Study On The Function Of Lactobacillus Rhamnosus Against Enteropathogenic Bacteria Infection Through Regulating Intestinal Homeostasis

Lactobacillus rhamnosus is one of the normal flora in human and animal intestines.It is an important probiotics with high intestinal adhesion rate and strong colonization ability.It can form intestinal biological barrier,improve non-specific immunity,promote intestinal homeostasis,and then enhance the ability of the body to resist intestinal pathogenic bacteria infection.However,at present,the mechanism of Lactobacillus rhamnosus regulating intestinal homeostasis against pathogenic bacteria infection is not completely clear,and the research on the interaction between Lactobacillus rhamnosus and host to regulate the immune mechanism lags behind.Therefore,in order to further recognize the probiotic characteristics of Lactobacillus rhamnosus and better serve the production practice,this study used Lactobacillus rhamnosus intestinal isolate CIQ249 as a model bacteria to explore the potential mechanism of Lactobacillus rhamnosus in regulating intestinal homeostasis against intestinal pathogenic bacteria.In the first part,the one-step growth curve,acid production ability,intestinal colonization ability,digestive environment tolerance and promoting animal growth of CIQ249 strain were determined.The specific contents and results are as follows: firstly,the preserved Lactobacillus rhamnosus isolate CIQ249 was confirmed by Gram staining,biochemical identification and 16 Sr RNA gene sequencing,and the one-step growth curve and the corresponding acid production level of the strain were further determined.The experimental results showed that the bacteria entered the logarithmic growth phase 4 h after being transferred to culture at a ratio of 1: 100,and reached the growth platform stage at 14 h.At the same time,the ability of acid productio n increased with the proliferation of bacteria,and there was a positive correlation between them,and the p H value tended to be stable when the p H value decreased to about 3.0.Using BALB/c mice as an animal model,the intestinal colonization ability of C IQ249 strain was evaluated by CFDA-SE fluorescent probe.The results showed that the detection ratio of labeled bacteria in jejunum,ileum and colon of mice was 75.03%,86.43% and 86.52% respectively on the first day after administration of CFDA-SE-labeled bacteria,and then decreased gradually.On the 7th day,the average detection ratio of labeled bacteria in jejunum,ileum and colon was about 50%.The detection ratio was still more than 20% on the 15 th day after administration,indicating that the strain had good intestinal colonization ability.The tolerance of CIQ249 strain to digestive environment was evaluated.The results showed that the strain had a certain tolerance to extreme digestive environments such as gastric juice of p H1.5,0.3% bile salt an d 9% sodium chloride(hypertonic environment).and showed a growth trend in simulated intestinal juice environment.In addition,oral administration of CIQ249 strain could increase the animal's average daily feed intake and average daily weight gain.In the second part,the bacteriostatic effect of metabolites of CIQ249 strain,the ability to protect animals against intestinal pathogenic bacteria and the effect on infection animal growth performance were evaluated.The specific contents and results are as fo llows: the bacteriostatic effect of metabolites of CIQ249 strain in vitro was tested by bacteriostatic zone method.The supernatant of cell culture centrifuged(not adjusted / adjusted to p H)against Escherichia coli K99,Escherichia coli O78,Escherichia coli O157,Enteropathogenic Escherichia coli,Streptococcus haemolyticus,Serratia marcescens,Staphylococcus aureus,Yersinia enterocolitica,Shigella,Salmonella typhimurium,Citrobacter freundii,Vibrio alginolyticus,Listeria monocytogenes,Vibrio cholerae,Pathogenic bacteria such as Vibrio mimicus and Vibrio parahaemolyticus showed certain inhibitory effect.At the same time,scanning electron microscope observation showed that the pathogenic bacteria showed obvious shrinkage,which indicated that the metabolites of CIQ249 strain had bacteriostatic effect,but had no inhibitory effect on Lactobacillus plantarum,Lactobacillus German,Enterococcus faecium and Lactobacillus Royale.Mice and chicks were given intragastric administration of CIQ249 strain,and then mice were orally infected with absolute lethal dose of Salmonella typhimurium and Escherichia coli K99,and chicks were orally infected with Salmonella typhimurium and Escherichia coli O78.At the same time,CIQ249 group,pathogenic bacteria group,CIQ249 and pathogenic bacteria mixed group and normal animal control group were set up.The results showed that oral CIQ249 strain could provide animals with a certain protective effect against intestinal pathogenic bacteria.The protection rate was up t o 60%.The survival rate of animals in the mixture group of strain CIQ249 and pathogenic bacteria was more than 50%,while all