| Background & objective:Acute pancreatitis(AP)is a common digestive tract disease caused by a variety of etiology,and the autoactivation of trypsin in vivo is considered to be one of the pathogenesis mechanisms.In recent years,the morbidity of AP has been on the rise,and about 30% of patients developed acute necrotic pancreatitis(ANC),and the morbidity of pancreatitis was significantly higher when secondary pancreatic infection or extrapancreatic infection occurred.The treatment of severe acute pancreatitis(SAP)is still mainly symptomatic treatment,if concurrent feasible minimally invasive surgical treatment or pancreatic necrosis infection,although the current treatments on survival are increased a few decades ago,later the patient's quality of life,the economic burden and the complications are still deeply threatened the SAP patients.Therefore,reducing the incidence and mortality of SAP complications is still one of the difficult problems facing clinicians.The severity of SAP is related to many factors,including intestinal flora imbalance,intestinal barrier damage,and activation of inflammatory signaling pathways,which are closely related to the severity of SAP.Whether early use of antibiotics to prevent melt SAP intensive has been in debate.Previous study found that the prophylactic use of antibiotics may improve the prognosis of patients with SAP,but still need to be more clinical and basic research.In this study,rifaximin was selected as a prophylactic antibiotic to improve the prognosis of SAP.Therefore,this study intended to observe the effect of the prophylactic antibiotic rifaximin in SAP mice,and to explore the possible mechanism of the effect of rifaximin.Materials and methods:?.To study the effects of rifaximin on the pancreatic severity of mice with severe acute pancreatitis1.A mouse model of SAP was established using Balb /c and C57/BL6 mice.Eight Balb /c and C57/BL6 mice were randomly divided into two groups: one group was a CER + BOM group(n=4),and the other group was a normal saline control group(n=4).The pathological damage degree and amylase lipase level of the pancreas of mice after CER + BOM modeling were observed to determine whether the mouse model of SAP could be established stably.2.In order to further clarify the effect of rifaximin on the severity of SAP,Balb/c and C57/BL6 mice were used to observe the therapeutic effect of rifaximin.The two mouse components were divided into four groups(Con group,Rif group,Cer +Bom group and Cer + Bom + Rif group),with 10 mice in each group.C57/BL6 mice were 5 in each group.Pathological HE staining was used to observe the degree of pancreatic injury.3.The levels of amylase and lipase in blood were detected by amylase and lipase ELISA kit to observe whether rifaximin could affect the levels of amylase and lipase in blood.4.The m RNA levels of inflammatory cytokines(IL-1?,IL-6 and TNF-?)in pancreatic tissues were detected by fluorescence quantitative PCR.The level of MPO(myeloperoxidase)in pancreatic tissue was detected by immunohistochemical staining to observe the therapeutic effect of rifaximin.5.The blood inflammation multi-factor kit was used to check the levels of inflammatory factors in the blood and observe whether rifaximin could reduce the levels of inflammatory factors in the blood.?.To investigate the changes of intestinal flora in the process of rifaximin affecting mice with severe acute pancreatitis1.The caecum tissues of mice after modeling were taken(grouping the same as before),and the caecum contents were sequenced and analyzed for 16 S intestinal flora.After DNA extraction and PCR amplification,Illumina Miseq sequencing and data processing were used to obtain the data.The results were analyzed by ? diversity,? diversity,intestinal flora composition and LEFSE analysis to screen key bacteria genus,to observe whether rifaximin regulates intestinal flora and affects the severity of SAP.?.To investigate the role of intestinal flora in the effect of rifaximin on severe acute pancreatitis in mice using pseudoaseptic mice and aseptic mouse models.1.Balb/c and C57/BL6 mice were used to construct a pseudoaseptic SAP mouse model and divided into four groups(Cer + Bom group,Cer + Bom + Rif group,ABX Cer + Bom group and ABX Cer + Bom + Rif group),with 10 mice in each group.C57/BL6 mice were divided into 5 mice in each group.The degree of pancreatic pathological injury was observed by pathological HE staining and pancreatic pathological score.2.The levels of amylase and lipase in blood were detected by amylase and lipase ELISA kit to observe whether rifaximin could affect the levels of amylase and lipase in blood of pseudosterile mice.3.The m RNA levels of inflammatory cytokines(IL-1?,IL-6 and TNF-?)in pancreatic tissues of pseudosterile mice were detected by fluorescence quantitative PCR.The level of MPO(myeloperoxidase)in the pancreatic tissues of pseudo sterile mice was detected by immunohistochemical staining to observe the therapeutic effect of rifaximin.4.Inflammatory cytokines in the blood of