| Background: Acute Pancreatitis (AP) is one of clinical common surgical Acute abdomen,The 2012 Atlanta classification criteria classified it as mild,severe,or severe acute pancreatitis.Now there are 15% ? 30% of AP patients may develop Severe Acute Pancreatitis (SAP) .According to the classification standard,SAP refers to persistent organ failure (more than 48h) accompanied by local complications,with a mortality rate of 10% to 30%.Studi es have shown that SIRS/Spesis caused by infection is the main cause of Multiple Organ Disfunction Syndrome (MODS) caused by SAP.Acute Lung Injury (ALI) is also the main cause of early high mortality.60-70% of SAP patients who died within 7 days of admission died mainly from respiratory failure.Intestinal microecological disorder and intestinal barrier dysfunction,accompanied by intestinal flora and endotoxin translocation,may be the major factors for the complications of SAP infection.The mechanism o f intestinal flora translocation is not yet clear,and it is generally believed that it is closely related to the destruction of intestinal barrier.Intestinal flora may be translocated by blood,lymph or directly across the intestinal wall.Chinese medici ne considers AP as a category of visceral diseases.A ccording to Chinese medicine,gastrointestinal fever is one of the main pathogenesis of AP acute stage,which is yangming disease,and is manifested as “pi” “man” “zao” “shi” “jian”.Synopsis of golden chamber records: According to the heart of the full an d pain,this is the real.Therefore,it should be given the method of attacking through it.Purging intestines and stomach,driving out evil spirits.Rhubar b and its derivatives have been studied on the treatment of SAP and have protective effect on intest inal barrier.Emodin (EMO) is derived from anthraquinones purified from rhubarb.Therefore,it is worth studying how emodin is involved in the regulation of intestinal bacteria flora.Objective: Taking rats with severe acute pancreatitis as the animal model,taking SAP intestinal barrier injury as the ent ry point and 16S rDNA high-throughput sequencing as the research method,the intestinal bacte ria flora changes of cecum contents and bacterial translocation of mesenteric lymph nodes were studied to explore the regulation effect of emodin on intestinal bacteria flora.Methods: A total of 60 healthy adult male SD rats without specif ic pathogen (SPF) grade (180g?230g) were randomly divided into two large groups.The first group was the time point group.The main purpose was to explore the changes of intestinal barrier injury in SAP.Control (CON) group,6h SAP,12 h SAP,24 h SAP and 36 h SAP,each group of six.The second largest group was the drug intervention group,whose main purpose was to explore the bacterial translocation of changes in the intestinal bacteria flora of SAP and the effect of emodin intervention.The 30 rats were randomly divided into 5 subgroup s: Sham group,SAP group,EMO group,Dexamethasone (DEX) grou p,Ampicillin (AMP) group,each group of six.SAP disease model was prepared by slow retrograde injection of 5% sodium taurocholate (1ml/kg) through the biliopancreatic duct.The CON group was not treated,and the Sham group only slightly flipped the pancreas three times aft er laparotomy.EMO group was given (40mg/kg) by gavage twice at 2h and 12 h after surgery.Dexamethasone injection (10mg/kg) was intraperitoneally injected 2h and 12 h after surgery in the DEX group.Ampicillin sodium solution (30mg/kg) was intraperitoneally injected 2h and 12 h after surgery in the AMP group.The physiological state and activity changes of the rats in each group were observed and recorded.The first group was anesthetized at ea ch time point and the second group was anesthetized and colle cted 24 hours after the operation.The gross anatomy of each group of rats was observed at the time of open abdominal sampling,and the blood of abdominal aorta was collected immediately,and the plasma was collected.The plasma amylase (AMY) ,endotoxin (ET) and diamine oxidase (DAO) was detected by ELISA.Pancreatic and intestinal tissues were collected,and tissue damage was observed by hematoxylin-eosin (HE) staining.In addition,cecum contents and mesenteric lymph nodes of all rats in the drug intervention group was collected and stored as required.Western