Changes Of The Expression Of Toll-like Receptors, Phenotype And Function Of Dendritic Cells In Experimental Colitis And Roles Of Probiotics

PARTⅠChanges of gut flora and plasma endotoxin levels in ratswith TNBS-induced colitis and effects of BificoObjectiveTo investigate the changes of gut flora and plasma endotoxin in ratswith TNBS-induced colits, and to evaluate the effects of Bifico on gutinflammation, flora and plasma endotoxin levels of experimental colitisrats.MethodsForty Wistar rats were randomly divided into normal control (NC) group,positive control (UC) group, Bifico-treated (BC) group and mesalazine-treated (MC) group, 10 rats in each group. The rat model of experimentalcolitis (IBD model) was induced by 2, 4, 6-trinitrobenzene sulfonic acid(TNBS)/ethanol enema(100mg/kg). Rats in BC group were fed by gavage withBifico[live probiotics of combined bifidobacterium(Bif),lactobacillus(Lac) and enterococcusl by 2.2×10~9 colony-formingunit(CFU)/rat and rats in MC group were fed by gavage withmesalazine(200mg/rat), once a day for 4 weeks. Inflammatory score wasmeasured, plasma endotoxin levels were tested, and enteric microorganismsin cecum were recruited with standard methods.Results1. Inflammatory scores were 4.35±0.88 in NC group, 10.25±1.36 in UCgroup, 7.94±0.85 in BC group and 6.78±0.71 in MC group. Inflammatoryscores in UC group increased significantly compared with those in NC, MCand BC groups (P＜0.01 and P＜0.05). Inflammatory scores in BC and MC groupswere higher than those in NC group(P＜0.01). Inflammatory scores in BCgroup were higher than those of MC group (P＜0.05).2. There was a significant alteration in the enteric microbial flora:Bif and Lac were decreased significantly(P＜0.01 and P＜0.05) but Bacillus coil and Mycetes were increased in UC group than those in NC andBC groups(P＜0.01 and P＜0.05). Bifico supplement ameliorated thisimbalance. Though the counts of Bacillus coli in BC group were higher thanthose in NC group(P＜0.01), there was no significance of the differencein Bif and Lac counts between Be and NC groups (P＞0.05). Bacillus colicounts in Me groupwere lower than those in UC group (P＜0.05), but therewere no significance in the difference of other three kinds of flora countsbetween in MC group and in UC group (P＞0.05). The counts of Bacillus coli,Bif and Lac in MC group were lower than those in Be group (P＜0.05). Staphylococci and Clostridium porfringens were not detected in fourgroups.3. Plasma endotoxin levels were (35.20±15.12)pg/ml in NCgroup, (121.25±39.07)pg/ml in UC group, (93.33±21.28) pg/ml in BC groupand (67.55±19.30) pg/ml in Me group. Plasma endotoxin levels in UC groupincreased significantly compared with those in NC, Me and Be groups(P＜0.01 and P＜0.05), which in BC group were higher than those in NC andMC groups (P＜0.01 and P＜0.05). Plasma endotoxin levels in MC group werehigher than those in NC group(P＜0.01).Conclusions1. There was imbalance in gut flora and changes of plasma endotoxinlevels in rats with TNBS-induced colitis.2. Bifico could attenuate inflammation, decrease plasma endotoxin level,and increase Bif and Lac numbers in colons of TNBS-induced colitis rats,its mechanisms of attenuating inflammation may be the reinstitution ofgut flora balance.PARTⅡChanges in phenotype and functions of dendritic cells inmesenteric lymphoid nodes of rats with TNBS-inducedcolitis and effects of BificoObjectiveTo investigate the expressions of 0X-62, CD83, MttC-Ⅱand costimulantory molecules CD86 and function of dendritic cells(DCs) inmesenteric lymphoid nodes. To study effects of Bifico on maturation andactivation of dendritic cells, and to explore its immunoregulatoryfunctions in rats with experimental colitis.Methods1. Method of establishment of the rat model of experimental colitis wasas in PARTⅠ(no MC