Alleviating Effects And Mechanisms Of Bacillus Smithii XY1 On Intestinal Inflammation

Inflammatory bowel disease(IBD)is an immune-mediated disease characterized by chronic inflammation of the gastrointestinal tract.It has become a global disease,and the medical burden continues to grow.The specific pathogenesis of IBD is still unclear,and the prevention and treatment methods are not yet perfect.The existing schemes generally have many problems,including side effects,a single treatment approach,and substantial development costs.Dietary modification,including probiotics,as an emerging intervention with multiple advantages,is beneficial for alleviating and preventing IBD symptoms.Experimental and clinical studies have found that various probiotics,especially lactic acid bacteria and some Bacillus,can relieve inflammation and clinical symptoms in IBD patients.The immune system,genes,gut microbes,and environment are recognized as the four major factors in the pathogenesis and progression of IBD.Probiotics also regulate IBD through various mechanisms.However,there are many problems in the research and application of probiotics against IBD,including a limited selection of strains,some strains studied do not meet the "probiotics" standard,low stability of anti-inflammatory effects,potential safety hazards for immunocompromised patients and Insufficient understanding of the mechanism,etc.Based on the above background,a new strain of Bacillus smithii XY1 with good antiinflammatory ability was obtained by screening.Its whole genome was analyzed,a preliminary safety evaluation was completed,and then the regulation of the bacteria was studied in animal models.Its mechanisms for alleviating intestinal inflammation include modulating oxidative stress,gut microbiota,and the gut-brain axis.The main results are as follows:1.We isolate 29 strains of lactic acid bacteria or Bacillus from samples such as fermented food and use 11 strains from the Culture Collection Center as a control.The in vitro hemolytic test,zebrafish acute toxicity test,and growth ability test were carried out sequentially on the bacteria,and finally,the anti-inflammatory ability of the strain was screened through DSSinduced zebrafish severe and moderate enteritis models.A total of 8 candidate probiotics Lacticaseibacillus rhamnosus GG,Lacticaseibacillus rhamnosus NBRC 3425,Lactobacillus parafarraginis XYRR2,Bacillus licheniformis XYT3,Bacillus coagulans NBRC 12583,Bacillus coagulans XY2,Bacillus smithii DSM 4 216 and Bacillus smithii XY1 showed anti-inflammatory ability,and B.smithii XY1 exhibited two anti-inflammatory abilities simultaneously.The bacteria reduced the expression of inflammatory factors il-1β,tnf-α,and nod2 genes and could alleviate DSS-induced intestinal damage.2.The results of whole genome sequencing showed that B.smithii XY1 has a variety of genes related to adhesion and stress resistance,a large number of antioxidant enzyme genes,and five secondary metabolite gene clusters(respectively β-propiolactone,poly Ketones,siderophores,terpenes,cyclic lactone auto inducible peptides),microbial-associated molecular model genes.Comparative genomics compared the genome of B.smithii XY1 with the genome of the other four strains with weak anti-inflammatory effects and obtained its unique 29 gene families.Among them,the adhesion ability,heat resistance ability,gastric juice and intestinal juice resistance,longterm storage ability,antioxidant ability and siderophore secretion of B.smithii XY1 were verified by experiments.In addition,two drug-resistant genes(bac A and fos B)were identified in the genome,and there was no risk of horizontal transfer after analysis.The strain was initially safe with the acute oral toxicity test results in mice(LD50 > 5000 mg/kg).3.B.smithii XY1 ameliorated the animal inflammation induced by DSS,manifested as improving pathological changes such as weight loss,DAI index increase and colonic tissue damage in mice,and decreased Il-6,Il-1β,Il-12 a in tissues Gene expression of inflammatory factors.At the same time,B.smithii XY1 can reduce the gene expression of oxidase MPO and i NOS in the colon of mice with colitis,reduce the content of intestinal free radicals,increase the activity of GSH-Px antioxidant enzymes in the colon tissue of mice and the activity of