| Background and Aims:Acute pancreatitis(AP)is a common clinical acute abdomen.With the improve-ment of people’s living standards,the incidence rate is increasing year by year.The mortality rate of severe acute pancreatitis(SAP)can be increased.Up to 30%-50%,seriously threatening human health.In recent years,studies have found that there is an imbalance of intestinal flora in AP patients and animal models.However,the role of intestinal flora in the development of AP and its possible mechanisms are still unclear.A meta-analysis suggested that 59% of SAP patients with intestinal dysfunction,and intestinal mucosal barrier function damage can cause bacterial translocation,which is the main cause of secondary infection of pancreatic necrotic tissue and aggravation of disease progression.Over-release of inflammatory factors is one of the important mechanisms of intestinal mucosal injury.NLRP3(NOD-like receptor protein 3)inflammatory corpuscle is a key component of the host’s innate immune system,and its excessive activation can cause IL-1β,IL-18.A large number of pro-inflammatory factors are released,mediating inflammatory immune responses.Our previous results showed that the relative abundance of Escherichia-Shigella,Enterococcus,Proteus and other pathogenic bacteria in the intestinal tract of SAP mice was positively correlated with the up-regulation of NLRP3 expression in small intestinal tissues.Therefore,this study intends to further investigate the effects of intestinal flora on severe acute pancreatitis and its relationship with intestinal NLRP3 activation using sterile mouse animal models and bacterial transplantation methods.Materials and Methods:1、A pseudo-sterile(Abx)C57BL/6J mouse model was constructed by adding a broad-spectrum antibiotic to the drinking water of mice for 3 weeks,and then constructing a SAP mouse model,ie intraperitoneal injection of cerulein and lipopolysaccharide,the end of modeling Mice were sacrificed 24 h later.The experiment was divided into four groups: blank control group(control group),SAP group,Abx control group(Abx group)and Abx-SAP group.2、An animal model of sterile(germ-free,GF)C57BL/6J mouse SAP was constructed as described above.The experiment was divided into four groups: blank control group(control group),SPF-SAP group,GF control group(GF group)and GF-SAP group.3、A model of recovery of intestinal sterile flora in pseudo-sterile mice was constructed.Three weeks after the mice were fed with mixed antibiotics,the intestinal flora of the mice was restored by transplanting normal mouse fecal homogenate,and then the SAP model was established.The method was the same as above.The experiment was divided into two groups: Abx-cont-SAP group(non-transplanted group)and Abx-treated-SAP group(transplanted group).4、H&E staining was used to observe the pathological changes of pancreas and immunohistochemistry to detect the expression of myeloperoxidase(MPO)in pancreas and lung tissue.5、The contents of amylase,lipase,inflammatory cytokines(IL-1β,IL-10,IL-6,TNF-α)and D-Lac(D-Lac)in the serum of mice were detected by ELISA.6、The expression of NLRP3 inflammatory body-associated proteins(NLRP3,IL-1β,Caspase-1 p20)and intestinal mucosal tight junction proteins(occludin,claudin-1)in small intestinal tissues were detected by Western blot.7、The expression of NLRP3 inflammatory body-associated proteins(NLRP3,ASC,IL-1β,IL-18)in small intestinal tissues was detected by PCR.8、Immunofluorescence was used to detect the expression of tight junction proteins(occludin,claudin-1)in intestinal mucosa of small intestine.Results:1.Effect of regulating intestinal flora on pancreatic injury in mice with severe acute pancreatitis.(1)The serum amylase and lipase levels in the SAP group were significantly higher than those in the normal control group(p<0.01),and the pancreatic tissue edema,inflammatory cell infiltration and necrosis were observed in the SAP group,indicating successful SAP modeling.(2)Compared with the SAP group,the amylase and lipase levels in the Abx-SAP group were significantly decreased(p<0.01),pancreatic tissue edema,inflammatory cell infiltration and necrosis were significantly reduced(p<0.01),and MPO expression was significantly reduced(p<0.01).).The results of GF mice were consistent with pseudo-sterility,that is,the serum amylase and lipase levels of GF-SAP group were significantly lower than those of SPF-SAP group(p<0.01),pancreatic lesions were significantly reduced(p<0.01),MPO expression.The amount was significantly reduced(p < 0.01).