| Background and objective:Acute pancreatitis(AP)is usually caused by structural obstruction of the biliary tract,alcohol consumption,endoscopic retrograde cholangiopancreatography,and drugs,which eventually lead to acinar cell death and induce local and systemic inflammation.About 15%-20% of patients will develop severe acute pancreatitis(SAP).Microcirculation damage and hypovolemia occur in the process of SAP,which can cause intestinal mucosal ischemia,followed by reperfusion injury,leading to intestinal barrier dysfunction and intestinal flora translocation.A key link in causing pancreatic infection,necrosis.The intestinal flora constitutes the biological barrier of the intestinal mucosa,which can defend against pathogen infection.In addition to the intestinal flora itself,the short chain fatty acids(SCFAs)metabolites of the flora,including acetic acid,propionic acid,and butyric acid,can be Intestinal epithelial cells provide energy and play an important role in protecting the normal physiological function of the intestinal mucosal barrier and epithelial integrity.SCFAs can increase the growth and diversity of gut microbiota by reducing intestinal p H,increasing fecal acidification.In addition,SCFAs can promote the proliferation of some beneficial bacteria,such as Bifidobacteria and Lactobacilli.These beneficial bacteria in turn stimulate the synthesis of short-chain fatty acids,lower intestinal p H,inhibit pathogenic bacterial colonization,improve intestinal microbial barrier function,and promote intestinal homeostasis.However,the mechanism of intestinal microbiota-SCFAs damage to the intestinal mucosal barrier during the development of SAP is still unclear.Therefore,from the perspective of regulating the metabolic imbalance of intestinal microbiotaSCFAs,correcting intestinal mucosal barrier dysfunction and reducing intestinal bacterial translocation are important for preventing SAP.Secondary pancreatic necrosis infection and improvement of patient prognosis have practical guiding significance.Materials and methods:(1)Detection and analysis of the changes of short-chain fatty acids in the pathogenesis of SAP.1.Detection of SCFAs content in feces of SAP patients and healthy people by GC-MS technology.2.The content of SCFAs in feces of SAP mice and normal control mice was detected by GC-MS technology.(2)To construct an animal model of SAP to explore the effect of short-chain fatty acids on the severity of SAP disease.1.To construct a cerulein-induced SAP mouse model,the experiment was divided into six groups: control group,cer group,SCFAs＿cer group,acetate＿cer group,propionate＿cer group and butyrate＿cer group,and compared the mice in each group The degree of pancreatic injury,pancreatic body weight ratio and pancreatic pathology score.2.In addition to the SAP model induced by cerulein,two other SAP animal models,namely Bombesin combined with cerulein model and M3/ptf1α mouse SAP model,were constructed to verify the role of SCFAs in SAP,and to compare the degree of pancreatic injury in mice in each group..3.The serum amylase and lipase levels of mice in each group were detected by ELISA,and the changes of the levels in SAP mice model induced by caerulein and the effects of SCFAs supplementation were observed.4.The expressions of MPO(neutrophils)and F4/80(macrophages)in the pancreatic tissue of mice in each group were detected by immunohistochemistry,and the effect of SCFAs on the infiltration of inflammatory cells in the pancreatic tissue of SAP mice was observed.5.The serum concentrations of IL-17 A,IL-1β,TNF-α,IL-6,IL-10 and INF-γwere detected by serum multi-factor ELISA,and the effect of SCFAs on the systemic inflammatory response of SAP mice was observed..6.In order to determine the role of SCFAs in the recovery process of SAP,this study chose to observe the effect of SCFAs on the repair of pancreatic injury on the third day after induction of SAP in two SAP mouse models(simple cerulein model and Bombesin combined with cerulein model)..7.Observing the changes of SAP mice after intestinal injury and supplementation with SCFAs by HE staining.8.In order to observe the effect of SCFAs on the intestinal inflammatory response of SAP mice,this study detected the expression of NLRP3 in the intestinal tract of mice in each group by immunofluorescence,and detected NLRP3,IL-18 and caspase in the intestinal tract of mice in each group by Western blot.