Study Of The Mechanism Of Lactobacillus Reuteri On Regulating Intestinal Mucosal Barrier In Chicks

Lactobacillus,as a probiotic of the body,plays an important role in the body,which depends on the good tolerance and excellent probiotic performance.The intestine is the main place for nutrient absorption and immune defense,and lactic acid bacteria can inhibit the adhesion and colonization of pathogens,and also secrete metabolites such as short-chain fatty acids to promote the absorption of nutrients.This study screened a strain of Lactobacillus reuteri with good tolerance and good bacteriostatic effect,and further explored the regulation mechanism of Lactobacillus on intestinal mucosal barrier of chicks.This study is divided into the following three parts:1.Screening and identification of LactobacilliLactic acid bacteria are widely used in poultry,and lactic acid bacteria have certain tolerance to the body’s stomach acid,bile,various enzymes and other physiological environments.In this study,the acid-resistant and bile-tolerant salts of 26 strains of lactic acid bacteria were tested by simulating the normal physiological environment of the organism.It was found that the relative survival rates of different strains varied greatly.Through SPSS 17.0 software data analysis.Eight strains with high survival rate were used for subsequent experiments.Next,the Oxford Cup perforated plate diffusion method was used to investigate the inhibitory effects of lactic acid bacteria on common pathogens in poultry(eg,Salmonella pullorum(ATCC 9120),Escherichia coli,Salmonella typhimurium(SL1344)The addition of MRS medium does not inhibit pathogenic bacteria,but there is an obvious inhibition zone after adding lactic acid bacteria liquid,but the size of the inhibition zone is different.The strain with strong inhibition ability will produce a large and clear inhibition zone.Among them,Lactobacillus reuteri 22 has the strongest antibacterial effect against Salmonella pullorum,and the antibacterial area is large and the inhibition zone is clear.In addition,the antibacterial effects of different strains are different,and the strains of the same genus sometimes have antibacterial effects.that is to say,the bacteriostatic performance of the strain has individual differences.In this experiment,Caco-2 was also used as an in vitro model for studying bacterial adhesion performance,and the results showed that 8 strains of bacteria were screened for Caco-2,There is a significant difference in adhesion performance.The strongest adhesion ability is strain No.22,which is identified as Lactobacillus reuteri by 16SRNA,and is named as Lactobacillus reuteri 22.2.Lactobacillus reuteri enhances intestinal immune function in chicksChicks can easily cause severe stress during transport,feeding,and highly intensive farming.This stress can cause the chick’s intestinal villi to shrink severely,and the intestinal pathogenic microorganisms have opportunities.This experiment was to explore the effect of Lactobacillus reuteri 22 on the intestinal barrier of chicks.The results showed that the body weight of Lactobacillus reuteri 22 group increased significantly(P=0.108);HE staining results showed that there was no significant change in the length of villus after lactic acid bacteria treatment and the depth of crypts increased.In addition,staining of chick goblet cells and detection of Muc-2,lyz-1 and other genes by fluorescent quantitative PCR revealed that the number of goblet cells was significantly increased after lactic acid bacteria treatment,and the expression level of Muc-2 gene was significantly increased.The Notch pathway was activated and the Hes1 gene was down-regulated,explaining the increase in the number of secretory cells(including goblet cells).At the same time,the expression of claudin-1 and occludin,a gene encoding the tight junction protein of the intestinal epithelium,also increased in the lactic acid bacteria treatment group.Simultaneous ELISA was used to detect IgA in jejunum and common inflammatory factors TNF-α and IL-1 β.The results showed that the vaginal secretory immunoglobulin IgA increased in the lactic acid bacteria treatment group;the inflammatory factors in the jejunum of the lactic acid bacteria treatment group decreased slightly.The above results indicate that Lactobacillus reuteri 22 promotes the development of the intestinal barrier of chicks and enhances intestinal immunity.3.L.re uteri activates Wnt/β-catenin pathway to promote intestinal epithelial cell proliferationThere are many studies on the mechanism by which lactic acid bacteria maintain the intestinal barrier and inhibit pathogens.Whether the lactic acid bacteria regulate intestinal mucosal barrier by regulating intestinal stem cells and promoting intestinal proliferation is unclear.Proliferation genes such as PCNA were detected by real-time PCR and fluorescence quantification.The results showed that the proliferating genes had a consistent proliferation trend after treatment with Lactobacillus reuteri.At the same time,Wnt/β-catenin related genes Wnt3、Axin2,Lrp5 and other genes were significantly increased,Lgr5+active stem cells showed a significant increase in intestinal gene expression level and there was no significant change in the expression level of Bmil in stationary intestinal stem cells.The above results indicate that L.reuteri 22 can activate Wnt/βcatenin to promote intestinal stem cell activation and maintain intestinal mucosal barrier.