The Protective Effect Of Vitamin D₃ Combined With Lactobacillus Rhamnosus And P40 On Colitis And Its Mechanism

Part 1 The Correlation between Vitamin D Level and Intestinal Microbiota in Patients with Inflammatory Bowel DiseaseObjective:To explore the correlation between serum total 25-hydroxyvitamin D(T-25(OH)D)level and fecal microbiota in patients with inflammatory bowel disease(IBD).Methods:The fecal microbiota of 23 IBD patients who completed the tests for serum T-25(OH)D level was studied using V4 hypervariable region of 16S ribosomal RNA(rRNA)gene sequencing.The patients were divided into three groups including VitD normal group(n=5),VitD insufficient group(n=5)and VitD deficient group(n=13)according to the level of serum T-25(OH)D.The diversity and composition of microbiota among these three groups were analyzed.Spearman correlation test was used to investigate the correlation between serum T-25(OH)D level and relative abundance of fecal microflora at phylum,family and genus levels.Results:1.There was no significant difference in ? or ? diversity among three groups.2.The relative abundance of Actinomycete was highest in VitD sufficient group,and that of Proteobacteria was highest in VitD deficient group.3.At phylum level,serum T-25(OH)D level was positively correlated with the relative abundance of Actinomycetes(r=0.447,P=0.033)and negatively correlated with the relative abundance of Proteobacteria(r=-0.445,P=0.033).At family level,serum T-25(OH)D level was positively correlated with the relative abundance of Lachnospiraceae(r=0.414,P=0.049),Bifidobacteriaceae(r=0.468,P=0.024)?Erysipelotrichacea(r=0.584,P=0.003)and Eggerthellaceae(r=0.507,P=0.014),but negatively correlated with the relative abundance of Aerococcaceae(r=-0.514,p=0.012).At genus level,serum T-25(OH)D level was positively correlated with the relative abundance of Blautia(r=0.459,P=0.028),Bifidobacterium(r=0.468,P=0.024),Faecalitalea(r=0.544.P=0.007),Anaerostipes(r=0.475,P=0.022),Romboutsia(r=0.510,P=0.013),Flavonifractor(r=0.455,P=0.029)and Erysipelatoclostridium(r=0.617,P=0.002).Conclusions:The composition of fecal microbiota was different in IBD patients with different serum T-25(OH)D levels.Serum T-25(OH)D level was negatively correlated with the relative abundance of harmful bacteria such as Proteobacteria,and positively correlated with the relative abundance of probiotics including Lachnospiraceae and Bifidobacteriaceae.Part 2 The Protective Effects and Mechanisms of Combination Treatment with Vitamin D3 and Lactobacillus rhamnosus GG on Dextran sodium sulfate-induced Acute Colitis in Mice.Background:Although the level of T-25(OH)D decreased in IBD patients,and it was negatively correlated with disease activity,VitD supplementation didn't significantly decrease disease activity of IBD patients as expected due to the low expression of vitamin D receptor(VDR)in colonic epithelial cells.Lactobacillus rhamnosus GG(LGG)was reported to have the ability to up-regulate the expression of VDR.Objective:This study aimed to evaluate the protective effects and mechanisms of combination treatment with Vitamin D3 and Lactobacillus rhamnosus GG on dextran sulfate sodium(DSS)-induced acute colitis in mice.Methods:1.Vdr+/+mice:Twenty-five Vdr+/+C57BL/6 male mice were randomly divided into 5 groups(n=5)including control group,DSS group,VitD3 group,LGG group,and VitD3 combined with LGG group.Acute colitis was induced in mice by administration of 2.5%DSS for 5 days.From 7 days pre-DSS modeling to 6 days post-DSS modeling,VitD3 100IU/d,LGG 1.95 × 10^8 CFU/d,and VitD3 100IU/d combined with LGG 1.95 × 10^8 CFU/d were given to the intervention groups correspondingly by gavage.The severity of colitis was evaluated by body weight loss,disease activity index(DAI),colon length and histological score.Blood samples were collected to detect the serum T-25(OH)D level and colon specimens were harvested for assessing the relative expression of colonic cytokines by qRT-PCR.VDR expression and distribution in colon were detected by qRT-PCR,Western blot and immunohistochemistry(IHC).Ki67 expression in colonic epithelium was assessed by IHC.2.Vdr-/-mice:Twelve Vdr-/-C57BL/6 mice were randomly divided into three groups including control group(n=3),DSS group(n=4)and VitD3 combined with LGG group(n=5).The methods of modeling,intervention and evaluation of the above three groups were the same as Vdr+/+ mice from control group,DSS group and VitD3 combined with LGG group,respectively.Results:1.Vdr+/+ mice:Compared to the mice in control group,the expression of total and nuclear VDR protein of colon decreased in mice from DSS group(P<0.05).Compared to the DSS mice without intervention,the DSS mice treated with VitD3 tended to have lower DAIs and histological scores with longer colon length(P>0.05).The DSS mice treated with LGG showed decreased body weight loss and DAIs(P<0.05),accompanied with increased