group).2. All mesenteric lymph nodes of rats in the three groups were removedunder aseptic condition at the time of sacrifice. One was kept forexpressions of 0X-62 and CD83 measured by immunohistochemical staininganalysis. DCs in mesenteric lymph nodes were freshly isolated andpulificated by density gradient centrifugation and panning. Expressionsof MHC-Ⅱand co-stimulantory molecules CD86 on DCs in mesenteric lymphnodes were measured by flow cetometry. The capacity of stimulating T cellswas determined by allogeneic mixed leukocyte reaction (MLR).Results1. Expressions of 0X-62 and CD83 on DCs in mesenteric lymph node ofNC group, UC group and BC group were 198.6±87.7, 1422.1±598.2, 805.7±298.8 and 87.6±24.4, 709.2±199.6, 461.7±172.3, respectively.Expressions of 0X-62 and CD83 on DCs in UC group were significantly higherthan those of rats in NC group (P＜0.01),and also higher than those ofrats in BC group (P＜0.05) .Expressions of 0X-62 and CD83 of rats in BCgroup were higher than those in NC group(P＜0.01).2. Expressions of MHC-Ⅱand CD86 on DCs in mesenteric lymph nodes ofNC group, UC group and BC group were (26.23±7.19)％, (84.55±9.38)%, (66.49±8.40)% and (20.46±7.69)%, (80.63±8.84)%, (59.79±10.03)%, respectively. Expressions ofMltC-Ⅱand CD86 on DCs in UC group weremarkedly higher than those in other two groups (P＜0.01 and P＜0.05), andthose in BC group were higher than those in NC group (P＜0.01).3. Stimulatory Capacity of allogeneic T cells of DCs in UC group andBC group were higher than that in NC group, which was decreased in BC groupthan that in UC group. Conclusions1. DCs in mesenteric lymph nodes of rats with TNBS-induced colitisare increased and in a state of maturation and activation, and theirstimulatory capacity of allogeneic T cells was enhanced indicating moreimmature DCs of rats becoming mature and activated and the enhancementof the function of antigen presenting and activating naive T lymphocytes.2. Bifico might decrease naive T lymphocytes activation throughpreventing immature DCs of rats becoming mature and activated suggestingBifico may regulate immune function of rats with TNBS-induced colitis.PARTⅢExpressions of TLR2, TLR4 and NF-κBp65 on colons ofrats with TNBS-induced colitis and effects of BificoObjectiveTo investigate the expressions of protein and mRNA of Toll-likereceptor 2(TLR2),Toll-like receptor 4(TLR4) and nuclear factorkappaBp65(NF-κB p65) on colons of rats with TNBS-induced colitis.Tostudy effects of Bifico on expressions of TLR2, TLR4 andNF-κBp65 andto explore its immunoregulatory functions in rats with experimentalcolitis.Methods1. Method of establishment of the rat model of experimental colitis wasas in PARTⅠ(no MC group).2. The whole colon including cecum in the three group rats were removedat the time of sacrifice. Total protein and mRNA of the colon wereextracted. Expressions of protein and mRNA of TLR2, TLR4 andNF-κB p65were measured by Western Blotting and real-time quantitative polymerasechain reaction(PeR),respectively.Results1. Protein expressions: Protein expressions of TLR2, TLR4 and NF- κBp65 on colons in NC, UC and BC groups were TLR2:10.26±4.24, 47.20±4.62, 36.64±3.67;TLR4:8.16±4.50, 25.84±4.46, 19.71±3.28, and NF-κBp65:3.16±1.52, 42.96±10.32, 30.74±7.71, respectively. Proteinexpressions of TLR2, TLR4 and NF-κBp65 on colons in NC group were verylow, but markedly increased in UC group(P＜0.01). Protein expressionsof TLR2,TLR4,NF-κBp65 in BC group were lower than those in UC group(P＜0.05), and higher than those in NC group (P＜0.01).2. mRNA expressions: mRNA expressions(copies) of TLR2,TLR4, NF-κBp65 in NC, UC and BC groups were TLR2: (0.24±0.14)×10~3, (10.57±2.69)×10~3, (6.64±1.42)×10~3;TLR4: (0.21±0.11)×10~3, (8.58±2.71)×10~3, (5.29±1.54)×10~3, and NF-κBp65: (0.17±0.07)×10~3, (9.69±2.71)×10~3, (6.21±1.33)×10~3, respectively, mRNA