Study On Immunomodulatory Mechanism Of Lactobacillus Reuteri SBC5-3 On Intestinal Epithelial Cells

Probiotics have shown many physiological functions,such as regulating intestinal and organism immunity,maintaining the balance of intestinal flora,preventing and treating diarrhea,and alleviating intestinal inflammation.Intestinal epithelial cells（IECs）are important sites for probiotics to exert their immunomodulatory effects.Numerous studies have confirmed that probiotics regulate intestinal immunity,prevent intestinal inflammatory diseases,and promote body health through interacting with intestinal epithelial cells.As a commensal species in animal gut,Lactobacillus reuteri（L.reuteri）remains to elucidate the mechanism underlying its immune-modulatory impact.In our previous study,L.reuteri SBC5-3 was isolated from fecal sample of free-range Saba pig in Chuxiong,Yunnan Province.In vitro experiments showed that SBC5-3 had the probiotic properties of gastrointestinal tolerance,effective adhesion to intestinal epithelial cells,inhibition of the proliferation of major intestinal pathogenic bacteria and antibiotic sensitivity,and it significantly reduced the m RNA expression level of inflammatory factor IL-8 in TNF-α-induced HT-29 IEC.In this study,based on the previous research,effects of L.reuteri on transcription profiling and NF-κB key inflammatory signaling pathway in HT-29 IEC was investigated mediated busing the transcriptome sequencing（RNA-seq）and western blotting,in order to elucidate the anti-inflammatory mechanism of L.reuter.The main research contents and results are as follows:（1）1.5×10⁶ of HT-29 IECs were seeded into 6-well culture plates and incubated in complete culture medium for 48 h at 37°C,5%CO₂.Then they were randomly divided into Control,TNF-α,SBC5-3+TNF-αand SBC5-3 groups.Total RNA was extracted for transcriptome sequencing to study the effect of L.reuteri SBC5-3 on the transcription of HT-29 IECs induced by TNF-α.The results of differential gene expression analysis showed that,the TNF-α＿vs＿Control group had 27 significantly differentially expressed genes,including 24 up-regulated genes and 3 down-regulated genes,the SBC5-3+TNF-α＿vs＿TNF-αgroup had 6961 significantly differentially expressed genes including 3276 up-regulated genes and 3685 down-regulated genes,the SBC5-3＿vs＿Control group had 6132 significantly differentially expressed genes with 2863 up-regulated genes and 3269 down-regulated genes.GO functional annotation analysis showed that after 3 h induction of TNF-α,the main cellular component of differential genes was extracellular space,the main biological process was signal transduction,and the main molecular function was cytokine activity,receptor ligand activity and receptor regulatory activity.After L.reuteri SBC5-3preincubation for 16 h and induction of TNF-αfor 3 h,the differential genes were mainly partially aggregated in intracellular part,the main biological processes involved biological regulation,and in molecular functions such as protein binding.KEGG analysis showed that after 3 h induction of TNF-α,8 genes were significantly clustered in NF-κB signaling pathway,and all of them were up-regulated genes.After 16 h of L.reuteri SBC5-3 preincubation and 3 h of TNF-αinduction,36 genes were significantly clustered in the NF-κB signaling pathway,including 18 up-regulated genes and 18 down-regulated genes.（2）According to the results of transcriptome sequencing,quantitative real-time polymerase chain reaction（RT-q PCR）was used to verify the expression of immune-related genes.HT-29 IEC were pretreated with L.reuteri SBC5-3（10⁸ CFU/m L）for 16h,then stimulated with 50 ng/m L,TNF-αand incubated for 3 h.And relative expression of IL-8,IL-1β,CXCL10,CCL20,NFKB1,NFKBIA,TNFAIP3 and PTGS2 were detected by RT-q PCR.The results showed that the m RNA expression levels of IL-1β,CXCL10,CCL20,NFKB1 and PTGS2 in TNF-α-induced HT-29 IEC treated with L.reuteri SBC5-3 were significantly decreased（P<0.05）,while m RNA expression level of negative regulatory gene NFKBIA in NF-κB pathway was increased（P>0.05）.These results suggest that L.reuteri SBC5-3 could inhibit the activation of NF-κB signaling pathway induced by TNF-αin HT-29 IEC by up-regulating the expression of NFKBIA,and then down-regulating the expression of inflammatory cytokines encoding genes IL-8,IL-1β,CXCL10 and CCL20,thereby reducing the inflammatory response in cells.（3）Effect of L.reuteri SBC5-3 on activation of NF-κB signaling pathway.HT-29IEC were pretreated with L.reuteri SBC5-3（10⁸ CFU/m L）for 16 h,then challenged with TNF-αwith a final concentration of 50 ng/m L,and incubation continued for 5,15,30 and 60 min,at which point HT-29 IEC were collected for total protein extraction.The phosphorylation levels of key upstream kinase of NF-κB signaling pathway,IκB kinase IKK and inhibitory protein IκBαwere detected by western blotting.The results showed that pretreatment with L.reuteri SBC5-3 could decrease the ratio of p-TAK1 to TAK1,p-IKKα/βto IKKα/βand p-IκBαto IκBαin HT-29 IEC induced by TNF-αfor5 and 15 min（P>0.05）,and significantly decreased the ratio of p-IκBαto IκBαat 30and 60 min（P<0.05）.These results indicate that L.reuteri SBC5-3 could inhibit the activation of NF-κB signaling pathway induced by TNF-αin HT-29 IEC by inhibiting the phosphorylation of the key upstream kinases TAK1 and IκB kinase IKK and inhibiting the degradation of inhibitory protein IκBα.In conclusion,L.reuteri SBC5-3 can downregulate TNF-α-induced expression of genes involved in inflammatory activity,including cytokines,chemokines and their receptors,and expression of genes involved in innate immune signaling pathways such as NF-κB of IECs.L.reuteri SBC5-3 suppressed the activation of NF-κB signaling pathway via up-regulating the expression of NFKBIA,inhibiting the phosphorylation of key upstream kinase and IκB kinase IKK,inhibiting the degradation of inhibitory protein IκBα,and then reduce the inflammatory response in IECs.