A Preliminary Investigation Of The Mechanism Of The Effect Of Different Lactic Acid Bacteria On Intestinal Mucosal Immunity In Mice With Cyclophosphamide-induced Intestinal Injury

Lactic acid bacteria are not only part of gut microbiota,but also found in other body parts,certain foods,and nutrient-rich niche.Most of them are often referred to as probiotics,and have the positive effects on regulation of gut microbiota,cholesterol,and intestinal immunity.Previously,our research team isolated six lactic acid bacteria-Lactobacillus(L.)acidophilus NCU433(NCU433),Lactococcus lactis NCU036105(NCU036105),L.paracasei NCU622(NCU622),L.plantarum NCU1125(NCU1125),L.rhamnosus NCU2217(NCU2217)and L.casei NCU011054(NCU011054)from different substrates,and identified the species of these strains.However,study on intestinal mucosal immunity of the six lactic acid bacteria has not yet been carried out.Based on these,the paper aimed to investigate the effects of the six lactic acid bacteria as the target bacteria on small intestinal and colonic mucosal immunity in the cyclophosphamide(CP)induced intestinal injury model,and to preliminarily explore the mechanism of action of NCU1125 and NCU011054 on colonic mucosal immunity.The main research contents and results are as follows:1.A CP-induced intestinal injury model was used to explore the effects of the six lactic acid bacteria on small intestinal mucosal immunity from the aspects of physical and immunologic barriers,and the potential mechanism.Results showed that the six strains could increase body weight,the immune organ indexes(except for NCU433,NCU2217 and NCU011054 showed the best effect)and hepatic oxidative stress capacity(NCU1125 shown the best effect)in mice,repair small intestinal morphological structure(NCU036105,NCU622,NCU1125 and NCU011054 shown the best),and enhance small intestinal physical barrier function(NCU1125 shown the best).In addition,NCU036105 induced Th1 and Th2 polarization by stimulating CD4~+T cells proliferation,promoting secretion of IL-4 and IL-12p70,and up-regulating gene expressions of T-bet and GATA-3,whereas the other bacteria could induce Th1,Th2,Th17 and Treg differentiation by increasing the number of CD4~+T cells,stimulating secretion of cytokines associated with Th cells differentiation(IL-2、IL-4、IL-6、TGF-β3、IL-12p70、IL-17)and up-regulating gene expressions of Th cells transcription factors(T-bet,GATA-3,RORγt,Foxp3).NCU433 could regulate small intestinal balance of Th17/Treg by decreasing the ratio of RORγto Foxp3,and the ratio of IL-17 to IL-10,whereas NCU622,NCU1125,NCU2217 and NCU011054 could restore small intestinal balance of Th1/Th2 and Th17/Treg by increasing the ratio of T-bet to GATA-3 and IFN-γto IL-4,and decreased the ratio of RORγt to Foxp3 and IL-17 to IL-10,respectively The correlation analysis showed that Th cells differentiation-related factors were positively correlated with key factors in TLRs/DCs and TLRs/NF-κB pathways(except for NCU036105),and the genes of physical barrier related proteins positively correlated with key factors in TLRs/My D88 pathway(except for NCU036105).Moreover,Only NCU1125 could up-regulate gene expressions of plasma cell transcription factors and s Ig A component proteins,and increase the levels of Ig A and s Ig A in small intestine.These results indicated that the six lactic acid bacteria could improve small intestinal mucosal immunity,with NCU1125 being the best.All of them could up-regulate the level of tight junction proteins through TLRs/My D88pathway(except for NCU036105)to alleviate the small intestinal mucosal injury,among which NCU1125 could also achieve this by promoting the secretion of s Ig A from plasma cells,and induce Th cells differentiation to exert small intestinal mucosal immunomodulation,which might be related with the activation of TLRs/DCs or TLRs/NF-κB(except for NCU036105)pathway.In addition,NCU433could maintain small intestinal mucosal immune homeostasis by modulating Th17/Treg balance,whereas NCU622,NCU1125,NCU2217 and NCU011054 did so by regulating small intestinal Th1/Th2 and Th17/Treg balance.2.Effects of the six lactic acid bacteria on colonic mucosal immunity in terms of physical,immunological and biological barriers,as well as potential mechanism,were investigated in the CP-induced intestinal injury model.Resulted showed that all lactic acid bacteria could improve colonic morphological structure,strengthen colonic physical barrier function(NCU1125 and NCU011054 shown best),induce Th cells differentiation by promoting CD4~+T cells proliferation,stimulating secretion of Th cells related cytokines and up-regulating the expressions of transcription factors associated with Th cells differentiation,among which,NCU622,NCU1125 and NCU011054 induced the differentiation of Th1,Th2,Th17 and Treg,NCU433induced the differentiation of Th cells except for Treg,and NCU2217 induced the differentiation of Th cells except for Th2,whereas NCU036105 only induced Th17differentiation(NCU1125 and NCU011054 shown the best),regulate the balance of Th1/Th2(NCU1125,NCU011054)and Th17/Treg(except for NCU433 and NCU036105);modulate diversity and composition of gut microbiota,and influence intestinal metabolism[short-chain fatty acids(except for NCU2217)and ammonia].Only NCU1125 and NCU011054 up-regulated the gene expressions of key factors of TLR4/My D88 and TLR4/NF-κB pathways.Correlation analysis showed that gene expressions of physical barrier-related protein genes in NCU622,NCU1125 and NCU011054 were mainly regulated by Bacteroides,Lactobacillus and Bacteroides-produced butyric acid,whereas NCU433 and NCU2217 were regulated by Bacteroides and Lactobacillus and NCU036105 was regulated by Bacteroides;gut microbiota and short-chain fatty acids produced by gut microbiota influenced Th cells differentiation in NCU036105(Lactobacillus,propionic