expressions of TLR2,TLR4, NF-κBp65 in UC group were significantly increased compared withthose in NC group (P＜0.02) and in BC (P＜0.05). mRNA expressions ofTLR2, TLR4 and NF-κBp65 in Be group were higher than those in NC group(P＜0.01).Conclusions1. Expressions of TLR2, TLR4 and NF-κBp65 were increasedsignificantlyon colons of rats with TNBS-induced colitis.2. Bifico might decrease expressions of protein and mRNA of TLR2 andTLR4, and downregulate expression of NF-κBp65 of rats withTNBS-induced colitis.PARTⅣof cytokines both at the mucosal and SystemicalChangeslevels in rats with TNBS-induced colitis and effects of BificoObjectiveImbalance between pro-inflammatory and anti-inflammatory cytokinesplays an important role in the onset and development of IBD. The purposeof the study was to investigate the changes of pro-inflammatory cytokinesincluding tumor necrosis factor alpha(TNF-α), interferon-γ(IFN-γ), interleukin-12(IL-12) and anti—inflammatory interleukin-10(IL-10) andtransforming growth factor beta(TGF-β) at mucosal and systemicallevels. We assessed the impacts of Bifico on cytokine production, bothat the mucosal and systemical levels.Methods1. Method of establishment of the rat model of experimental colitiswas as in PARTⅠ(no MC group).2. The spleens of all rats were removed under aseptic condition at thetime of sacrifice, and the splenocytes were isolated and cocultured withthe standard Bacillus coli strain (1×10~6 cells/ml) for 72 hours at 37℃. Supernatants were harvested and stored at -20℃for cytokineanalysis by using enzyme immunoassay (EIA). Cytokines analyzed were TNF-α, FN-γ, IL-12, IL-10 and TGF-β.3. The small intestines of the rats were removed and the lymphoidfollicles of the Peyers' patches (PP) carefully removed from theintestinal serosal side with curved scissors. Seven to ten Peyers'patches were obtained per rat, and immunocytes at Peyers' patches wereisolated and cocultured with the standard Bacillus coli strain (1×10~6 cells/ml) for 72 hours at 37℃. Supernatants were collected and storedat -20℃for analysis of TNF-α, FN-γ, IL-12, IL-10 and TGF-β. Cytokineproduction was measured by EIA.Results1. Cytokine analysis was performed in splenocyte supernatants by EIAfollowing stimulation in vitro with standard Bacillus coli strain. TNF—αand IFN-γwere increased significantly in UC group than those inNC group(P＜0.01) and in BC(P＜0.05), IL-10 were decreased(P＜0.01 andP＜0.05). TNF—αand IFN-γin BC group were higher and IL-10 was lowercompared with those in NC group(P＜0.03). There was no significance indifference of IL-12 among the three groups(P＞0.05). Moreover,production of TGF-βwas maintained in three groups(P＞0.05).2. Cytokine analysis was performed in supernatants of Peyers'patches cells by EIA following stimulation in vitro with standard Bacillus coli strain. TNF—α, IFN-γand IL-12 were significantlyincreased in UC group compared with those in NC group and in BC group(P＜0.01 and P＜0.05), but IL-10 was markedly reduced compared withthose in other two groups (P＜0.01 and P＜0.05). TNF—α,IFN-γandIL-12 in BC group were higher than those in NC group (P＜0.01), but IL-10was lower (P＜0.02). The difference of TGF-βin three groups was slightand didn't reach significance (P＞0.05).Conclusions1. Imbalance between pro-inflammatory and anti-inflammatory cytokineswas existed in rats with TNBS-induced colitis, both at the mucosal andsystemical levels.2. Following challenge with Bacillus coli in vitro, there were reductionin Th1 cytokines (TNF-α, IFN-γ, and IL-12) and increase in Th2 cytokine(IL-10) in the Bifico group whereas production of the immunoregulatorycytokine TGF-βwas maintained.3. The Bifico could improve the imbalance between pro-inflammatorycytokines and anti-inflammatory cytokines at the mucosal and systemicallevels.