NRF2-,FOXO-expression of genes related to the antioxidant defence system,showing the ability to reduce oxidative stress caused by intestinal barrier breakdown.Experiments in the germ-free nematode model found that the ability of B.smithii XY1 to reduce oxidative damage can play a direct role without the help of intestinal flora.Adding this bacterium can alleviate the intestinal barrier damage induced by DSS and activate SKN-1,DAF-16,SOD-3,and GST-4 protein expressions.Further experiments with daf-2 and daf-16 mutant nematodes found that B.smithii XY1 alleviated the oxidative damage caused by DSS by activating the DAF-16/FOXO pathway.4.B.smithii XY1 can regulate the abundance and function of intestinal flora related to inflammation.16 S r RNA results showed that B.smithii XY1 could improve the decrease of intestinal flora diversity in mice with colitis and reverse the increase in the abundance of proinflammatory bacteria such as Fusobacterium,Clostridium difficile,Desulfovibrioceae,Heterobacterium,and Ruminococcus.In addition,biomarkers of B.smithii XY1 group such as Escherichia-Shigella,Bacteroides,etc.,and biomarkers of the DSS group,such as Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿UCG-001,Alloprevotella,etc.There is a clear negative correlation between them.B.smithii XY1 can significantly increase the enrichment of KEGG pathway and COG function of intestinal flora in colitis mice in terms of antiinflammation,anti-cancer,anti-oxidation,anti-foreign body,and prolonged life while reducing harmful metabolites,pro-inflammatory,enrichment of infection-related functions.Among the common opportunistic pathogens,B.smithii XY1 had the most significant inhibitory ability against the genus Burkholderia in the intestinal flora.Through B.smithii XY1 and the model bacteria Burkholderia cenocepacia H111 in vitro co-cultivation,hole diffusion,inhibition of microcolony formation,etc.,and its effects on biofilm formation,group dynamics and zebrafish of two quorum sensing deficient strains(ΔCep I and ΔRpf F)The research on virulence,colonization and pro-inflammatory ability found that B.smithii XY1 can weaken the virulence of the bacterium and alleviate the intestinal inflammation caused by it by inhibiting the Cep IR quorum sensing system of the bacterium.5.B.smithii XY1 can regulate the gut-brain axis in animals.Animal behaviour experiments found that B.smithii XY1 could significantly alleviate the decrease in light and dark sensitivity of zebrafish and the decrease in nematode foraging ability after DSS treatment.Neuronal injury experiments showed that B.smithii XY1 improved DSS-induced reduction in the expression of excitatory glutamatergic neurons and transmitter transport-related genes in the intestine of nematodes,as well as the inhibition of γ-aminobutyric acid(GABA)increased neuronal expression.Similarly,abnormalities in the GABAergic nervous system were significantly attenuated by B.smithii XY1 in the colon tissue of colitis mice,which significantly reduced the expression of GABA synthesis genes gad1,gad2 and A,B,and C receptor genes.At the same time,the aberrant expression of GABA-related genes and proteins related to intestinal barrier homeostasis was alleviated,including intestinal epithelial cell renewal and apoptosis,intestinal permeability and tight junction proteins,suggesting that B.smithii XY1 mediates colitis in animals.The GABAergic nervous system maintains intestinal permeability homeostasis,thereby relieving inflammation.The genome of B.smithii XY1 contains a complete GABA degradation pathway,and in vitro tests have confirmed that it has higher GABA-T enzyme activity than the control bacteria,indicating that the bacteria mediate the GABAergic nervous system may be completed by degrading GABA.This paper discusses the possible anti-inflammatory mechanism of B.smithii XY1 from three perspectives.The interaction of the three mechanisms may be the reason why B.smithii XY1 has better anti-inflammatory ability than other strains.This paper lays the foundation for the research on B.smithii XY1 in probiotics,further deepens our understanding of the anti-inflammatory mechanism of probiotics,and provides a reference for the subsequent development of special functional foods for the IBD population with probiotics as functional additives.