-1 The expression of p20 and TLR2,and the relative expressions of NLRP3,IL-1β,IL-6 and TNF-α in the intestinal tract of mice in each group were detected by PCR.9.In order to observe the effect of SCFAs on the intestinal barrier function of SAP mice,in this study,the expression of occludin and claudin-1 in the intestines of mice in each group and the content of serum D-lactic acid were detected by immunofluorescence and Westren blot.Effects of rat intestinal permeability.(3)To investigate the effect of changes in gut microbiota induced by short-chain fatty acid supplementation on the severity of SAP.1.In order to observe the effect of SCFAs on the intestinal flora of the mice in each group,we detected the intestinal flora of the mice in each group by 16 S r RNA,and compared the α diversity and β diversity of the intestinal flora of the mice in each group.and differential bacterial composition.2.In order to observe the role of the intestinal flora in the mice treated with SCFAs in SAP,we transplanted the intestinal flora of the mice treated with SCFAs by the method of flora transplantation,and observed the pancreatic damage of the mice in each group.,Pancreatic tissue inflammatory cell infiltration,serum amylase lipase and serum inflammatory factor levels.We used two mouse models to verify this effect:the normal SPF mouse model and the pseudo germ-free mouse model.3.In order to observe the role of the intestinal flora of mice treated with SCFAs in the intestinal injury of SAP mice,we observed the effect of transplanting the intestinal flora of mice treated with SCFAs on the intestinal pathology of SAP mice.The expressions of occludin and claudin-1 in the intestinal tract of the mice in each group were detected by immunofluorescence and Westren blot,the serum D-lactic acid level of the mice in each group was detected by ELISA,and the intestinal tract of the mice in each group was detected by fluorescence quantitative PCR.Relative expression of inflammatory factors in the tract.4.In this study,the differences in intestinal flora of mice in the normal control group and the SAP model group were compared by the method of comparative analysis between groups.The results showed that the abundance of Lactobacillus＿murinus and Bifidobacterium＿pseudolongum was significantly decreased in SAP mice,and the abundance of Lactobacillus＿reuteri and Bifidobacterium＿pseudolongum was significantly increased in the short-chain fatty acid group after supplementation with SCFAs.These bacteria are closely related to the production of short-chain fatty acids.5.Supplementing with Bifidobacterium＿pseudolongum can significantly alleviate pancreatitis in mice,which is manifested as decreased pancreatic edema,inflammation and necrosis.On the third day after modeling,the mice in the supplemented Bifidobacterium＿pseudolongum group recovered significantly,and the area of pancreatic damage was significantly reduced.6.Supplementation with Bifidobacterium＿pseudolongum can significantly reduce the intestinal barrier damage in SAP mice.Western blot results showed that supplementation with Bifidobacterium＿pseudolongum could up-regulate the expressions of occludin and claudin-1 in the small intestine.7.Supplementation with Bifidobacterium＿pseudolongum can significantly reduce the systemic inflammatory response in SAP mice.Supplementation with Bifidobacterium pseudolongum decreased the serum levels of pro-inflammatory factors IL-17 A,IL-6 and TNF-α,and increased the level of anti-inflammatory factor IL-10 in SAP mice.(4)To explore the possible mechanism of short-chain fatty acids affecting the intestinal barrier function of SAP mice.1.In this study,RNA-seq was used to analyze the changes of gene transcription levels in the small intestine tissue of SAP mice,and the effect of SCFAs intervention on the gene transcription levels of SAP mice small intestine tissue,and screened out transcriptome differential genes.2.In this study,the differential gene sets of the normal control group and the SAP group,and the differential gene sets of the SAP group and the SAP supplemented SCFAs group were subjected to KEGG functional enrichment analysis and GSEA analysis method to investigate whether the expression levels of genes related to related pathways in different groups.do not have significant differences.3.In order to verify the role of IL-17 signaling pathway in SAP and the effect of SCFAs on this pathway,this study first verified the effect of SCFAs on IL-17 A activation in intestinal epithelial cells,and then verified the effect of each group by Western blot experiments.Expression of IL-17 A and STAT1,p-STAT1 and ROR-γt in