Ameliorated Effect Of Aplysin On Pancreatic Necrosis And Inflammatory In NOD Mice

Objective To observe the protective effect of Aplysin on spontaneous pancreatic necrosis and inflammation in non-obese diabetic(NOD)mice,and to preliminarily explore its possible mechanism of intestinal mucosal barrier stability and regulation of intestinal microbial composition.Methods1.Animal experiment intervention plan: Six-week-old female NOD mice(n=30)were randomly divided into two groups after 2 weeks of adaptive feeding: Aplysin group(n=15)and Control group(n=15).Aplysin group was given aplysin(150 mg/kg)and the Control group was given soybean oil(0.1 m L)by gavage once a day.Mice were weighed,measured fasting blood glucose,and recorded the food intake once a week.Four weeks later,mice were anesthetized with pentobarbital sodium(40 mg/kg)by intraperitoneal injection,and blood samples,pancreas tissues,jejunum tissues,colon tissues,and colon contents were collected for analysis.During the experiment,the mice were placed in the Specific Pathogen-Free Animal Center of Qingdao University,where they could freely obtain standard irradiated rodent feed and sterile tap water.2.Hematoxylin-Eosin(HE)staining was used to observe the pathological changes of the pancreas.3.Transmission electron microscope(TEM)was used to observe the ultrastructural changes of pancreas,jejunum and colon.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum endotoxin and insulin levels.5.Immunofluorescence was used to observe the distribution of insulin in pancreas tissue and the expression of claudin-1,occludin,and Zonula occludens 1(ZO-1)proteins in jejunum and colon tissues.6.Western blot was used to detect the protein levels of toll-like receptor 4(TLR4)and its downstream Myeloid differentiation protein 88(My D88),TNF receptor-associated factor 6(TRAF-6),TRIF-related adaptor molecule(TRAM),TIR domain-containing adaptor inducing interferon-(TRIF),nuclear factor-κB(NF-κB p65),interferon regulatory factor 3(IRF-3),interleukin 1β(IL-1β),interferon β(IFN-β)in pancreas tissue and claudin-1,occludin and ZO-1 in the jejunum and colon tissue.7.EZ-link Sulfo-NHS biotin tracer experiment was used to evaluate the intestinal permeability.8.16 S r RNA gene sequencing technology was used to analyze the differences in the intestinal flora of the two groups of mice.Results1.The body weight and the food intake were not changed significantly in the two groups(P>0.05).2.The spontaneous diffuse necrosis of pancreatic acinar cells was significantly improved in the Aplysin group than that in the Control group,and the pancreatic pathology score was significantly lower in the Aplysin group than that of the Control group(P<0.05).3.The level of fasting blood glucose was not changed significantly in the two groups(P>0.05),and both were within the normal reference range.Immunofluorescence showed that the expression of insulin in pancreatic tissue in the Aplysin group was higher than that in the Control group.However,the level of serum insulin was not changed significantly in the two groups(P>0.05).4.ELISA showed that the level of serum endotoxin was significantly reduced in the Aplysin group than that in the Control group(P<0.05).5.Western Blot showed that the acinar cell membrane protein level of TLR4 was significantly decreased in the Aplysin group than that in the Control group(P<0.05).And its downstream cytoplasmic protein levels of My D88,TRAF-6,TRAM,TRIF,IL-1β,IFN-β,and the nuclear protein levels of NF-κB p65 and IRF-3 were significantly decreased(P<0.05).6.TEM and the tracer experiment showed that the mucosa structure of the jejunum and colon was more complete in the Aplysin group.Immunofluorescence showed that the expression of claudin-1,occludin and ZO-1 in intestinal epithelial cells in the Aplysin group was higher than that in the Control group.Western Blot showed that the protein levels of claudin-1,occludin,and ZO-1 in jejunum and colon tissue were significantly higher in the Aplysin group than those in the Control group(P<0.05).7.16 S r RNA sequencing showed that compared with the control group,the diversity,and composition of the intestinal flora of NOD mice were changed in the Aplysin group.Conclusion1.Aplysin can improve significantly pancreatic necrosis and inflammation in NOD mice.2.Anti-inflammatory effect of Aplysin may be achieved by inhibiting TLR4 and its downstream My D88 and TRIF-dependent signaling pathways.Aplysin can improve significantly pancreatic necrosis in NOD mice,its mechanism may be related to repair the intestinal mucosal barrier and regulate the composition of intestinal flora,prevent intestinal leakage of endotoxin.