(3)The transplanted normal mouse fecal homogenate was used to restore the intestinal flora of pseudo-sterile mice,and then induced SAP found that the serum lipase level of the transplanted group was significantly higher than that of the non-transplanted group(p<0.05),and there was no difference in amylase level..The pathological changes of pancreas and the expression of MPO in the transplanted group were significantly aggravated compared with the non-transplanted group(p<0.05).It indicated that the pancreatic injury of mice was alleviated after the intestines were aseptic or antibiotics,and the pancreatic damage was aggravated after transplanting the normal mouse fecal homogenate to restore the intestinal flora.2.Effect of regulating intestinal flora on systemic inflammatory response in mice with severe acute pancreatitis.(1)The expression of MPO in the lung tissue of the SAP group was significantly higher than that of the normal control group(p<0.01),while the Abx-SAP group was significantly lower than the SAP group(p<0.01).The results of the sterile mice were consistent with the pseudo-sterility,that is,the expression of MPO in the lung tissue of the GF-SAP group was significantly lower than that of the SAP group(p<0.01).(2)The levels of serum inflammatory factors IL-1β,TNF-α,IL-6 and IL-10 in SAP group were significantly higher than those in normal control group(p<0.01),while those in Abx-SAP group were significantly lower than those in SAP group(p<0.01).(3)After transplanting normal mouse fecal homogenate to restore the intestinal flora of pseudo-sterile mice,SAP was found to have the expression of MPO in the lung tissue of the transplanted group(p<0.05)and serum inflammatory factor levels IL-1β and TNF-α.There was a significant increase in the transplantation group(p<0.05),and there was no significant difference between the two groups in IL-6 and IL-10 levels.3.Possible mechanisms of regulation of intestinal flora in the development of severe acute pancreatitis in mice.(1)The protein expression and mRNA levels of NLRP3(p<0.01),IL-1β(p<0.05)and caspase-1 p20(p<0.01)in the small intestine of SAP group were significantly higher than those in the normal control group;the NLRP3 inflammation in the Abx-SAP group compared with the SAP group.The activation of corpuscles decreased,NLRP3(p<0.01)and caspase-1 p20(p<0.01)were lower than those in SAP group,and there was no significant difference in IL-1β.The results of partial experiments in sterile mice were similar to those in the pseudo-sterile section.GF-SAP group had less activation of NLRP3 inflammatory bodies than SPF-SAP group,NLRP3(p<0.01),IL-1β and caspase-1 p20(p<0.01).Both showed a decrease in expression compared to the SPF-SAP group.(2)The activation of NLRP3 inflammatory bodies was increased in the transplanted group after transplantation of normal mouse fecal homogenate to restore SAP in the sterile sterile mouse intestinal flora,NLRP3(p<0.01),IL-1β and caspase-1 p20(p< 0.01)Protein expression and mRNA levels were lower than in the non-transplanted group.(3)The expression of occludin(p<0.01)and claudin-1(p<0.01)in SAP group and D-Lac level(p<0.01)were significantly higher than those in normal control group;the tight junction protein of intestinal mucosa in Abx-SAP group and SAP group There was no significant difference in expression and D-Lac levels.The results of partial experiments in sterile mice were similar to those in the pseudo-sterile section.There was no significant difference in the expression of tight junction proteins between the GF-SAP group and the SPF-SAP group.The level of D-Lac in the GF-SAP group was significantly lower than that in the SPF-SAP group.p<0.05).(4)The expression of tight junction protein and D-Lac in the intestinal mucosa of the transplanted group was significantly higher than that in the non-transplanted group(p<0.01)after transplantation of normal mouse fecal homogenate to restore SAP in the intestinal flora of pseudo-sterile mice.Conclusions:1.Regulation of intestinal flora can affect pancreatic pathological damage and systemic inflammatory response in mice after induction of SAP,suggesting that intestinal flora imbalance may play an important role in the development of SAP.2.Intestinal flora imbalance may promote the severity of AP by activating NLRP3 inflammatory bodies and impairing intestinal mucosal barrier function. |