mouse small intestine.4.In order to observe the effect of the intestinal flora of mice treated with SCFAs on the IL-17 signaling pathway,this study verified the expression of IL-17 A,STAT1,p-STAT1 and ROR-γt in the small intestinal tissues of mice in each group by Western blot experiment.Express.5.In order to observe the effect of supplementing Bifidobacterium pseudolongum on the IL-17 signaling pathway,this study verified the expression of IL-17 A and STAT1,p-STAT1 and ROR-γt in the small intestinal tissues of mice in each group by Western blot experiment.Results:(1)Decreased fecal short-chain fatty acid content in severe acute pancreatitis disease state.1.Collect the feces of SAP patients and healthy people,and use GC-MS technology to detect and quantitatively analyze the fecal SCFAs content of the two groups.The results showed that the content of total acid,acetate,propionate and butyrate in fecal SCFAs of SAP patients was significantly lower than that of healthy people.2.The mouse SAP model was constructed,and the content of SCFAs in the feces of the two groups of mice was detected by GC-MS.The results also found that the total acid,acetic acid,propionic acid and butyric acid of the fecal SCFAs of the SAP mice were significantly lower than those of the normal mice.(2)Short-chain fatty acids can alleviate acute pancreatitis in mouse model.1.Comparing the pancreas histopathology of six groups of mice in control group,cer group,SCFAs＿cer group,Acetate＿cer group,Propionate＿cer group and Butyrate＿cer group,it was found that the pancreatitis in the mixed SCFAs treatment group was significantly alleviated,and the pancreatic edema,inflammatory cell infiltration and acinar cells The necrosis was significantly reduced,and the pancreatitis of the mice treated with acetic acid,propionic acid and butyric acid alone was slightly alleviated,but the effect was not as obvious as that in the three mixed treatment groups.2.The results of Bombesin combined with cerulein model and M3/ptf1α mouse SAP model also confirmed that SCFAs can reduce pancreatic edema,inflammatory cell infiltration and cell necrosis in mice.3.The serum amylase and lipase contents of the mice in each group were detected by ELISA.The results showed that the serum amylase and lipase contents of the mice in the simple SAP model group were significantly increased,while the serum amylase and lipase contents of the mice in the SCFAs supplemented group were significantly increased.content was significantly reduced.4.The expression of MPO(neutrophils)and F4/80(macrophages)in mouse pancreas was detected by immunohistochemistry.The results showed that,compared with the pure SAP model group,MPO in the pancreatic tissue of mice in the SCFAs supplementation group was increased.and F4/80 expression was significantly reduced.5.The serum concentrations of IL-17 A,IL-1β,TNF-α,IL-6,IL-10 and INF-γ were detected in this study by serum multi-factor detection technology.Compared with the mice in the SCFAs supplemented group,serum IL-17 A,IL-1β,TNF-α and INF-γ were significantly decreased,and there was no significant difference in IL-6 and IL-10 compared with the simple model group.6.Observing the effect of SCFAs on the repair of pancreatic damage on the third day after modeling in two SAP models(the cerulein model alone and the Bombesin combined with cerulein model).Large areas of pancreatic tissue damage were still seen in the pancreatic tissue,and normal acinar tissue was relatively small.However,large areas of normal pancreatic tissue were seen in the SAP mice in the SCFAs-supplemented group on the third day.7.Observing the intestinal pathology of mice in different treatment groups,it was found that the small intestinal tissue of the mice in the pure cerulean model group was shorter than that of the normal control mice.However,the damage to the small intestinal villi was alleviated after SCFAs supplementation.8.The expression of NLRP3 in the intestine of mice in each group was detected by immunofluorescence technique.It was found that the expression of NLRP3 was obvious in the nucleus and cytoplasm of the intestinal tissue of the mice in the cerulein model group.After supplementing short-chain fatty acids,the SAP mice were reduced.Intestinal NLRP3 expression;Western blot results showed that compared with normal control mice,the expression of small intestinal tissue proteins NLRP3,IL-18,caspase-1 p20 and TLR2 were significantly up-regulated in the pure cerulein model group,while After supplementation with SCFAs,the expressions of these proteins were significantly decreased(NLRP3,IL-18,caspase-1 p20,TLR2).