| BackgroundUlcerative colitis (UC) is one of inflammatory bowel disease (IBD) with pathogenesis unknowned. UC is a refractory chronic non-specific inflammatory bowel disease, regarded as closely correlated to colorectal cancer. Worryingly, the incidence of UC is increasing in this decade. Although the etiology and pathogenesis of it are not quite clear, the immune response against self antigens play a significant role in the development of the disease process. More evidence was found to support the disease autoimmune damage theory. It has been reported that the imbalance of Th17/Treg, Thl/Th2transformation may be an important factor in the pathogenesis of IBD.As a vital immunocyte, B cells can not only mediate humoral and cellular immunity, but also play a positive role in immune regulation. Recently studies shown that, regulatory B lymphocytes (Bregs), one type of B cell subsets, also control negative regulation in immune responses. Bregs regulate the inflammatory process, inhibit excessive inflammation, reduce transplant rejection, and protect immune cells in autoimmune diseases. Studies of Bregs mainly focus on animal models at present, but rarely focus on humans and nonhuman primates. And It remains unknown whether there are any imbalance factors to the translation of the responsive B cellsand regulatory B cells in the process of UC pathogenesis. Preliminary studies show that, Bregs have a good mitigation for intestinal inflammation induced by DSS in mice. Some studies also found that, Bregs in patients with autoimmune disease (such as systemic lupus erythematosus, etc.) are disfunction. Base on the review of the current studies, Bregs have a variety of phenotypes in different diseases. Even the same phenotype will have different functions in different diseases. Recently, studies of Bregs are mainly focus on the autoimmune disease, and its role in the pathogenesis of UC remains to be explored. Recently there is a subpopulation of Bregs marked as CD19+CD5+CDld+B cell has been found to protect the inflamed colon in DSS induced colitis in mice. In this study, we focused on CD19+CD5+CDld+B cell, and to explore its relationship with UC.The regulatory function of Bregs are mainly relayed on IL-10secretion, which is TLRs/MyD88signaling pathway dependent. LPS can effectively promote the secretion of IL-10by stimulating the cells from mouse spleen. So, as LPS ligand, TLR4/MyD88signaling pathways correlates more closely to the IL-10excretion. Meanwhile, TLRs/MyD88signaling pathways mediate cell-specific signals, by which way dendritic cells can promote T cell activation. But B cells provided a surroundings riching in IL-10and inhibited T cell proliferation and differentiation.At present, adverse reactions of the first-line drug of UC are obvious. Patients with chronic and critical inflammation are relied on the drugs with more serious side effects. So we need to explor some more new tolerant medicine for UC. In the theory of Traditional Chinese medicine, UC belongs to the category of "Dysentery","Changpi""Changfeng""diarrhea", etc,. Baicalin is one of the main active ingredient in Scutellaria, one of traditional Chinese medicine, plays a role in anti-inflammatory,-infective,-allergic,-oxidation,-liver fibrosis,-hyperglycemic,-tumor,-cell damage and so on, and it is widely used in clinic in China. Studies had showed that, the anti-inflammatory effect of baicalin is to reduce the pro-inflammatory cytokines of IL-1β and TNF-a, and reduced NF-κB activity in LPS-activated macrophage and so on. At the same time, baicalin influenced on leukocytes via inhibiting the biosynthesis of leukotriene B4and C4, inhibiting Ca2+production stimulated by fMLP and promoting the intracellular levels of cAMP. So, it is worth to explore the anti-inflammatory effects of baicalin in IBD by regulating Breg cells activation.In this study, UC patients and colitis mouse model were taken as the research object, healthy human as control, and irritable bowel syndrome (IBS) patients as disease control, the correlation of CD19+CD5+CDld+B cells and UC, and the regulation of baicalin to CD19+CD5+CDld+B cells as well as TLR/MyD88signaling pathway were explored.Objective1. To explore the frequency of CD19+CD5+CDld+B cells in peripheral blood of UC patients, as well as the cytokine’s expression levels in serum, and analysis the correlation between CD19+CD5+CDld+B cells and UC.2. To explore whether baicalin will up-regulate the frequency of CD19+CD5+CDld+B cells and the expression of intracellular IL-10of CD19+CD5+CDld+B cells in UC patients. The effect of baicalin act on TLR4/MyD88signal pathway in CD19+CD5+CDld+B cells were explored as well.3. To explore the effects of baicalin on the frequency of CD19+CD5+CDld+B cells and them related signaling proteins. The efficacy of baicalin treatment in mice colitis induced by DSS were also discussed.Methods1. The correlation analysis of CD19+CD5+CDld+B cells and UC(1) Patients access and grouping Inclusion criteria:UC patients:using the diagnostic criteria descripted in The Chinese Standard Diagnosis and Treatment Consensus Opinion of Inflammatory Bowel Disease (2008).IBS patients:using the diagnostic criteria descripted in The Diagnosis and Treatment Consensus Opinion of Irritable Bowel Syndrome in traditional Chinese combined western medicine (2011). Grouping:Healthy control group(n=36);UC group(n=36); IBS group(n=27)。