Effects Of Two Strains Of Lactobacillus From Pigs On Intestinal Inflammation And Its Mechanism

Modern intensive and large-scale breeding mode increases the risk of intestinal diseases in pigs at all growth stages,the most important one is diarrhea.The frequent occurrence of piglet diarrhea has seriously restricted the efficient production of pig industry.Therefore,it is of great significance to study the prevention and treatment measures of intestinal inflammation for improving the growth performance of piglets and achieving efficient production.Intestinal microorganisms are closely related to the regulation of intestinal inflammation.Lactobacillus is an important probiotic in the intestine,which plays an important role in the regulation of intestinal microenvironment homeostasis.In this study,two strains of Lactobacillus bacteria isolated from healthy pig feces were selected in the laboratory in the early stage,and molecular identification was performed on them,and their metabolic activity was tested through in vitro culture to evaluate their antibacterial effects on E.coli and Salmonella.Secondly,the mouse enteritis model induced by dextran sodium sulfate(DSS)was used to study the effect of the lactobacillus on intestinal inflammation and its mechanism of regulating the intestinal epithelial barrier.Finally,the role of the lactobacillus in the Salmonella infection model was studied.The study provides reference basis and theoretical guidance for its application in pig production.The thesis research is mainly divided into the following three parts:1.Bacteriostatic experiment of two Lactobacillus strains in vitroTwo strains of Lactobacillus from pigs were identified as Lactobacillus reuteri strain DSM(LR)and Lactobacillus taiwanensis strain BCRC(LT).The results of growth curve showed that the growth of LR and LT in the first 10 hours was in the logarithmic growth stage,and the growth rate began to slow down after 10 hours,and the growth tended to be stable after 25 hours,entering the plateau stage.The yield of lactic acid,the change of p H value and the content of SCFAs were synchronous with the growth rate.Bacteriostatic test showed that LR and LT could effectively inhibit the growth of Escherichia coli and Salmonella typhi2.Effects of Lactobacillus on DSS-induced colitis in miceThe experiment selected 40 C57BL/6J male mice of similar body weight at 9 weeks of age and randomly divided them into 4 groups(10 replicates in each group): normal control group,DSS group,LR bacteria treatment group(DSS+LR),and LT bacteria treatment group(DSS+LT).The acclimatization period was 7 days and the test period was 16 days,after which the mice were sacrificed and sampled.The results showed that compared with the DSS group,the weight loss of the mice in the LR and LT groups was not significantly alleviated,but the disease activity index(DAI)score was significantly decreased;and the length of colon was significantly increased and the histopathological score of colon was significantly reduced in LT group(P < 0.05).The test results of colonic myeloperoxidase(MPO)activity showed that compared with the DSS group,the MPO activity of the LR and LT groups decreased,and the effect of LT bacteria on reducing the MPO activity was more obvious(P < 0.05).Further ELISA testing of colon and plasma inflammatory factors found that LR bacteria significantly reduced the levels of colonic pro-inflammatory factors TNF-α and IL-22,as well as plasma IL-1β levels,and increased plasma anti-inflammatory factors IL-10 levels;LT bacteria significantly decreased colon and plasma TNF-α,IL-1β,IL-6 and IL-22 levels increased colon and plasma IL-10 levels(P < 0.05).Immunohistochemistry and ELISA methods were used to detect barrier-related proteins.The results showed that treatment with LR and LT bacteria can up-regulate the expression of Claudin-1,Muc-2,Occludin,ZO-1 and protein in the colon.Among them,Claudin-1,Occludin,ZO-1 The difference is significant(P < 0.05).Finally,16 S r DNA sequencing technology detects the composition of colonic flora.PCoA analysis shows that the normal control group and the DSS group are significantly separated,and the LT treatment group is closer to the normal control group.At the phylum level,DSS significantly increased the abundance of the Proteobacteria(P < 0.05),and treatment with LT bacteria could reduce its abundance,but it did not reach a significant level(P > 0.05).At the genus level,DSS significantly reduced Muribaculaceae＿norank and increased the abundance of Bacteroides.Treatment with LR or LT bacteria increased the Muribaculaceae＿norank abundance and decreased the abundance of Bacteroides,but the difference was not significant(P > 0.05).3.Effects of Lactobacillus on inflammation in mice infected by SalmonellaThe experiment selected 40 C57BL/6J male mice with similar body weight and randomly divided them into 4 groups(10 replicates in each group): normal control group,Salmonella group(ST),LR bacteria treatment group(ST+LR)and LT bacteria treatment group(ST+LT).The adaptation period is 7 days.On 0-13 days of the test,normal saline or LR,LT bacteria are intragastrically administered,and normal saline or Salmonella typhimurium is administered on the 7th day,and the mice are sacrificed and sampled at 13 days.The results showed that the number of cecal chyme Salmonella in the ST group was significantly increased;compared with the ST group,LR and LT bacteria treatment reduced the number of Salmonella(P < 0.05).Treatment with LR and LT bacteria did not alleviate the weight loss caused by ST bacteria and the increase in colonic histopathological scores(P > 0.05).Among the blood routine indexes,ST infection significantly decreased platelets and significantly increased the percentage of monocytes(P < 0.05).Treatment with LR and LT bacteria did not alleviate this effect.However,treatment with LR and LT bacteria can significantly alleviate the increase in colonic MPO levels caused by ST(P < 0.05).Moreover,compared with ST group,LR and LT bacteria treatment significantly increased the levels of Occludin,ZO-1 and Muc-2(P < 0.05).In summary,this study identified two strains of Lactobacillus: Lactobacillus reuteri and Lactobacillus taiwanensis,which can inhibit the growth of Escherichia coli and Salmonella typhi in vitro.In vivo experiments show that Lactobacillus reuteri and Lactobacillus taiwanensis can strengthen the intestinal epithelial barrier,reduce the level of proinflammatory factors,increase the level of anti-inflammatory factors,and play a certain role in alleviating the intestinal inflammation caused by DSS;among them,the protective effect of Lactobacillus taiwanensis on the intestinal epithelial barrier is more obvious.In a mouse model of Salmonella infection,the bacteria did not alleviate the weight loss and blood immune indexes,but reduced the colonic MPO level and enhanced the intestinal epithelial barrier function.