A Study On Protective Effection On Mongolian Medicine“Babu-7” Compound On Intestinal Mucosal Barrier In Mice With Diarrhea Induced By Escherichia Coli

Diarrhea caused by E.coli is an infectious disease with a high mortality rate,causing serious economic losses in the livestock industry.Previous studies have evaluated the clinical efficacy of BBS against diarrhea.However,there are few studies evaluating the effect of BBS on intestinal function in E.coli induced dirrhea.Accordingly,in order to study the effect of BBS on intestinal barrier function in E.coli-induced diarrhea.The present study have five experiments as follow.Experiment 1:In order to establish diarrhea model,90 mice were divided into 5groups.Vehicle group(VG,oral instillation of saline,n=10),model I group(MG I,intraperitoneal injection of 1.0×10⁹CFU/m L E.coli O₁,n=20),model II group,(MG II,oral instillation of 1.0×10⁹CFU/m L E.coli O₁,n=20),model III group,(MG III,single oral instillation of 2%Na HCO3 and 1.0×10⁹CFU/m L E.coli O₁,n=20),model IV group,(MG IV,consecutive 1 time/3 d instillation of 2%Na HCO3 and 1.0×109CFU/m L E.coli O1,n=20).The mice were observed for 7 d after the attack for diarrhea rate,mortality,DAI score,intestinal lesions and detection of inflammatory factors.The results showed that MG?mice showed diarrhea symptoms and mortality rate reached 75%.MG?mice showed no abnormality;MG?mice showed diarrhea rate was 60%and mortality rate was 15%,and diarrhea symptoms resolved spontaneously after 24 h of attack.The diarrhea rate of MG?mice reached 65%and the mortality rate reached 15%.The symptoms of diarrhea,depression and anorexia in surviving mice could last for more than 72 h,and the intestinal pathological changes and inflammatory factors were significantly higher than those in the normal control group.The results showed that 3 d consecutive(1 time/d)instillation of 2%sodium bicarbonate solution and 1.0×10⁹CFU/m L of pathogenic E.coli O₁could successfully establish a mouse diarrhea model.Experiment 2:To study the effect of BBS on the mechanical barrier of intestinal mucosa in mice with E.coli-induced diarrhea.125 mice were divided into 9 groups:normal control group(VG,n=10),model group(MG,n=10),positive control group(CG,n=15),high,medium and low dose groups of the Mongolian medicine Babu-7(BBS-H,BBS-M,BBS-L,n=15),high,medium and low dose groups of Emodin(Em-H,Em-M,Em-L,n=15).The mice were treated with gavage for 7 d.On the 8th day,killed the mice and collected samples.The pathological damage of mouse intestine was observed by light microscopy and calculated the villus height/Crypt depth(V/C)ratio;the ultrastructure of mouse intestine was observed by transmission electron microscopy;the genes and proteins of Claudin-1,Occludin,ZO-1,MLCK and JAMA were detected by RT-PCR,immunohistochemistry and Western blot expression.ELISA method for the determination of DAO,D-LA and ET in mouse serum.The results showed that the intestinal villi in each intestinal segment of MG were broken,inflammatory cell infiltration and V/C ratio were decreased(P<0.05),the expression of tight junction-related proteins Claudin-1,Occludin,ZO-1,MLCK and JAMA were decreased(P<0.05),and the serum levels of DAO,D-LA and ET were significantly increased(P<0.05).However,Mongolian medicine BBS-H group and EM-H group can alleviate intestinal injury(P<0.05),increased V/C value and tight junction associated protein expression(P<0.05)and decrease the content of DAO D-LA ET in serum(P<0.05).It was shown that the Mongolian medicine BBS-H and Em-H could reduce intestinal mucosal permeability by increasing the expression of tight junction-related proteins,thus to repair the intestinal mechanical barrier damage caused by pathogenic E.coli O₁(P<0.05).Experiment 3:To study the effect of BBS on the immune barrier of intestinal mucosa in mice with E.coli-induced diarrhea.The percentages of CD3⁺T,CD4⁺T,CD8⁺T,CD19⁺B cells and TH1 and TH2 cells were measured by flow cytometry and calculated the CD4⁺/CD8⁺ratios;the immune-related cytokines IL-1?,IL-6,IL-8,TNF-?,IL-2,IL-4,IL-10,IFN-?,TGF-?,IL-17,IL-23,and SIg A in the duodenum,immunoglobulin Ig M,Ig G,lg A in serum was measured by ELISA.The results showed that pathogenic E.coli O₁decreased the percentage of CD3⁺T,CD4⁺T,CD19⁺B and CD4⁺/CD8⁺ratio(P<0.05),elevated IL-1?,IL-6,IL-8,NO,TNF-?,IL-17,IL-23(P<0.05)contents,decreased IL-2,IL-4,IL-10,IFN-?,TGF-?,SIg A,Ig M,Ig G,lg A contents(P<0.05).However,BBS-H and Em-H can increased the percentage of CD3⁺T,CD4⁺T,CD19⁺B and CD4⁺/CD8⁺ratio(P<0.05),decreased IL-1?,IL-6,IL-8,NO,TNF-?,IL-17,IL-23 content(P<0.05),increased IL-2,IL-4,IL-10 IFN-?,TGF-?,SIg A,Ig M,Ig G,lg A contents(P<0.05).Thus,BBS-H and Em-H could repair the immune barrier damage caused by E.coli O_1.Experiment 4:To study the effect of BBS on the chemical barrier of intestinal mucosa in mice with E.coli-induced diarrhea.The intestinal epithelial cup cells were observed by PAS staining;the MUC-2 content in mouse duodenum was detected by immunofluorescence;and the intestinal trefoil factor,lysozyme,disaccharidase activity in duodenum were detected by ELISA.The results showed that pathogenic E.coli O₁decreased the number of global cells and MUC-2,TFF3 content,and attenuated lysozyme,disaccharidase activity(P<0.05).However,BBS-H and Em-H significantly increased the number of global cells and MUC-2,TFF3 content,and increased lysozyme,disaccharidase activity(P<0.05).Thus,BBS-H and Em-H can repair the intestinal chemical barrier damage caused by pathogenic E.coli O_1.Experiment 5:To study the effect of BBS on the biological barrier of intestinal mucosa in mice with E.coli-induced diarrhea.The 16S rDNA sequencing technology was used to sequence the microorganisms in the mouse cecum.The effects of BBS on the intestinal mucosal barrier of diarrhea mice induced by E.coli O₁were analyzed comprehensively by?diversity and?diversity.The results showed that E.coli O_1.invasion decreased Chao1,Ace,and Shannon indices(P<0.05)and increased Simpson index(P<0.05),whereas BBS and EM treatment increased Chao1,Ace,and Shannon indices and decreased Simpson index(P<0.05).Invasion of E.coli O₁reduced the diversity and abundance of intestinal flora and decreased the relative abundance of the phylum Bacteroides,the order Bacteroides,the genus Bacteroides,the phylum Verruciformes,the order Verruciformes,the order Lactobacillus,and the genus Lactobacillus(P<0.05).Increased the relative abundance of Bacillus phylum,Bacillus phylum,Bacillus phylum,Clostridium phylum,Clostridium phylum,Enterobacter phylum,and Enterococcus phylum was increased(P<0.05).In contrast,BBS-H and Em-H treatments could restore the flora structure and increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria.