The Immuno-modulation And Gut Microbiome Alteration Associated With Long-term Dietary Supplementation Of Lactobacillus Rhamnosus CGMCC In SD Rats With Colorectal Cancer

Probiotics are commensal microorganisms colonizing naturally gastrointestinal tract and many other vital organs.They are contributing as an important bio-factors producing metabolites and helping in digestion and boosting immune-system.There are many probiotics identified and used safely as food including many Lactobacillus spp such as yogurt and other fermented food.Lactobacillus rhamnosus CGMCC one of the new identified Chinese sub-spp.,Lactobacillus rhamnosus strains which culturing and maintaining in our lab,in the present study we have assessed and intensively studied the safety and microbiome structure modification along with serum cytokines and other serum lipoprotein profile in chemically induced colon cancer rat model.During the tumorigenic processes,the tumor cells employs several intrinsic escaping mechanisms to avoid recognisation and elimination by immune cells.The present study aimed at investigating the possible immunomodulation associated with gut microbiota alteration in mediated the antitumor response of Lactobacillus rhamnosus CGMCC against Di-methyl hydrazine?DMH?induced colon cancer in Sprague dawley?SD?rats.Methods Lactobacillus rhamnosus Chinese general microbial collection strain 1.2134?CGMCC?was growing and sub-cultured in de Man,Rogosa and Sharpe?MRS?broth medium at 37?C,for oral inoculation about 1x10⁹ CFU Lact/ml were daily given orally using gavage for 2 weeks prior to tumor induction followed by intraperitoneal injection of chemical carcinogen DMH 40mg/kg rats wt weekly once for ten consecutive weeks with continuous daily oral LBR administration up to terminated of the experiment at 24 weeks.Half of the animals were sacrificed at the week 10 for aberrant crypt foci assessments which demonstrated as early tumor development.And the rest of animals were sacrificed at week24 from the first DMH injection.The colon tissues were assessed for apoptosis using Terminal deoxynucleotidyl transferase dUTP nick end labeling?TUNEL?assay and western blotting protein markers for apoptosis Bax,Bcl2,and caspase3.In addition,the immune response including Th1,Th2,Th17 cytokines?IL-2,IL-4,IL-6,IL-10,TNF?,IFN?,and IL-17a?were analyzed at week 10 and 24 using flowcytometry cytokines beads array,and the serum lipoproteins profile were measured.Colon tissues were analyzed for inflammatory?NFkB-p65,Cox2,TNF??,and tumor markers?-catenine with western blotting,quantitative PCR,and histological assessments at both intervals.Fecal samples were collected at both intervals and subjected to genomic microbiome DNA extraction followed by 16S rRNA sequencing using Illumina Hi-Sequencer.The sequencing data were analyzed for assuming operational taxonomic units OTUs.The sequence reads?2x150bp paired end?.Effective tags with C97%similarity given same operational taxonomic unit?OTU?.Furthermore,to put all sequences in to their respective OTUs GreenGene ribosomal database project?RDP?algorithm classifier were used,both alpha and beta diversity were processed using Quantitative Insights Into Microbial Ecology?QIIME?software,and metabolome functions further analyzed with the Phylogentic Investigation of Communities by Reconstruction Unobserved State?PICRUSt?and the statistical Analysis of Metagenomics Profiles?STAMP?software to annotate KEGG Orthologs?KO?level2,level3,and Clusters of orthologs?COG?to investigate the functional metabolites differences between the groups.Results At the early stage after tumor induction?10 weeks of tumor induction?LBR treated groups showed significant decrease in total number of ACF's in GLC treated group?149±68?compared with?269.5±137?in GCA group,and we noted a remarkable increased Bax,P53,and Caspase-3 proteins expression with significant attenuation of NFkB-p65,Cox2 and?catenin expression compared to GCA group.Serum cytokines array at the week10 revealed significant elevation of IL-2,IL-6,and IFN-?,as well as decreased serum level of IL-10 in GCL treated animals.The 16S rRNA fecal microbiome genomic analysis showed that LBR alters the gut microbiome structure,composition and functions at phylum,family and genus level.Collectively,at this time point we found LBR significantly reduces colon ACFs incidence and multiplicity via inhibition of pro-carcinogenesis biomarkers induced epithelial cells apoptosis and modulate the gut microbiota population.At the 24^th week of tumor induction LBR treatment significantly reduced the tumor incidence volume and multiplicity.The total number of tumor/rats in GCL treated groups had significant?p value 0.016?decreased compared to GCA group.Also,the tumor volume has a significantly?p value 0.05?difference between GCA and GCL.We have found that at this stage the tumor microenvironment in GCL treated group shown upregulated of apoptotic cells as seen in TUNEL assay and further confirmed using western blot results.In addition,increased macrophages and fibroblasts cytokines markers and possibly activated T-cells immuno-response in the tumor-microenvironment leads to upregulate cancer cells apoptosis.Moreover,LBR protects the gut integrity and attenuates epithelial mesenchymal transition.16S rRNA analysis revealed Lactobacillus rhamnosus ameliorated the gut microbiome structure and enhanced the gut metabolites and functions.Conclusion The dietary intervention of Lactobacillus rhamnosus possess a protective activity in early tumors development via boost the immune response in the tumor microenvironment as well as ameliorates the gut-microbiome population,communities and metabolome profile.