Protective Effect Of Lactobacillus Paracasei From Koumiss On The Intestinal Mucosal Barrier Of Diarrhea Mouse Models Caused By E. Coli O8

Diarrhea caused by pathogenic E.coli is very common in livestock production.Infected animals have many symptoms such as diarrhea,elevated body temperature,and dehydration,calves are the most susceptible and cause about 30%mortality in infected calves.Antibiotics can effectively treat this disease,but long-term use of antibiotics can easily lead to a series of problems such as bacterial resistance,intestinal flora destruction and cattle production performance decline.Studies have shown that probiotics have a good effect on inhibiting the growth of pathogenic microorganisms and have the potential to replace antibiotics.Inner Mongolia traditional food koumiss has a variety of therapeutic effects since ancient times,and probiotics play an irreplaceable role.Therefore,the study intends to find a probiotic with bacteriostatic effect from koumiss,and to study the effect of this probiotic on the intestinal microbial environment and intestinal mucosal barrier.Based on this,the following experiments were carried out:1.Study on purification and probiotic properties of probiotics from koumissSeven probiotic strains were isolated from koumiss using MRS and PDA medium,including five lactic acid bacteria（L1,L2,L3,L4 and L5）and two yeasts（Y6 and Y7）.The tolerance of 7 strains of probiotics to acid,bile salt,artificial gastric juice and artificial intestinal juice was tested.The hydrophobicity and self-polymerization ability of lactic acid bacteria were determined and the antibacterial ability was tested.The strain with good probiotic effect were identified.The test results show that:（1）7 strains of probiotics showed good acid resistance when p H=4.When p H increased to 2,L4 and L5 showed better acid resistance than other strains;（2）When the bile salt concentration was 0.5%,L4 and L5 bile salt tolerance was the better than other strains;（3）The tolerance of artificial gastric juice and artificial intestinal juice showed that L4 and L5 strains was better than other strains;（4）The hydrophobicity and aggregation ability of L3 and L4 were better than other strains.The antibacterial ability of strain L4 was significantly stronger than other strains.Based on the above probiotic performance tests,it was found that L4 may have the potential to adapt to the gastrointestinal environment and be used as a probiotic.L4was identified and found to belong to the subspecies of Lactobacillus paracasei.Therefore,L4 was named Lactobacillus paracasei（L.paracasei）.2.Establishment of diarrhea model of pathogenic Escherichia coliSeventy-two SPF Kunming mice were randomly divided into 6 groups,with 12mice in each group.Five strains of Escherichia coli（E.coli O1,E.coli O2,E.coli O8,E.coli O78 and E.coli O86）isolated from the feces of clinical diarrhea calves were intragastrically administered at 3×10¹³ CFU/m L for 7 days.The serum of mice was collected on the 3 th,5 th and 7 th day,and the duodenal tissue and cecal contents were collected on the 7 th day.The contents of serum,duodenal tissue and cecal contents were used to detected IL-6,IL-1β,TNF-α,Ig A,Ig G,Ig M,the pathological changes of duodenum and the changes of cecal contents.The results show:（1）The five strains of E.coli all caused diarrhea in mice.According to histopathological sections,it was found that the duodenal intestinal villi of mice were broken and the intestinal villi gap increased after E.coli challenge,especially E.coli O8 caused the most serious damage;（2）ELISA results showed that the levels of IL-6,IL-1βand TNF-αin serum of mice after challenge were significantly increased,and the content of immunoglobulin was decreased;（3）The abundance of Bacteroidetes,Proteobacteria and Actinobacteria increased in the intestine,especially E.coli O8.Therefore,a mouse diarrhea model was established by continuous challenge with3×10¹³ CFU/m L E.coli O8 for 7 days.To provide an animal model for studying the effect of L.paracasei on Escherichia coli diarrhea.3.Effect of L.paracasei on intestinal mucosal barrier in diarrhea miceSixty