(2) The frequency of CD19+CD5+CDld+B cells in peripheral blood were detected by flow cytometry.(3)The frequency of CD19+CD5+CDld+B cells in colon tissue were detected by laser scanning confocal microscope.(4)Serum levels of IL-6, IL-10, IL-13, IL-17, TNF-a and TGF-β of UC patients were detected by enzyme-linked immunosorbent assay.2. The study of the in vitro effect of Baicalin to CD19+CD5+CDld+B cells(1) Cells inclusion and grouping Inclusion criteria:peripheral blood monouclear cells of moderate UC patients. Grouping:Control group, LPS group, LPS+Baicalin group.(2) Isolation of peripheral blood monouclear cells of UC patients included.(3) Peripheral blood mononuclear cells cocultured with PBS, LPS and LPS+Baicalin.(4) The frequency of CD19+CD5+CDld+B cells in peripheral blood were detected by flow cytometry after cocultured, the expression of IL-10、TLR4and MyD88in CD19+CD5+CDld+B cells were detected.(5) The effect of different stimili on the frequency of CD19+CD5+CDld+B cells.(6) The expression of IL-10, TLR4and MyD88in CD19+CD5+CDld+B cells between different groups.3. The study of the effect of baicalin on CD19+CD5+CDld+B cells and inflamatory signaling proteins in mouse colitis induced by DSS(1) Mice Grouping:32C57BL/6mice were randomly distributed into4groups (n=8):Control group, DSS group, DSS+Baicalin group (DSS+BCLN), DSS+Mesalazin group (DSS+MSLZ).(2) Mice colitis modeling:mice colitis were induced by normal drinking water containing3%(w/v) dextran sulfate sodium (DSS, molecular mass36to50kDa) for7days.(3) Mice treaments:Control group were normal feeding, Baicalin and mesalazine were used to treat DSS colitis in modeling groups, respectively. Treatment programs are as follows:Baicalin, mesalazine were intragastric administration at the dose of100mg/kg0.3ml for7days after modeling success.Statistical AnalysisExperimental data were showed as mean±standard. Statistical software SPSS19.0was use for statistical analysis. ANOVA (one-way ANOVA) method was used for multiple comparison. Firstly, data was tested for homogeneity of variance. If the variance is homogeneity, LSD method was used for multiple comparisons. If the variance is heterogeneity, Welch test was used, and Dunnett’s T3method was used for multiple comparisons. P<0.05was defined as statistically significant.Results1. The correlation analysis of CD19+CD5+CDld+B cells and UC1.1There was no statistically significant difference in frequency of CD19+CD5+CDld+B cells in each group (F=0.402, P=0.670).1.2Serum level of IL-6, IL-10, IL-13, IL-17, TNF-a and TGF-p:(1) IL-6:There was stastically significant difference between the3groups (F=0.402, P=0.013). UC group was higher than the control and IBS group (P=0.577, P=0.381). The control group showed no statistically significant difference with IBS.(2) IL-10:There was stastically significant difference between the3groups (Welch=129.474, P=0.000). UC group was significantly higher than IBS group or control group (P=0.000, P=0.000). IBS group was lower than control group (P=0.002);(3) IL-13:There was stastically significant difference between the3groups (F=4.288, P=0.016). IBS group was significantly higher than control group (P=0.019, P=0.010). There was no signification difference between UC group and IBS group.(4) IL-17:There was stastically significant difference between the3groups (F=27.27, P=0.000). UC group was significantly higher than IBS group and control group (P=0.000). IBS group was significantly lower than control group (P=0.040). (5) TNF-a:There was stastically significant difference between the3groups (F=16.669, P=0.000). UC group was significantly higher than IBS group or control group(P=0.000, P=0.011). IBS group was significantly higher than control group (P <0.001).(6) TGF-P:There was stastically significant difference between the3groups (Welch=71.804, P=0.000). UC group was significantly higher than IBS group or control group (P=0.000). IBS group was significantly higher than control group (P=0.021).1.3Expression of TLRs in colon tissue(1) TLR2:There was stastically significant difference between the3groups (F=10.399, P=0.001). UC group was significantly higher than IBS group and control group (P=0.000, P=0.001). There is no stastically significant difference between IBS group and control group.