Effects And Mechanisms Of Lactobacillus And Bifidobacterium On Alleviating Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a systemic autoimmune disease with unclear pathogenesis and characterized by symmetrical pain in facet joints,cartilage and bone erosion.Methotrexate(MTX)is the first-line drug for the treatment of RA.The onset of RA is accompanied by imbalanced intestinal immune responses and damage of immune tolerance,which are closely related to gut microbiota dysbiosis.The genus of Lactobacillus was increased while Bifidobacterium was decreased in pre-clinical and early RA patients.It has been found that supplementation of Lactobacillus can reduce systemic inflammation in RA animals,but the effects on gut microbiota and gut immune are unclear.Few studies are focusing on the effects of Bifidobacterium on RA.This study first analyzed the characteristics of the gut microbiota of RA patients that treatment with short-term and low-dose MTX,especially the distribution of the species of Lactobacilli and Bifidobacteria.Then Lactobacillus and Bifidobacterium species with the potential to alleviate RA were screened using RA rats.Lastly,the mechanisms of Lactobacillus and Bifidobacterium on alleviating RA were explored through regulating the composition and function of the gut microbiota,intestinal metabolites,and colonic gene expression.The results are summarized as follow:1.High-throughput sequencing and gas chromatography-mass spectrometry technology were used to analyze and compare the gut microbiota and short-chain fatty acids(SCFAs)between the RA patients treated with MTX(MTX-RA)and that of healthy controls(HCs).The intestinal microbial compositions in MTX-RA changed when compared with HCs,including the significantly increased relative abundance of Bifidobacterium and Rumenococcus,and the significantly reduced of Dorea and Erysipelotrichaceae?unclassified,as well as the relative abundance of L.delbrueckii,L.enterobacillus and L.manibotivorans were decreased.The fecal acetae,propionate,butyrate and pentanoate in the MTX-RA were1.038,1.16,0.93 and 1.09 times to those in the HCs,respectively,which with insignificant differences.In addition,low-dose and short-term MTX did not affect the diversity of gut microbiota and the levels of SCFAs in RA patients.2.The effects of L.casei,L.plantarum,L.fermentum,L.rhamnosus,L.salivarius,and L.reuteri on alleviating RA were compared to selecte effective Lactobacillus species,using bovine type-?-collagen-induced-arthritis(Collagen-induced Arthritis,CIA)rats.Six species of Lactobacillus reduced the levels of auto-antibodies and pro-inflammatory cytokines in serum.L.casei reduced levels of Th1 cell-related immune cytokines,inhibited cell infiltration and synovial hyperplasia,and reduced damage and swelling of joint tissue.L.reuteri and L.rhamnosus reduced the levels of Th1 and Th17 cell-related immune cytokines,respectively.L.plantarum inhibited Th1 and Th17 cell-related immune cytokines levels.And the three species reduced synovial tissue damage without obviously alleviating joint swelling.L.salivarius was ineffective in reducing Th1 and Th17 cell-related cytokines levels and had no alleviating effect on joint synovial inflammation and joint swelling.The six species of Lactobacillus used did not affect the diversities of gut microbiota of CIA rats,and while affect the composition of gut microbiota.In summary,the effects of Lactobacillus on alleviating CIA were species-depended in rats,and L.casei showed best relieved effects.3.The effects of B.breve,B.adolescentis and B.bifidum species on alleviating CIA were compared in rats.The arthritic phenotypes and pathological characteristics of joint were used as indicators for screening effective species.The effects of Bifidobacteria species on the serum levels of auto-antibodies and pro-inflammatory cytokines Treg cells in intestine,the expression of tight junction genes in ileum,gut microbiota and SCFAs were explored in CIA rats.The three species of Bifidobacterium reduced the levels of auto-antibodies.B.breve reduced synovial inflammation and joint swelling,significantly reduced pro-inflammatory cytokines,increased the proportion of Treg and the expression of tight junction genes.B.adolescentis showed a certain effect of relieving arthritis,which only significantly reduced TNF? and increased the expression of tight junctions genes.B.bifidum was useless in alleviating CIA in rats.The three species of Bifidobacterium did not affect the diversity of gut microbiota and suppresse the increasing of harmful bacteria Alitipes in CIA rats.Only B.breve increased the levels of intestinal SCFAs.Therefore,the effects of Bifidobacterium on alleviating CIA rats were species-depended,and B.breve showed best relieved effects.4.The effects of three strains of L.casei and five strains of B.breve on alletiving CIA were compared in rats.Among them,L.casei CCFM1074 and B.breve CCFM1078 effectively reduced synovial inflammation,joint swelling and erosion,and reduced the levels of auto-antibodies and IL-1?,as well as increased the proportion of Treg cells.L.casei CCFM1074 and B.breve CCFM1078 affected the compositions and structure of gut microbiota,inhibited the increase of harmful bacteria Alitipes.B.breve CCFM1078 promoted the recovery of the beneficial bacteria including Turcibacter,Parasutterella and Erysipelotrichaceae.These results showed that L.casei CCFM1074 and B.breve CCFM1078 alleviated CIA were closely related to the regulation of gut microbiota in rats.5.To analyze the mechanisms of L.casei CCFM1074 and B.breve CCFM1078 on alleviating CIA,the metagenomic and non-targeted metabolome techniques were used to analyze the function of gut microbiota and metabolites,and the transcriptome technique was used to analyze the expression and functions of genes in the colon.Both L.casei CCFM1074 and B.breve CCFM1078 regulated the composition and functions of gut microbiota,and significantly down-regulated the expression of gut microbial genes relating to fatty acid metabolism,ketone synthesis and degradation,styrene degradation and xylene degradation.L.casei CCFM1074 significantly affected aminoacyl-t RNA biosynthesis,arginine and proline metabolism,while B.breve CCFM1078 significantly affected the vitamin B6 metabolism and aminoacyl-t RNA biosynthesis when using enrichment analysis of differential metabolites.The differential expressed genes in B.breve CCFM1078 mainly involved in immune system pathways,while the differential expressed genes in L.casei CCFM1074 were not enriched in any pathways.