| Objective: To study the changes of microbiota in the colonic mucus layer of rats with severe acute pancreatitis(SAP),and to further explore the influence of the intervention of Dai-Huang-Fu-Zi-Tang on the interaction between mucus layer and microbiota,so as to reveal the mechanism of Dai-Huang-Fu-Zi-Tang regulating the microbiota in the colonic mucus layer of SAP,and provide experimental basis and theoretical basis for the clinical use of Dai-Huang-Fu-Zi-Tang.Methods: Eighteen SPF grade healthy male SD rats were randomly divided into 3 groups after weighing: Sham operation group(Sham group,n=6),severe acute pancreatitis model group(SAP group,n=6)and Dai-Huang-Fu-Zi-Tang therapeutic group(DHFZT group,n=6).In Sham group,the pancreas was turned several times after laparotomy and the abdomen was closed.SAP model was induced in the SAP group and DHFZT group by slow retrograde injection of 4% sodium bezoar cholate into the pancreatic bile duct.After the model was successfully established,the Sham group and the SAP group were intragastrically given 1.5ml normal saline 6h and 12 h after surgery,while the DHFZT group was intragastrically given1.5 ml DHFZT 6h and 12 h after surgery.All moulding was done by the same person.The rats in each group were anesthetized with isoflurane 24 hours after modeling.Blood samples were collected through abdominal aorta,and the contents of amylase,endotoxin,TNF-α and IL-6 in rats were determined by ELASA method.Histopathological changes of lung,pancreas and colon were detected by HE staining.16 SrRNA gene sequencing was used to analyze the structural changes of colonic mucus layer bacteria.Liquid-mass spectrometry(LC-MS/MS)was used to study the microbiological metabolomics of the mucus layer.Results:(1)The serum levels of amylase,endotoxin,TNF-α and IL-6 in SAP group were remarkably increased compared with those in Sham group,while DHFZT group were significantly lower than SAP group.(2)There was no obvious damage in lung,pancreas and colon in Sham group.The SAP and DHFZT groups had different degrees of damage to the lung,pancreas and colon.The SAP group had more severe injuries,and the DHFZT group had less injuries.Histopathological scores in SAP group were significantly lower than Sham group,DHFZT group was significantly higher than SAP group.(3)There were significant differences between groups at both phylum level and genus level,among which Verrucomicrobia had the highest significant difference between groups at the phylum level,while Akkermansia had the highest difference between groups at the genus level.Compared with Sham group,the structure of colonic mucus microflora in SAP group was significantly different.Compared with SAP group,the species diversity of colonic mucus layer in DHFZT group was significantly increased.(4)The metabolites varied significantly between groups.Compared with the Sham group,the content of butyric acid and intermediate products of butyric acid metabolism(-3-Hydroxybutyric acid)decreased in the SAP group,which plays a key role in the Butanoate metabolism.Primary and secondary bile acids were significantly reduced in the DHFZT group compared with the SAP group.Conclusion:(1)The rat model of severe acute pancreatitis was successfully established by retrograde cholangiopancreatobiliary injection of 4% sodium bezoar cholate;(2)Intestinal barrier function was damaged in early SAP rats,and the structure of colonic mucus microflora was changed.After treatment with Dai-Huang-Fu-Zi-Tang,the damage of intestinal mucosa epithelium was alleviated,the species diversity of colonic mucus layer was increased,and the abundance of intestinal mucosa epithelium was increased.(3)Changes in the microorganism structure of the mucus layer in the colon of SAP rats affected butyric acid synthesis and metabolism,and butyric acid content decreased.After treatment with Dai-Huang-Fu-Zi-Tang,the diversity of mucus layer bacteria mainly affected the formation of bile acid,and the content of bile acid was significantly reduced. |
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