acid),NCU622(Lactobacillus,Bacteroides,propionic and butyric acids),NCU1125(Lactobacillus,Bacteroides,propionic and butyric acid)and NCU011054(Lactobacillus,Bacteroides,acetic,propionic and butyric acids),whereas Th cells differentiation was induced by Lactobacillus and Bacteroides in NCU433,and by Lactobacillus in NCU2217;Th17/Treg balance was regulated by Lactobacillus in NCU622 and NCU2217,and Th1/Th2 and Th17/Treg balance were regulated by Bacteroides,Lactobacillus,and gut microbiota-produced propionic and butyric acids in NCU1125;and Lactobacillus,Bacteroides and short-chain fatty acids regulated the balance of Th1/Th2(Lactobacillus,acetic,propionic and butyric acids)and Th17/Treg(Lactobacillus,Bacteroides,acetic,propionic and butyric acids).The above results indicated that all the six lactic acid bacteria could improve the colonic mucosal immunity with some differences,among which NCU1125 and NCU011054 were the best.They could alleviate CP-induced colonic mucosal injury by enhancing the colonic physical barrier function,regulate colonic mucosal immunity by inducing Th cells differentiation,and maintain colonic mucosal immune homeostasis by regulating the balance of Th1/Th2 and/Th17/Treg(except for NCU433 and NCU036105),which might be the result of their direct action or indirectly by regulating gut microbiota/gut microbiota-produced short-chain fatty acids.Moreover,gene expressions of physical barrier-associated proteins and Th cells differentiation by NCU1125 and NCU011054might be related with the TLR4/My D88 and TLR4/NF-κB pathways,respectively.3.Effects of the six lactic acid bacteria on colonic metabolites were explored by UPLC-triple-TOF/MS in mice with CP-induced intestinal injure.These results showed that the six lactic acid bacteria were mainly involved in the regulation of pyrimidine/purine metabolism pathways in gut.In pyrimidine metabolism pathway,NCU433 and NCU2217 could increase the contents of orotic acid and thymine while decreasing the content of deoxyuridine,and NCU036105 could enhance the content of thymine while decreasing the contents of uridine and deoxyuridine.In the purine metabolic pathway,NCU622 could enhance the levels of deoxyinosine,xanthosine,inosine and d IMP,and inhibited the production of hypoxanthine;NCU1125 could up-regulate the contents of d IMP,inosine and xanthoside,and down-regulate the content of uric acid,and NCU011054 could increase the contents of d IMP,deoxyinosine and inosine,and decrease the contents of hypoxanthine and uric acid.These results revealed that NCU433,NCU036105 and NCU2217 could alleviate colonic pyrimidine metabolism disorder caused by CP,whereas NCU622,NCU1125and NCU011054 could regulate colonic purine metabolism disorder induced by CP.4.A pseudo-germ-free mouse model of CP-induced intestinal injury was used to further verify the regulatory mechanism of NCU1125 and NCU011054 in colonic mucosal immunity.Previous studies have shown that NCU1125 and NCU011054could induce Th cells differentiation and regulate Th1/Th2 and Th17/Treg balance in colon,but whether these effects were independent on gut microbiota was still unclear.Based on these,NCU1125 and NCU011054 were selected as the target bacteria for further study in pseudo-sterile mode.In pseudo-sterile mode,the target bacteria improved body weight,increased spleen index,alleviated colonic injury,and up-regulated the gene expressions of physical barrier related proteins,suggesting that the target bacteria could alleviate CP-induced colonic mucosal injury by improving colonic physical barrier function.Additionally,the target bacteria also stimulated the proliferation CD4~+T cells,promoted secretion of Th cells-related cytokines,up-regulated the gene and protein expressions of Th cells transcription factors and key factors of TLR4/NF-κB pathway,elevated the ratio of T-bet to GATA-3 and IFN-γto IL-4,and decreased the ratio of RORγt to Foxp3,suggesting that they could enhance colonic mucosal immunity by inducing colonic Th cells differentiation and stimulating secretion of Th cell-related cytokines.At the same time,they could maintain colonic mucosal immune homeostasis by regulating the balance of Th1/Th2and Th17/Treg,which might be related with the activation of TLR4/NF-κB pathway.The results of gut microbiota showed that antibiotic combined with CP treatment decreased the relative abundance of Lactobacillus to approaching zero and reduced the diversity of the bacterial community,while NCU1125 and NCU011054 could significantly increase the relative abundance of Lactobacillus and diversity of gut microbiota.The above results demonstrated that NCU1125 and NCU011054 could enhance colonic mucosal immunity by directly inducing Th cells differentiation independently of gut microbiota,and maintain colonic mucosal immune homeostasis by regulating Th1/Th2 and Th17/Treg balance,which might be associated with the activation of TLRs/NF-κB pathway.In summary,six strains of lactic acid bacteria could modulated intestinal(small intestine and colon)mucosal immunity and differed.Some lactic acid bacteria could enhance intestinal physical barrier function possible through the TLR/My D88pathway;improve intestinal mucosal immunity by activating TLR/NF-κB or TLR/DCs pathways to induce naive T cell differentiation;maintain intestinal mucosal immune homeostasis by regulating Th1/Th2 and Th17/Treg balance in intestine.Additionally,NCU1125 and NCU011054 could be independent of gut microbiota to induce Th cells differentiation possible through the activation of TLR4/NF-κB pathway,and regulate colonic Th1/Th2 and Th17/Treg balance.The above studies not only provided theoretical support for the study of lactic acid bacteria on alleviating intestinal mucosal injury caused by CP,but also provided new ideas for the development and mechanism of probiotics.