bolt was used to detect the expression of tight junction protein ZO-1 and Occludin,and 16S rDNA high-throughput sequencing was used to detect the changes of intestinal bacteria flora in the cecum contents and the bacterial translocation and changes in the mesenteric lymph nodes.Result: Compared with CON group and Sham group,the general performance of the SAP group at each time point was worse,and the general cond ition of the abdominal cavity was more serious.The pancreatic and ileum tissues in HE sections showed different degrees of patho logical changes.The pathological score was significantly increased (p<0.05) ,and the content of plasma AMY,ET and DAO was significantly increased (p<0.05) .The condition of the rats gradually worsened with the change of time,and reached a peak at 24 h.Compared with Sham group,the expression of tight junction proteins ZO-1 and Occludin-1 in intestinal tissues in 24 h SAP group was significantly decreased (p<0.01) .Compared with the SAP group,the general performance of rats in the three EMO,DEX,AMP groups was significantly improved,and the ileal and pancreatic tissue pathological scores of the plasma AMY and DAO and the expres sion of tight junction protein ZO-1 and Occludin-1 were alleviated to different degrees (p<0.05) .In addition,ET detection results showed that compared with SAP,EMO and DEX could significantly reduce their expression levels (p<0.05) ,while there was no statistical difference in the regulatory effect of AMP on ET (p>0.05) .The 16S rDNA sequencing results showed that there were bacteria in the cecum contents and mesenteric lymph nodes,and the composition of the two groups of bacteria was significantly different.The main dominant phylum were Firmicutes,Bacteroides and Proteobacteria,but the species composition ratio was significantly different,which may be related to the different int ernal environment.Compared with the Sham group,the pathogenic bacteria of the SAP group such as Erysipelotrichia,Helicobacter,Clostridia and Lachnospiraceae increased,while the common bacteria such as Lactobacillus,Bifidobacteria and Aminoacidcoccus decreased.Compared with the SAP group,the pathogenic bacteria such as Erysipelotrichia significantly decreased in the EMO group,while the common bacteria such as Lactobacillus increased.The change of intestinal bacteria flora in DEX group was not obvious.AMP group showed a decreasing effect on both beneficial and harmful bacteria.Conclusion:1.The rat model of severe acute pancreatitis can be successfully prepared by retrograde injection of sodium beguile cholate through the biliopancreatic duct.The condition of SAP rats is time-dependent,with significant destruction of the intestinal mucosal barrier,increased intestinal permeability and a large amount of endotoxin into the blood within 24 hours.2.The intestinal bacteria flora in the cecum contents of SAP rats changed to different degrees and the bacteria translocated in the mesenteric lymph nodes,which was manifested by the increase in the abundance of pathogenic bacteria such as Erysipelotrichia,Helicobacter,Clostridium and Lachnospiraceae,and the decrease in the abundance of common bacteria such as Lactobacillus,Bifidobacteria and Aminoacidcoccus.3.Emodin has a significant protective effect on the intestinal mucosal barrier and reduce endotoxin into the blood,which can up-regulate the beneficial bacteria mainly Lactobacillus and down-regulate Enterobacter and other pathogenic bacteria.What's more,emodin can correct the imbalance of intestinal bacteria flora and inhibit bacterial translocation to a certain extent.Dexamethasone can also relieve the SAP condition in rats,assist to reduce intestinal mucosal barrier damage and endotoxin into the blood,but has no obvious regulatory effect on intestinal flora disorders.Antibiotics can slightly reduce the tissue damage of SAP and delay the disease,but cannot effectively protect the intestinal mucosal barrier or reduce the endotoxin into the blood.It can not only reduce the pathogenic bacteria but also the beneficial bacteria,killing bacteria while producing endotoxin while killing bacteria,which may increase the endotoxin entering the blood circulation and produce endotoxemia when the intestinal barrier function is impaired. |
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