-6.TNF-α was significantly higher than that in the normal control group,and the level of inflammation in the mice in the SCFAs supplementation group decreased,which was manifested as a significant decrease in the expressions of pro-inflammatory factors IL-1β,IL-6,and TNF-α,as well as the expression of NLRP3 inflammasome.decreased,and the expression of anti-inflammatory factor IL-10 increased.9.The detection results of immunofluorescence and Westren blot showed that the expressions of claudin-1 and occludin proteins in the intestines of mice in the pure SAP model group were significantly down-regulated compared with the normal group,and SCFAs supplementation could increase the protein expressions of claudin-1 and occludin;detection Compared with the simple SAP model group and the supplemented SCFAs model group,the serum D-lactic acid content in the SCFAs supplemented group was significantly decreased.(3)Short-chain fatty acid supplementation alleviates severe acute pancreatitis in mice by affecting the intestinal flora of mice.1.The intestinal flora of mice in each group was detected by 16 S r RNA,and it was found that the Shannon index and Chao index of SAP mice were lower than those of normal control mice,while the Shannon index and Chao index of the intestinal flora of SAP mice after supplementing SCFAs Chao index increased significantly.Continuing to analyze and observe the similarities and differences of the intestinal flora of the mice in each group,it was found that the intestinal flora of the three groups of the normal control group,the SAP group and the SCFAs＿SAP group had a clear trend of separation,indicating that there were obvious differences among the three groups.;Further LEf Se analysis was used to screen the differential bacteria in the intestinal flora of the normal control group and the SAP group,and to observe the changes of the differential bacteria in the SAP mice after supplementation with short-chain fatty acids.Group relative abundances were significantly reduced,whereas supplementation with SCFAs increased the relative abundances of these bacteria in SAP mice.2.The intestinal flora of mice treated with SCFAs can also alleviate pancreatitis in mice.The pancreatic edema,inflammatory infiltration and necrosis area of mice were significantly reduced,the levels of serum amylase and lipase were significantly reduced,and the levels of MPO and F4/ The expression of 80 was also significantly reduced,indicating that the number of inflammatory cells infiltrated in pancreatic tissue was significantly reduced;the results of serum multi-factor detection showed that the intestinal flora of mice transplanted with SCFAs treated serum inflammatory factors IL-17 A,IL-1β,TNF The levels of-α and INF-γ were significantly decreased,indicating that the systemic inflammatory response of the mice in this group was milder.3.Observing the intestinal pathology of mice in different treatment groups,it was found that the intestinal flora of the mice treated with SCFAs was reduced in the SAP group,and the intestinal damage was reduced by thicker mucus layer,longer small intestinal villi,and reduced villus apical defect;and The expression of occludin and claudin-1 in the small intestinal tissue was higher,and the serum D-lactic acid level was significantly decreased.The results of real-time quantitative PCR showed that the intestinal flora of mice after transplantation of SCFAs could also reduce intestinal inflammation in SAP mice,which showed that the expression of pro-inflammatory factors IL-17 A,IL-1β,IL-6,and TNF-α was significant.decreased,and the expression of NLRP3 inflammasome was also significantly decreased.4.In this study,the differences in intestinal flora of mice in the normal control group and the SAP model group were compared by the method of comparative analysis between groups.The results showed that the abundance of Lactobacillus＿murinus and Bifidobacterium＿pseudolongum was significantly decreased in SAP mice,and the abundance of Lactobacillus＿reuteri and Bifidobac-terium＿pseudolongum was significantly increased in the short-chain fatty acid group after supplementation with SCFAs.These bacteria are closely related to the production of short-chain fatty acids.5.Supplementation with Bifidobacterium＿pseudolongum can significantly alleviate pancreatitis in mice,which is manifested as decreased pancreatic edema,inflammation and necrosis.On the third day after modeling,the