agarose gel electrophoresis and immunofluorescence staining.Results:Mouse pancreatic organoids were successfully established, which could be stably passaged for 10 generations. The organoids grew spherically and formed a duct-like structure. The internal cavity corresponded to the lumen of pancreatic duct tissue. The pancreatic organoids stably expressed stem progenitor cell marker gene SOX9 and ductal epithelial cell-specific gene KRT19, which were both localized in the epithelium. The organoids did not express amylase. The organoids maintained stable expression of epithelial ion channels Clcn1, Kcnma1, CFTR, Slc12a5, Slc26a3, Slc26a6 and Scnn1a, low expression of Ano1 and no expression of Clcn3, Kcna1, Kcna2, Kcnd3, Kcnh1, Atp12a, Slc4a4, Slc9a1, Slc12a2 and Slc26a11; and CFTR highly expressed in epithelial cells. The organoids maintained high expression of antimicrobial peptides Reg3a, CRAMP and glycoprotein 2, low expression of Defb1, Defb2, and Defb3 and no expression of Defa1 and Defa4; and both CRAMP and Reg3a were expressed in the epithelial cells and secreted into the lumen of the organoids.Conclusions:Mouse pancreatic organoids are successfully established, which can be stably passaged. The organoids maintain the characteristics of ductal epithelial cells and can be used as an in vitro model to study the physiology of pancreatic ducts.

Background:Intestinal microbiota dysbiosis and impaired intestinal barrier,leading to bacterial translocation,are involved in acute pancreatitis(AP)exacerbation.Antimicrobial peptides participate in the regulation of intestinal microbiota,yet their changes and roles in the course of AP are unclear.Aims:To investigate the dynamic changes and significance of antimicrobial peptides in the ileum and pancreas among three classical AP models.Methods:Three AP mouse models were established by using cathelicidin plus lipopolysaccharide(CAE+LPS),sodium taurocholate(N-Tau)and L-arginine(L-Arg),respectively.The pathological changes of pancreatic and ileal tissues were observed and scored.Real-time PCR was applied to detect the expression levels of the proinflammatory cytokines,and antimicrobial peptides including lysozyme(LZM),secretory phospholipase A2(sPLA2),angiogenin 4(Ang4),regenerating islet-derived protein 3(REG3)family,β-defensin family,cathelicidin-related antimicrobial peptide(CRAMP),and glycoprotein 2(GP2)in ileum and/or pancreas.The association between expressions of antimicrobial peptides and the injuries of pancreas and ileum was analyzed.Results:Pancreatic and ileal injuries could be observed in all three AP models in different time points with various degrees.The pathological scores of the CAE+LPS and N-Tau models reached the highest level and then declined from 0-72 h,while those of L-Arg model progressively increased within 72 hours.Compared with the corresponding controls,the mRNA levels of LZM,sPLA2,and Ang4 in ileal tissue,and the mRNA levels of CRAMP,GP2,and β-defensins in pancreatic tissue,were generally downregulated in all three AP models(all P<0.05).CAE+LPS and N-Tau models showed a trend of initial decrease followed by partial recovery,while L-Arg model exhibited a gradual downregulation trend.The mRNA levels of REG3β and REG3γ in ileum upregulated and reached the peak at 48 h or 24 h and downregulated significantly at 72 h in all three AP models(all P<0.05);while in pancreatic tissue,both REG3β and REG3γ were generally upregulated in all three AP models(all P<0.05),but fell back in CAE+LPS and N-Tau models at 72 h.The mRNA levels of ileal β-defensins upregulated significantly in the early stage of the disease(12 h)in all three AP models(all P<0.05),and then gradually decreased.Spearman correlation coefficient analysis showed that the expressions of ileal LZM,sPLA2,and Ang4,as well as the pancreatic CRAMP,GP2,and β-defensins,were significantly negatively correlated with the pancreatic and ileal pathological scores in all three AP models(all P<0.05);but the expression of REG3β in the pancreas was significantly positively correlated with the pancreatic and ileal pathological scores(all P<0.05).Conclusions:The expressions of LZM,sPLA2,and Ang4 in the ileum,as well as the expressions of CRAMP,GP2,β-defensin family,and REG3β in the pancreas of the three classical AP models,dynamically changed with the severity of the disease.Ileal and pancreatic antimicrobial peptides may affect the injuries of pancreas and intestine during AP by regulating intestinal microbiota.