(2) TLR4:There was stastically significant difference between the3groups (F=18.699, P=0.000). UC group was significantly higher than IBS group and control group (P=0.000, P=0.000). There was no stastically significant difference between IBS group and control group.(3) TLR9:There was stastically significant difference between the3groups (F=41.533, P=0.000). UC group was significantly higher than IBS group and control group (P=0.000, P=0.019). IBS group was significantly higher than control group (P=0.000)1.4The frequency of CD19+CD5+CDld+B lymphocytes in colon tissueThere was no stastically significant difference between the3groups (F=0.728, P=0.486)2. The study of the in vitro effect of baicalin on the frequency of CD19+CD5+CDld+B lymphocytes.2.1The frequency of CD19+CD5+CDld+B cells in PBMC was up-regulated after baicalin treatments.There was stastically significant difference between the3groups (F=16.361, P=0.000). Compared to LPS group and control group, the frequency of CD19+CD5+CDld+B cells in PBMC were higher in LPS+baicalin group(P=0.004, P=0.026). Compared to PBS group, there was significantly higher in LPS group (P=0.033).2.2The expression of MyD88, TLR4and IL-10on CD19+CD5+CDld+B cells after baicalin treatments (F=6.575, P=0.045).(1) TLR4:There was stastically significant difference between the3groups (F=6.575, P=0.006). The expression of TLR4on CD19+CD5+CD1d+B cells increased after LPS stimulation (P=0.002), ther was no stastically significant difference between LPS+BCLN and PBS group (P=0.154);(2) MyD88:There was no stastically significant difference between the3groups (F=1.745, P=0.199).(3) IL-10:There was stastically significant difference between the3groups (Welch=4.577, P=0.035).The expression of IL-10in CD19+CD5+CD1d+B cells increased after LPS stimulation, but decreased after baicalin intervention, the difference of them was statistically significant (P=0.045, P=0.041).3. The effect of baicalin on CD19+CD5+CD1d+B cells and inflamatory signaling proteins in mouse colitis induced by DSS3.1Mice general condition changes after modeling(1) Mice in control group have a smooth coat and normal responsibility, activity, eating and excretion.(2) Modeling group began abnormalities three days after DSS administration (lazy to move, hogback, gather together), weight loss, fur ruffled, diet reduced, and diarrhea. The stool occult blood test were positive and the mice undergoed mucous blood stool apead at day4, this symton reach the peak at day7and can not alleviate until day12.(3) Mice in DSS+BCLN group persistent diarrhea, bloody stool or occult blood untill day8. Bloody stool began to improve at day10and the symton had been generally alleviating at the day12.(4) Mice in DSS+MSLZ group persistent diarrhea untill day8. And bloody stool or occult blood remission at day11. The general conditions were much better than DSS group.3.2Mice mDAI scoresBy repeated measures analysis of variance test, the mDAI score in modeling group (DSS group, DSS+BCLN group and DSS+MSLZ group) changed with the changed of time, time The time factor showed the main effect on various factors of mDAI (F=556.751, P=0.000). There is a main effect between the moduling groups (F=439.921, P=0.000). Compared with modeling group and control group, the difference was statistically significant (P=0.000) after day3. No statistically significant difference between the modeling groups (P=0.418, P=0.260, P=0.059). DSS administration stoped at day8, and the treatment group was given the appropriate medications, mDAI score of DSS+BCLN group and DSS+MSLZ group changed with changed of time, The time factor showed the main effect on various factors of mDAI (F=108.323, P=0.000), There is a main effect between each groups (F=1004.906, P=0.000). mDAI score in DSS+BCLN group and DSS+MSLZ group were lower than DSS group, the difference was statistically significant (all P=0.000); DSS+MSLZ group were lower than DSS+BCLN group, the difference was statistically significant (P=0.016)3.3Frequency of CD19+CD5+CDld+B cells in mice PBMC in each groupThere was no statistically significant difference among DSS group, DSS+MSLZ group, DSS+BCLN group and control group (F=1004.906, P=0.249).3.4Frequency of CD19+CD5+CDld+B cells on colon tissue of miceThere was no statistically significant difference among DSS group, DSS+MSLZ group, DSS+BCLN group and the control group (F=0.932, P=0.438).3.5. Expression of TLR4, MyD88and IL-10on CD19+CD5+CDld+B cells in colon tissue(1) TLR4:There was statistically significant difference between each group (F=4.626, P=0.009). DSS group was significantly higher than control group, DSS+BCLN group and DSS+MSLZ (P=0.01, P=0.001, P=0.026). There was no significant difference between DSS+BCLN group and DSS+MSLZ group.