The Effect Of Lactobacillus Plantarum A3 From Equine On Ulcerative Colitis In Mice With Intestinal Microflora Disorder

Inflammatory bowel disease(IBD),including Crohn’s disease(CD)and ulcerative colitis(UC),is a kind of disease that can lead to abdominal pain and diarrhea,and can induce colon cancer in severe cases.It seriously endangers human and animal health.It gets more and more attention because of the increasing incidence in the United States,Europe and countries with rapid economic development.Recent studies have shown that antibiotic therapy in human life increases the incidence of inflammatory bowel disease,meanwhile,they found that antibiotic increases susceptibility to DSS-induced colitis.Therefore,if we know intestinal bacteria disorders by antibiotics in humans and animals in the early life,we will need use appropriate drugs or agents to reduce the incidence or symptoms of inflammatory bowel disease.In this study,we used Lactobacillus plantarum A3 from equine to intervene a mouse colitis model of intestinal bacterial disorders,which has been screened for its therapeutic effect on ulcerative colitis in mice.The therapeutic effect for A3 was evaluated by clinical indicators,biochemical indexes and inflammatory cytokines.Besides,the changes of intestinal microflora and metabolites were analyzed by microbiological analysis and untargeted metabolomics which mean the potential biomarkers and therapeutic targets may be obtained.The research methods and results are as follows:1.Establishment of a mouse model of intestinal flora disturbance and the regulation of Lactobacillus plantarum A3Five-week-old male C57BL/6 mice were randomly divided into three groups:control-antibiotic group(C＿LCM group),antibiotic intervention group(LCM group)and Lactobacillus plantarum A3-antibiotic group(LP＿LCM group).On 1-5d,the LCM group and LP＿LCM group were given Lincomycin Hydrochloride(30 mg/10 g bodyweight).On6-12d,the LCM group was given normal saline to recover spontaneously,and the LP＿LCM group was given Lactobacillus plantarum A3(5×10~8CFU/10 g bodyweight).The C＿LCM group had been given normal saline during the whole experiment time as a blank control.At the end of the experiment,the intestinal contents of the duodenum,jejunum,ileum,cecum,colon,and rectum in each group were taken for 16S amplicon sequencing analysis to explore the changes of intestinal bacteria in mice.The results showed that after the mice were given Lincomycin Hydrochloride,the number of flora in the duodenum,jejunum and ileum were changed little,the number of flora in the duodenum even increased.In contrast,the number of flora in the cecum,colon and rectum decreased substantially,it is reflected inα-diversity decreased significantly(p<0.05),β-diversity had a significant difference(p<0.05)and a large number of genus disappeared,but the number of Enterobacter increased significantly(p<0.05);When Lactobacillus plantarum A3 intervention,the number of flora in the cecum,colon and rectum increased slightly,α-diversity increased significantly(p<0.05),β-diversity had a significant difference(p<0.05),the abundance of flora in Enterobacter was down-regulated and Bacteroides was up-regulated.2.The effect of Lactobacillus plantarum A3 from equine on ulcerative colitis in mice with intestinal microflora disorderFive-week-old male C57BL/6 mice were randomly divided into four groups:control-DSS group(Control group),DSS group,Lactobacillus plantarum A3-DSS group(LP＿DSS group)and buytrate-DSS group(BA＿DSS group).On 1-5d,all groups except the Control group were given Lincomycin Hydrochloride(30 mg/10 g bodyweight).On6-19d,The LP＿DSS group was administered intragastrically with Lactobacillus plantarum A3(5×10~8CFU/10 g bodyweight);the BA＿DSS group was administered intragastrically with equal volume buytrate(30mg/10g body weight)as the positive control and the DSS group was given equal volume of saline by gavage,too.The Control group was given the same amount of saline throughout the experiment as the blank control group.During13-19d,except the control group,which maintained normal drinking water,the other three groups were given drinking water containing 3%(w/v)DSS for seven days,in which freshly prepared DSS solution was changed every two days.The results showed that when DSS began to intervene in mice,the body weight of mice gradually decreased and reached the lowest level at 19d,which was significantly lower than that of the Control’s mice(p<0.001);The disease activity index(DAI)of mice began to increase gradually and reached the highest on the 19th day(p<0.001).In addition,compared with the control group,the spleen index of DSS group was significantly increased(p<0.01)and the colon length was significantly shortened(p<0.01).It was noteworthy that the colon contents were red-black color.Further analysis for the results of H&E staining of colonic tissue revealed that more colonic epithelial cells were detached,a large number of inflammatory cells were infiltrated in the mucosal and submucosal layers;Serum biochemical indexes showed a significant increase in myeloperoxidase(MPO)and malondialdehyde(MDA)(p<0.001),except the superoxide dismutase(SOD)decrease significantly(p<0.001).The expression of inflammatory cytokines IL-1β,IL-6,TNF-αand IFN-γwere significantly increased(p<0.05),the expression of Foxp3 and IL-10 was significantly decreased(p<0.05).Although the LP＿DSS and BA＿DSS group did not significantly improve body weight,they significantly delayed the DAI index’s increase,especially at the 19d(p<0.05).The spleen index and colon length of mice were recovered,and the results of H&E staining of colon tissue also showed a decrease which in the number of inflammatory cell infestation.Besides,both serum biochemical indexes and inflammatory cytokines of the two groups were closer to the values of the Control group.In order to further study the regulatory effect of Lactobacillus plantarum A3,the colonic contents of the Control,LP＿DSS and DSS groups were subjected to 16S amplicon sequencing and untargeted metabolomics analysis.The results of microbiological analysis showed that,compared with the DSS group,the LP＿DSS group’sα-diversity was significantly increased(p<0.001)andβ-diversity was also had significantly different(p<0.01);In addition,at the phylum level,the LP＿DSS group down-regulated the flora abundance of Bacteroidetes(p<0.05),up-regulated the flora abundance of Firmicutes(p<0.05)and Verrucomicrobia(p<0.05);At the genus level,it down-regulated the flora abundance of Bacteroides(p<0.05)and up-regulated the flora abundance of Blautia(p<0.05),Akkermansia(p<0.05),and Citrobacter(p<0.05).The results of untargeted metabolomics analysis showed that the contents of metabolites such as Arachidonoyl ethanolamide phosphate(AEA-P)(p<0.001),Cortisone(p<0.001),Stercobilin(p<0.001),Pantethine(p<0.001)and Choline bitartrate(p<0.001)were up-regulated after L.plantarum A3 intervention,espeically Arachidonic acid ethanolamine phosphate had the highest foldchange(28.70).It significantly affects metabolic pathways such as Ubiquinone and other terpenoid-quinone biosynthesis(Impact=0.5,p<0.05,Fold Enrichment=26.24),Phenylalanine,tyrosine and tryptophan biosynthesis(Impact=0.25,p<0.05,Fold Enrichment=19.68).In summary,in this study,we used Lincomycin Hydrochloride to establish a mouse model of intestinal bacteria disorder and found that Lincomycin Hydrochloride had an effect on mouse intestinal microflora and a worse effect on the flora of the large intestine.Besides,Lactobacillus plantarum A3 from equine had a certain recovery effect on intestinal microflora.In addition,A3 had a regulatory effect on colitis in mice with intestinal bacteria disorders,reflected in clinical indicators,biochemical indexes and inflammatory cytokines.It also had a certain recovery ability for intestinal microflora damaged by antibiotics and DSS,up-regulate metabolites like Arachidonoyl ethanolamide phosphate and Cortisone which had anti-inflammatory and analgetic effect,and affect metabolic pathways such as terpenoid-quinone biosynthesis and amino acid biosynthesis.