pseudoseptic mice were examined by blood inflammation multi-factor kit to observe whether rifaximin could reduce the level of inflammatory cytokines in the blood of pseudoseptic mice.5.A sterile SAP mouse model was established using Balb/c sterile mice.Twelve mice were divided into two groups: one was a sterile model group(GF Cer+ Bom,n=6)and the other was a sterile rifaximin group(GF Cer+ Bom + Rif,n=6).Pathological HE staining was used to observe the degree of pathological damage of the pancreas of sterile mice and pancreatic pathological score.6.The levels of amylase and lipase in blood were detected by amylase and lipase ELISA kit to observe whether rifaximin could affect the levels of amylase and lipase in blood of sterile mice.7.The m RNA levels of inflammatory cytokines(IL-1?,IL-6 and TNF-?)in the pancreatic tissues of sterile mice were detected by fluorescence quantitative PCR.?.Intestinal transcriptome sequencing analysis was conducted to explore the possible mechanism of rifaximin affecting the prognosis of mice with severe acute pancreatitis.1.Intestinal transcriptome sequencing analysis of ordinary Balb/c mice was conducted and divided into four groups,namely Con group,Rif group,Cer + Bom group and Cer + Bom + Rif group.Intestinal RNA sequencing samples were prepared according to the requirements,and RNA sequencing results were analyzed after RNA extraction,quality assessment and purification.After obtaining the results,identification,comparison,differential gene screening,GO and KEGG enrichment analysis were carried out to screen out the relevant signal pathways,then PCR and WB methods were used for verification.Result:?.To study the effect of rifaximin on pancreatic severity in mice with severe acute pancreatitis1.After the modeling of two kinds of mice,edema,inflammation and necrosis were observed in the Cer + Bom group compared with the Con group,and amylase and lipase were significantly higher than those in the Con group.After repeated experiments,it was confirmed that the mouse model of SAP had been stably established.2.The gross pancreas volume of the Cer + Bom group was significantly increased compared with the control group,and the pancreas weight was reduced after preventive treatment with rifaximin.The pancreas weight ratio of the Cer + Bom group was significantly increased compared with the normal group.Pancreatic weight was reduced after prophylactic use of rifaximin compared with the Cer + Bom group.Pancreas of Balb /c and C57/BL6 mice showed significant edema,inflammation and necrosis of the pancreas in the Cer + Bom group,and pancreas edema,inflammation and necrosis were significantly reduced after rifaximin intervention.Pathological score showed that pancreas edema,inflammation and necrosis in the rifaximin intervention group.The necrosis and total score were lower than those of the Cer +Bom group(P < 0.05),indicating that prophylactic use of rifaximin can reduce the severity of pancreatic pathology in SAP mice.3.Amylase and lipase in Balb /c and C57/BL6 mice were significantly increased in the Cer + Bom group,and significantly decreased in the rifaximin intervention group(Cer + Bom + Rif)compared with the Cer + Bom group,suggesting that prophylactic use of rifaximin can reduce the secretion of enzymes in SAP mice.4.The m RNA levels of various inflammatory cytokines(IL-1?,IL-6,TNF-?)in BALB /c and C57/BL6 mice model group(Cer + Bom)were significantly increased,while the m RNA levels of three inflammatory cytokines in rifaximin intervention group(Cer + Bom + Rif)were decreased.The neutrophils(MPO)content was significantly increased in the Cer + Bom model group and decreased in the rifaximin intervention group(Cer + Bom + Rif).These results confirm that prophylactic use of rifaximin can reduce the levels of pancreatic inflammatory factors in mice with SAP.5.The serum IL-6 content in the Cer + Bom group was significantly higher than that in the control group(Con),and the serum L-6 level was decreased after the prophylactic use of rifaximin(P < 0.05),indicating that the prophylactic use of rifaximin can also reduce the content of inflammatory factors in the blood.?.To investigate the changes of intestinal flora in mice with severe acute pancreatitis affected by rifaximinAfter preliminary screening and quality control,a total of 556 OTUs were obtained from all the samples after removing rare OTUs and sequence pumping treatment.Results such as sparse curves suggest that the current 16 S sequencing depth and data are sufficient to reflect the composition of most of the flora of cecal content species.The ? diversity analysis of the intestinal flora indicated that the composition of the intestinal flora after rifaximin intervention was significantly lower than that of the non-intervention group,which confirmed that the composition of the intestinal flora was affected by rifaximin during the disease progression(P < 0.05).The results of ?