(2) MyD88:There was statistically significant difference between each group (F=3.705, P=0.023). DSS group was significantly higher than DSS+MSLZ group (P=0.002). There is no statistically significant difference among control group, DSS+BCLN group and DSS+MSLZ group.(3) IL-10:There is no statistically significant difference among control group, DSS group, DSS+BCLN group and DSS+MSLZ group (F=0.766, P=0.523).3.6Expression of TLRs on mice colon(1) TLR2:There was statistically significant difference between each group (F=38.862, P=0.000). DSS group was significantly higher than control group, DSS+BCLN group and DSS+MSLZ (all P=0.000). DSS+BCLN group was higher than control group (P=0.000), There is no statistical difference between Control group, DSS+MSLZ group (P=0.055).(2) TLR4:There was statistically significant difference between each group (F=29.428, P=0.000). DSS group was significantly higher than Control group, DSS+BCLN group and DSS+MSLZ group (all P=0.000). There was no significant difference among control group, DSS+BCLN group and DSS+MSLZ group (P=0.454, P=0.140).(3) TLR9:There was statistically significant difference between each group (F=4.581, P=0.010). DSS group was significantly higher than Control group, DSS+BCLN group and DSS+MSLZ group (P=0.006, P=0.025, P=0.002). There is no significant difference among the control group, DSS+BCLN group and DSS+MSLZ group (P=0.538, P=0.698).3.7Expression of signaling protein TLR4, MyD88and NFκB-p65in colon tissue(1) TLR4:There was statistically significant difference between each group (F=5.391, P=0.003). DSS group was significantly higher than DSS+BCLN group, DSS+MSLZ group and the control group (P=0.000, P=0.010, P=0.006). There was no significant difference between DSS+MSLZ group and DSS+BCLN group (P=0.599, P=0.551).(2) MyD88:There was statistically significant difference between each group (F=5.809, P=0.003). DSS group, DSS+BCLN group and DSS+MSLZ group was significantly higher than control group, respectively (P=0.001, P=0.003, P=0.004). There was no significant difference among DSS group, DSS+MSLZ group and DSS+BCLN group.(3) NFKB-p65:There was statistically significant difference between each group (F=30.219, P=0.000). DSS group was significantly higher than DSS+BCLN group, DSS+MSLZ group and control group (all P=0.000). There was no significant difference between DSS+MSLZ group and DSS+BCLN group (P=0.017, P=0.018).3.8Expression of IL-6, IL-10, IL-13and TNF-a mRNA in colon tissue(1) IL-6:There was statistically significant difference between each group (F=9.084, P=0.000). DSS group was significantly higher than DSS+BCLN group, DSS+MSLZ group and the control group (P=0.000, P=0.001, P=0.000).(2) IL-10:There was statistically significant difference between each group (F=19.575, P=0.000). DSS group, DSS+BCLN group and DSS+MSLZ group was higher than control group, respectively (all P=0.000). DSS+BCLN group was higher than DSS group and DSS+MSLZ group (P=0.003, P=0.002).(3) IL-13:There was statistically significant difference between each group (F=10.423, P=0.000). DSS group was higher than control group, DSS+MSLZ group, and DSS+BCLN group (P=0.001, P=0.000, P=0.000).(4) TNF-α:There was statistically significant difference between each group (F=6.613, P=0.002). DSS group was significantly higher than DSS+BCLN group, DSS+MSLZ group and the control group (P=0.000, P=0.001, P=0.005).Conclusion1. There was no significant difference in.the frequency of CD19+CD5+CDld+B cells in UC patients, healthy donor and IBS patients group, suggesting that, there was no significant relation between CD19+CD5+CDld+B cells and UC;2. After stimulated by baicalin in vitro, the frequency of CD19+CD5+CDld+B cells in PBMC of UC patients were upregulated, but the expression of TLR4, MyD88and IL-10were failed to increase.3. After intragastric administration, baicalin failed to increase the frequency of CD19+CD5+CDld+B cells in peripheral blood and colon tissue of mice with colitis induced by DSS, and it failed to up regulate the expression of IL-10in CD19+CD5+CDld+B cells via TLR4/MyD88signal protein up-regulate. suggesting that baicalin failed to up-regulate the regulatory function of CD19+CD5+CDld+B cells through TLR4/MyD88;4. Baicalin treatment up regulated the transcriptional levels of IL-10in colon tissue, and also it can reduced IL-6, IL-13and TNF-a transcripttional levels, consequently remit the inflammation of DSS-induced colitis. The efficacy of Baicalin in DSS induced colitis may be associated with the down-regulation TLR4/NFκB signaling pathway protein in inflammatory cells in the colon tissue of mice. |
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