Effect Of DHFZT On Intestinal Microbiota And Intestinal Mechanical Barrier In Rats With Severe Acute Pancreatitis

Objective: Intervention of Dai-Huang-Fu-Zi-Tang in rats with severe acute pancreatitis to observe changes in intestinal microbiota.To determine the expression of tight junction proteins Claudin-1,occludin and ZO-1 in intestinal mucosa,the expression of these proteins in SAP rats and the changes after Dai-Huang-Fu-Zi-Tang intervention,To explore the effect and significance of Dai-Huang-Fu-Zi-Tang on gut microbiota and intestinal barrier mechanical barrier function in rats with severe acute pancreatitis.Methods: A total of 24 SPF male SD rats weighing 230-270 g were randomly divided into 4 groups: sham group(n=6),Dai-Huang-Fu-Zi-Tang sham group(n=6),SAP group(n=6),and Dai-Huang-Fu-Zi-Tang treatment group(n=6).Anesthesia with isoflurane;searched the pancreas of sham group and Dai-Huang-Fu-Zi-Tang sham group,turned it over several times after laparotomy.SAP models were established by slow retrograde injection of 4% sodium taurocholate(1ml/kg)into the pancreatic duct.The sham group and SAP group were retention enema with 2 ml of normal saline(about 38 ?)at 12 hours,24 hours and 36 hours after operation.The Dai-Huang-Fu-Zi-Tang sham group and treatment group were treated with 2 ml of Dai-Huang-Fu-Zi-Tang(about 38 ?)at 12 hours,24 hours and 36 hours after operation.Blood samples were taken through the abdominal aorta after 48 h.Plasma endotoxin,blood amylase,serum calcium,serum DAO and inflammatory factor IL-1? were detected by ELISA.Morphological changes of HE staining in lung,pancreas and ileum by microscopic observation.The pathological scoring of lung tissue in each group was performed by Smith's scoring method,to ensure that the SAP model was successfully built.The pathological scoring of pancreatic tissue in each group by modified Schmidt scoring criteria.The pathological evaluation of ileum tissue was scored by Chiu's scoring method.The expression of intestinal mechanical barrier tight junction proteins ZO-1,occludin and claudin-1 in ileum tissue were detected by immunohistochemistry and immunofluorescence.Using Semi-quantitative analysis of tight junction proteins ZO-1,occludin and Claudin-1 by Western-blot.Analysis of gut microbiota by 16 S rRNA gene sequencing.Results: 1.Compared with the sham group the levels of serum endotoxin,amylase,DAO and IL-1? in the Dai-Huang-Fu-Zi-Tang sham group showed no significant changes.The levels of serum endotoxin,amylase,DAO and IL-1? in the SAP group were significantly higher than those in the sham group,and serum calcium levels were significantly lower than those in the control group.Serum levels of endotoxin,amylase,DAO and IL-1? in the treatment group were lower than those in the SAP group,and serum calcium levels were higher than those in the SAP group,with statistically significant differences(P<0.05).2.The structure of pancreatic tissue,lung tissue and ileum were all destroyed after SAP,and the expression of tight junction proteins ZO-1,occludin and Claudin-1 in intestinal epithelium was weakened.After treatment with DHFZT the destruction of pancreatic tissue,lung tissue and ileum was repaired.The expression levels of tight junction proteins ZO-1,occludin and Claudin-1 in intestinal epithelium were significantly increased compared with SAP group.3.The samples of DHFZT sham group were contaminated and is not available for analysing.The other results of ileal tissue analysis showed that there was no significant change in the microbiota richness and diversity of the SAP group compared with the sham group.Dai-Huang-Fu-Zi-Tang treatment significantly increased the abundance(P<0.05,except for the phylum level)and the diversity of the microbiota(P<0.05).There were significant differences in the structure of the three groups of the sham,SAP and Dai-HuangFu-Zi-Tang treatment.According to the analysis of the composition of the flora,the Firmicutes,Bacteroidetes and Proteobacteria are the three dominant bacteria in the relevant flora,especially Firmicutes.There was no significant change in the level of dominant bacteria in the SAP group.Proteobacteria was significantly increased in the Dai-Huang-FuZi-Tang treatment group.At the genus level,Lactobacillus was the dominant genus in the sham group,and its relative abundance decreased significantly after the occurrence of SAP.The dominant position was the relative increase in the relative abundance of unclassified_f__Peptostreptococcaceae.Lactobacillus in the treatment group was significantly increased compared with the SAP group to become the dominant genus.Conclusion: After the occurrence of severe acute pancreatitis,intestinal flora environment changed,can further lead to intestinal structure damage,closely connection expression level lower intestinal mucosal mechanical barrier,intestinal epithelial permeability increase,endotoxin and other harmful substances into the bloodstream,elevated inflammatory factor levels in the blood appears "class" waterfall effect,aggravating illness.After treatment with rhubarb aconite soup,serum endotoxin and IL-1? in blood were reduced,which effectively inhibited pancreatic enzyme secretion and cascade activation of inflammatory factors.At the same time,Dai-Huang-Fu-Zi-Tang promotes the increase of probiotics such as Lactobacillus,and promotes the expression of tight junction proteins in intestinal epithelium,improves the mechanical barrier of intestinal mucosa,and achieves the effect of relieving the disease.