mice in the supplemented Bifidobacterium＿pseudolongum group recovered significantly,and the area of pancreatic damage was significantly reduced.6.Bifidobacterium＿pseudolongum supplementation can significantly reduce the intestinal barrier damage in SAP mice.Western blot results showed that supplementation of Bifidobacterium＿pseudolongum can up-regulate the expressions of occludin and claudin-1 in the small intestine.7.Supplementation with Bifidobacterium＿pseudolongum can significantly reduce the systemic inflammatory response in SAP mice.Supplementation with Bifidobacterium pseudolongum decreased the serum levels of pro-inflammatory factors IL-17 A,IL-6 and TNF-α,and increased the level of anti-inflammatory factor IL-10 in SAP mice.(4)Short-chain fatty acids may alleviate intestinal barrier damage in SAP mice by regulating IL-17 signaling pathway.1.The RNA-seq method was used to analyze the changes of gene transcription levels in the small intestine of SAP mice,and the effect of SCFAs intervention on the gene transcription levels of SAP mice.The results showed that there were 223 differential genes between SAP and the control group,84 genes were up-regulated and 139 genes were down-regulated in the SAP group(Fig.5.1).Compared with SAP,a total of 314 genes were significantly different in SAP mice after SCFAs supplementation,including69 up-regulated genes and 245 down-regulated genes.2.The results of KEGG functional enrichment analysis showed that the significant enrichment pathways of differential gene sets between the control group and the SAP group were mainly "Cholesterol metabolism","Fat digestion and absorption","IL-17 signaling pathway","PPAR signaling pathway" ","AMPK signaling pathway","Insulin signaling pathway" and other KEGG pathways.We continued to analyze the differential gene sets in the small intestine tissue of the mice in the SAP group and SCFAs＿SAP group for KEGG functional enrichment analysis.The main enriched pathways included"Fat digestion and absorption","IL-17 signaling pathway","PPAR signaling pathway","Cholesterol metabolism","Vitaimin digestion and absorption","Estrogen signaling pathway" and other KEGG pathways.The results of further GSEA analysis showed that the genes of the "IL-17 signaling pathway" pro-inflammatory response pathway can be up-regulated during the development of SAP,and the supplementation of SCFAs in SAP mice can cause the "IL-17 signaling pathway" pro-inflammatory response.Downregulation of genes in response pathways.3.SCFAs inhibit the up-regulation of IL-17 A in intestinal epithelial cells.The role of SCFAs in the IL-17 A signaling pathway was verified in the SAP animal model.The results showed that the SAP group mice IL-17 A,STAT1,p-STAT1 and ROR-γt The expression was significantly higher than that of normal mice,and supplementation of SCFAs could significantly reduce the expressions of IL-17 A,STAT1,p-STAT1 and ROR-γt in the intestinal tissue of SAP mice.4.The results of verifying the effect of the intestinal flora of mice treated with SCFAs on the intestinal IL-17 signaling pathway of SAP mice found that the intestinal flora of mice transplanted with SCFAs treated mice IL-17 A,The protein expressions of STAT1,p-STAT1 and ROR-γt were significantly decreased.5.Western blot detection and supplementation of intestinal tissue proteins of Bifidobacterium＿pseudolongum＿SAP mice and SAP mice showed that Bifidobacterium＿pseudolongum could down-regulate the expressions of IL-17 A,STAT1,p-STAT1 and ROR-γt.Conclusions:1.In the process of severe acute pancreatitis,the content of fecal SCFAs was significantly lower than that in the normal state.2.SCFAs supplementation can significantly reduce intestinal inflammation and intestinal mucosal barrier damage caused by pancreatic pathology and SAP in mice.3.SCFAs supplementation can affect the intestinal flora of SAP mice,and transplantation of the intestinal flora of mice treated with short-chain fatty acids and supplementation of Bifidobacterium＿pseudolongum can significantly reduce the pancreatitis in mice,so it can be speculated that SCFAs supplementation may be through the regulation of SAP The intestinal microflora in mice thus play a role in reducing pancreatitis in mice.4.Supplementation with SCFAs and Bifidobacterium＿pseudolongum can downregulate the activation of IL-17 signaling pathway in SAP mice,and inhibition of IL-17 signaling pathway may provide a new idea for the treatment of severe acute pancreatitis. |
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