SPF Kunming mice were randomly divided into 5 groups:normal control group（CK）,model group（NC）,positive control group（CIP）,L.paracasei prevention group（PE）and L.paracasei treatment group（TE）,with 12 mice in each group.The experiment lasted for 15 days.From day 1 to day 7,mice in NC,CIP and TE groups were intragastrically administered with E.coli O8 to establish diarrhea model.CK group was given 0.9%normal saline by gavage;PE group was treated with L.paracasei.On 8-14 d,CK and NC groups were given 0.9%normal saline by gavage;CIP group was given 125μg/m L ciprofloxacin by gavage;PE group was given E.coli O8;TE group was given L.paracasei by gavage.Six mice in each group were sacrificed on day8 and day 15,and serum,small intestinal tissue and cecal contents were collected for subsequent experiments.The body weight,diarrhea rate and diarrhea index of mice in each group were counted.The cecal contents of mice were analyzed using 16s r RNA sequencing.The serum levels of DAO,zonulin,D-LA,EGF,TGF-β,ITF,IL-6,IL-1β,TNF-α,Ig A,Ig G,Ig M and s Ig A were detected by ELISA.RT-PCR was used to detect the expression of IL-6,IL-1β,TNF-α,IL-10,CXCL2 and CCL2 in ileum tissue.HE staining was used to observe the structural changes of duodenum,jejunum and ileum.The number of goblet cells was observed by AB-PAS staining.The expression of MUC2 was observed by immunohistochemistry.Western blot and immunohistochemistry were used to observe the expression of claudin-1,occludin and ZO-1 in ileum.Immunofluorescence was used to observe the expression of CD3⁺,CD4⁺and CD8⁺.The results show:（1）The body weight growth rate of mice in the challenge group was slow,and diarrhea symptoms appeared.The diarrhea rate and diarrhea index were increased.Preventive use of L.paracasei did not cause diarrhea in mice,and had a certain preventive effect on diarrhea caused by E.coli O8.In addition,the therapeutic effect of L.paracasei on diarrhea after challenge was better than that of the untreated group;（2）Alpha-diversity and Beta-diversity were significantly decreased after E.coli O8 treatment,L.paracasei increasedα-diversity andβ-diversity,and CIP treatment significantly decreased the diversity.This shows that L.paracasei can improve the diversity of flora to some extent,but the use of CIP reduced the diversity of flora to some extent.At the same time,E.coli O8 significantly increased the abundance of Proteobacteria,Stenotrophomonas and Enterobacter,and the intervention of L.paracasei increased the abundance of flora producing short-chain fatty acids such as Roseburia,Parabacteroides,Alistipes and Odoribacter;（3）After E.coli intervention,the expression of DAO,zonulin,D-LA,TGF-β,IL-6,IL-1βand TNF-αin mice were significantly increased,and the number of goblet cells,MUC2,claudin-1,occludin,ZO-1,EGF,ITF,IL-10,Ig A,Ig G,Ig M and s Ig A were decreased.E.coli increased intestinal permeability,stimulated the body to produce inflammatory response and destroyed intestinal epithelial tight junction;（4）E.coli O8 intervention reduced the content of CD3⁺,CD4⁺,CD8⁺and CD4⁺/CD8⁺,but the difference between the groups after treatment was not significant.L.paracasei can prevent the damage of intestinal mucosal barrier caused by E.coli O8,and has a certain therapeutic effect,but the therapeutic effect is not necessarily better than the long-term preventive effect.4.L.paracasei mediates intestinal mucosal repair by inhibiting NF-κB-MLCK and MAPK signaling pathwaysWestern blot was used to study the protein levels of TLR4,IκB-α,p-65,MLC2,MLCK,ERK,p-ERK,p38,p-p38 and JNK in the ileum of mice.Research shows that:（1）E.coli significantly increased the expression of p65,MLC2,MLCK,JNK,p-ERK/ERK and p-p38/p38,while the intervention of L.paracasei had no significant effect,but decreased the expression of IκB and p65;（2）L.paracasei as a preventive drug can effectively inhibit the increase of p65,MLC2,MLCK,JNK,p-ERK/ERK and p-p38/p38 expression and the decrease of IκB after E.coli infection.Therefore,the therapeutic or preventive effect of L.paracasei on E.coli diarrhea may be achieved through the intervention of NF-κB-MLCK and MAPK signaling pathways.