Ki67 expression in colonic epithelium(P=0.038).While the DSS mice treated with VitD3 and LGG displayed less severe colitis compared to DSS mice without interwention,with decreased DAIs and histological scores and increased colon length(P<0.05).Decreased expression of TNFa mRNA(P=0.028)and increased expression of IL-10 mRNA(P=0.016)in colon were also observed in DSS mice treated with VitD3 and LGG.Besides,the serum level of T-25(OH)D was increased significangtly in DSS mice from this group(P<0.001),accompanied with the increased relative expression of the VDR mRNA and protein in colon(P<0.05),as well as the nuclear VDR protein in colonic epithelium(P=0.024).What's more,the expression of Ki67 in colonic epithelium also increased(P<0.05)in mice from VitD3 combined with LGG group.2.Vdr-/-mice:There were no significant differences in weight loss,DAI,colon length,histological scores,relative expression of colonic TNFa a mRNA or Ki67 expression in colonic epithelium between DSS mice without intervention and mice treated with VitD3 and LGG.Conclusions:Coadministration of VitD3 and LGG synergistically alleviates the DSS-induced acute colitis in mice through increasing the expression of VDR in colonic mucosa,promoting the proliferation of colonic epithelial cells and regulating the expression of colonic cytokines.Part 3 The Protective Effects and Mechanisms of Combination Treatment with Vitamin D3 and Lactobacillus rhamnosus GG Secreted Protein p40 on Dextran sodium sulfate-induced Acute Colitis in Mice.Background:VitD3 combined with LGG can play a synergistic anti-inflammatory role by upregulating the expression of VDR in colon.However the mechanism is still unclear.p40 is a soluble protein with anti-inflammatory effects secreted by LGG.Objective:This study aimed to explore the effects of p40 on VDR expression in colon,and the effects and mechanisms of VitD3 combined with p40 on DSS-induced acute colitis in mice.Methods:1.In vitro:After stimulation of HCT116 with different concentrations(0-200ng/ml)of p40 for 48h,and different duration(0-48h)of p40 100ng/ml,qRT-PCR and Western blot were used to measure the expression of VDR.After stimulation of HCT116 with p40 100ng/ml for 48h,Western blot was used to assess the nuclear and cytoplasma VDR expression.After stimulation of HCT116 with different p40 concentrations(0-100ng/ml),cell growth curve was assessed by MTT.2.In vivo:Twenty-five Vdr+/+ C57BL/6 male mice were randomly divided into 5 groups(n=5)including control group,DSS group,VitD3 group,p40 group,VitD3 combined with p40 group.Acute colitis was induced in mice by treating with 2.5%DSS for 6 days.From the first day of DSS modeling to one day after DSS withdrawal,VitD3 100IU/d gavage,p4020?g/d enema,VitD3 100IU/d gavage combined with p40 20?g/d enema were given correspondingly to intervention groups.The severity of colitis was evaluated by body weight loss,DAI,colon length and histological score.Serum T-25(OH)D level was measured.VDR expression and distribution in colon were detected by qRT-PCR,Western blot and IHC.Ki67 expression in clonic epithelium was also assessed by IHC.Results:1.In vitro:Compared to control group,the relative VDR protein expression of HCT116 increased after p40 50ng/ml,100ng/ml and 200ng/ml stimulation for 48 hours,as well as p40 100ng/ml stimulation for 36 h and 48 h(P<0.05).After p40 100ng/ml stimulation for 48 h,the expression of nuclear VDR protein tended to increase,while no change of cytoplasmic VDR protein expression was observed.The number of HCT116 living cells increased after p40 20ng/ml stimulation for 24 hours,p40 1-50ng/ml stimulation for 48h and p40 1-100ng/ml stimulation for 72 hours.2.In vivo:Compared to DSS mice without intervention,body weight loss decreased(P=0.036)and serum T-25(OH)D level increased(P<0.001)in DSS mice treated with VitD3.The DSS mice treated with p40 tended to have lower body weight loss and histological scores,accompanied with longer colon length than mice from DSS group without significance(P>0.05).However,both of the expression of total colonic VDR protein and nuclear VDR protein in colonic epithelium increased significantly(P<0.05)in mice from p40 group.The mice treated with VitD3 combined with p40 displayed less severe colitis than mice from DSS group,with decreased body weight loss and DAIs and longer colon length(P<0.05).Besides,the serum level of T-25(OH)D was also increased(P<0.001),accompanied with the increased relative expression of colonic VDR mRNA and protein,as well as the nuclear VDR protein expression in colonic epithelium(P<0.05)in mice treated with VitD3 combined with p40.In addtion,the expression of Ki67 detected by IHC also increased in mice from this group(P=0.032).Conclusions:VitD3 combined with p40 can synergistically alleviate DSS-induced acute colitis by upregulating the expression of VDR and promoting the proliferation of colonic epithelium in mice.