animals in the group infected with pathogenic bacteria died.The results of intestinal pathological observation of experimental animals showed that intestinal pathogenic bacteria infection could lead to serious pathological damage of intestinal mucosa,while oral administration of CIQ249 strain could effectively reduce intestinal mucosal injury,promote the growth of intestinal villi,increase the ratio of villus length to crypt depth,reduce the clinical symptom score of infected animals and improve the growth performance of animals infected by pathogenic bacteria.In the third part,we evaluated the effect of CIQ249 strain on the e xpression of tight junction protein,promoting the secretion of immune-related cytokines and regulating the secretion of mucosal antibody Ig A.Specific contents and results are as follows: indirect immunofluorescence,q PCR and Western blot method was used to detect the effects of strain CIQ249 on the expression of intercellular tight junction proteins Zo-1 and Claudin-1 in vitro and in vivo.The results showed that CIQ249 strain could significantly promote the m RNA transcription and protein expression of intercellular tight junction proteins Zo-1 and Claudin-1 genes in porcine intestinal epithelial cells(PIEC)in vitro.The results are the same after feeding mice and chicks.The m RNA transcription and protein levels of Zo-1 and Claudin-1 genes in the pathogenic bacteria group were down-regulated,while the levels of pathogenic bacteria Zo-1 and Claudin-1 were not down-regulated in experimental animals after feeding CIQ249 strains,indicating that Lactobacillus rhamnosus could resist the infection of pathogenic bacteria by enhancing the tight junction between cells.The levels of cytokines IFN-?,IL-2,IL-4,IL-17 and IL-27 in serum and intestinal mucus of mice fed with CIQ249 strain were detected by ELISA kit.Compared with the blank control group,the levels of these cytokines showed a significant upward trend.The levels of Ig A antibody in intestinal mucus and serum of mice fed with CIQ249 strain were detected by ELISA kit.The results showed that feeding CIQ249 strain could significantly promote the secretion of non-specific Ig A antibody.The mechanism of Ig A production regulated by Lactobacillus rhamnosus was preliminarily explored.The results showed that Lactobacillus rhamnosus could activate intestinal dendritic cells,promote the expression of Bcl-6 in CD4+T cells of Peyer's collecting lymph nodes,and then induce CD4+T cells to differentiate into CXCR5+CD4+Thf cells.at the same time,B cells gradually differentiated and matured into B220 murine Ig A + plasma mother cells mediated by Thf cells.In the fourth part,in order to further analyze the mechanism of Lactobacillus rhamnosus regulating immunosuppression of intestinal pathogenic bacteria,In this study,with normal mice were used as controls(group C),oral CIQ249 mice(CL group),mice infected with Salmonella typhimurium(CP group),and mice oral CIQ249 + infected Salmonella typhi(CLP Group)of the intestinal transcriptome was sequenced,Compared with group C,728 differentially expressed genes were obtained in CL group(251 up-regulated and 477 down-regulated),149 differentially expressed genes were obtained in CP group(38 up-regulated and 111 down-regulated).Compared with CP group,5326 differentially expressed genes were obtained in CLP group(4019 up-regulated and 1307 down-regulated).Among them,significantly differentially expressed genes in CL group were mainly enriched in signal pathways regulating cell growth,apoptosis and anti-inflammation,such as p53 signaling pathway,MAPK signaling pathway,NLRs signaling pathway,etc,while significantly differentially expressed genes in CP group were mainly enriched in salmonella infected(Salmonella infection)signal pathway.When interfered with Lactobacillus rhamnosus,the number of significantly differentially expressed genes enriched in this s ignaling pathway significantly decreased.In addition,the differentially expressed genes MX2,Rnasel,IL-6,Il-13ra2,Il-22ra1,Pla2g2 a,Anpep,Rap1 gap,Galnt6,IGF2,Rsl1d1,Cfh,Masp2 and Bdkrb1 related to host immunity in CP group vs.CLP group were randomly selected for RT-q PCR compliance verification.The results showed that the gene expression trend of RT-q PCR was consistent with that of RNA-seq sequencing,indicating that RNA-seq sequencing was reliable.The above sequencing results provide basic data for further understanding the mechanism of Lactobacillus rhamnosus regulating host immunity and promoting anti-intestinal pathogenic bacteria infection.In summary,this study shows that Lactobacillus rhamnosus has good intestinal colonization,digestive environment tolerance,promoting animal growth and other probiotics.It can effectively regulate intestinal homeostasis(physical barrier and immune barrier)to enhance the ability of animals and resist intestinal pathogenic bacteria infection.The result s provide theoretical and data support for the further development of Lactobacillus rhamnosus microecological biological agents.