-diversity analysis showed that the microflora structure of the rifaximin intervention group and the model group were distributed in different quadrants,suggesting that the intestinal microflora structure of the rifaximin intervention group was significantly different from that of the model group.At the phylum level,the relative abundance of Firmicutes and Bacteroidetes increased in the normal group,while the number of Proteobacteria increased significantly in the rifaximin intervention group.Veen analysis found 219 common OUT in the modeling group and the rifaximin intervention group.LEFSE analysis showed that there were a total of 50 species with significant differences among groups at the phylum,class,order,family and genus levels(P < 0.05),and the dominant flora was different among groups.?.The effects of rifaximin on severe acute pancreatitis in mice were investigated in pseudoaseptic mice and aseptic mice.1.After prophylactic administration of rifaximin,the volume of pseudoaseptic SAP mice decreased significantly,and the results of pancreatic weight ratio analysis were consistent with that of the naked eye.The results of HE staining indicated obvious edema,inflammation and necrosis in the pancreatic tissue of the pseudoseptic SAP mice.The pathological score analysis showed that rifamximin also alleviated the severity of the pseudoseptic SAP mice.2.The levels of amylase and lipase decreased after rifaximin intervention in pseudoseptic SAP mice,which confirmed that rifaximin reduced amylase and lipase contents in pseudoseptic SAP mice,indicating that intestinal flora largely did not affect the effects of rifaximin.3.The m RNA content and MPO level of pancreatic inflammatory cytokines(IL-1?,IL-6,TNF-?)in rats with pseudoseptic SAP after rifaximin intervention were significantly lower than those in mice with pseudoseptic severe acute pancreatitis(ABX Cer+ Bom)(P < 0.05).It was confirmed that rifaximin also reduced the level of pancreatic inflammation in pseudoseptic SAP mice,suggesting that the effect of rifaximin was not entirely dependent on intestinal flora.4.The level of IL-6 in the blood of mice with pseudoseptic SAP after rifaximin intervention was significantly lower than that of mice with pseudoseptic SAP,suggesting that rifaximin can also reduce the content of inflammatory factors in the blood of mice with pseudoseptic SAP.5.The pancreas weight ratio analysis results show that after the intervention of sterile in SAP in mice pancreas weights are sterile in SAP group decreased obviously,pancreatic pathology HE staining results suggest after the intervention of sterile degree of SAP group in mice pancreas pathology of SAP group decreased,It suggested that rifaximin could reduce the severity of pancreas in sterile mice.6.Amylase and lipase levels decreased after rifaximin intervention in mice with severe aseptic acute pancreatitis,confirming that rifaximin reduced amylase and lipase levels in aseptic SAP mice.7.The m RNA levels of pancreatic inflammatory factors(IL-1?,IL-6,TNF-?)in sterile SAP mice after rifaximin intervention were significantly lower than those in pseudoseptic SAP mice(P < 0.05),which confirmed that rifaximin alleviated pancreatic inflammation in sterile SAP mice.?.Intestinal transcriptome sequencing analysis to investigate the effect of rifaximin on the prognosis of mice with severe acute pancreatitisThe purity and integrity of the extracted intestinal RNA were evaluated,and the results indicated that the RNA had high purity and no other protein contamination.The results of transcriptome sequencing were compared with the reference sequence,and the results indicated that the transcriptome sequencing results had high uniformity and coverage.Compared with the control group,there were 905 differentially transcriptional genes in the model group of SAP,among which 663 genes were up-regulated and 242 genes were down-regulated.Compared with the SAP model group,there were 818 transcriptional differential genes in the rifaximin intervention group,including 187 up-regulated genes and 631 down-regulated genes.Among the identified differentially transcribed genes,a total of 409 differentially overlapping genes were found between the up-regulated SAP-VS-Con genes and the down-regulated SAP-VS-Con genes,and 44 differentially overlapping genes were found between the down-regulated SAP-VS-Con genes and the up-regulated SAP-VS-Con genes.The GO and KEGG analysis suggested that multiple genes and signaling pathways were involved.Finally,TLR-4/ NF-?B pathway was selected as the differential signaling pathway.PCR fluorescence quantification and WB detection of the pathway suggested that TLR-4/ NF-?B pathway played a role in the progression of rifaximin in the intervention of SAP.Conclusion:1.Rifaximin alleviated pancreatic pathological injury and inflammatory response in SAP mice.2.Rifaximin reduced pancreatic injury in normal,pseudoseptic and aseptic SAP mice,suggesting that rifaximin does not completely regulate SAP severity through intestinal flora.3.Rifaximin can affect the severity of SAP through TLR-4/ NF-?B signaling pathway. |
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