| BackgroundUlcerative colitis (UC), a chronic relapsing non-specific inflammatory bowel disease, is a condition in which the inflammatory response and morphologic changes remain confined to the colon. The most consistent feature of UC is the presence of blood and mucus mixed with stool, accompanied by lower abdominal cramping which is most intense during the passage of bowel movements. Ulcerative colitis and Crohn’s disease are chronic, relapsing, immunologically mediated disorders that are collectively referred to as inflammatory bowel diseases (IBD).The prevalence of IBD rapidly increased in Europe and North America and is becoming more common in the rest of the world as different countries adopt a Western lifestyle.In Asia, UC is more prevalent than CD. Unfortunately,the currenttherapeutic strategycould ameliorate the inflammation, but neither to cure the disease nor change the underlying disturbances of immune regulation, which leads to a lifelong chronic, recurrence or accelebrate condition. Under the theory of Troditional Chinese medicine, UC was mainly due to dampness pathogen, and associated with intestines, liver, kidneysclosely. At the onset of UC, it’s syndrome are mainly as interior dampness-heat, impairment of the spleen and kidney over time,and invading collaterals, the viscera, Qi, blood, Yin Yang. UC was located in bowels and blood-aspect.It is argued that UC is caused by dampness-heatsyndrome in active stage, spleen-kindney deficiency and heat, dampness in remission.Chinese classics side HQT for UC have a good effect, therefore, development of natural medicine compound or monomer is expected to open up a new way for treating UC.The etiology of UC most likely involves a complex interaction of genetic, environmental, and immunoregulatory factors. Current hypotheses propose that damage to the bowel mucosa occurs as a result of a dysregulated immune response to multiple mucosal antigens comprised within the constituents of the "normal" intestinal microbial flora. Studies have shown that CD4+T cells, which was mainly comprised of Thl, Th2, Th17and Treg, is closely related to the pathogenesis of UC.The colon homing of CD4+T cells by chemokines is an important stepto induce a local immune response in UC. Adhesion molecules on thesurface of T lymphocytes control the cells into the lymphatic and non-lymphatic tissue exudate, and integrin plays an important role in this process.For lymphocytes, adhesion to the vascular wall is partly controlled by β1integrin (also known as CD29) which identify its ligands VCAM-1. Studies have shown that VCAM-1plays a central role in leukocyte recruitment in colitis and blockade of this adhesion molecule has therapeutic effect in experimental model. Our previous study also found that VCAM-1(CD29ligand) in colons of damp-heat rats and patients with UC were increased. Inflammation in UC is limited to the mucosa and superficial submucosamay be associated with expression of VCAM-1and homing of CD4+CD29+T cells by chemokine.CD4+CD29+T cells is lowered insecondary immune responsedeficiency diseases(such as atopic dermatitis, and chronic granulomatous disease), and increased in gingivitis or acute hepatitis C or other over response inflammatory diseases. Our previous studies have observed that CD4+CD29+T cells of peripheral blood in damp-heat rats and patients with UC were increased. Therefore, we believe CD4+CD29+T cell may be related with UC over immune response.As we all know, UC is an intractable diseases that repeatedly remit and relapse throughout life, It is also known that extraintestinal complications, such as primary sclerosing cholangitis, develop in some cases of ulcerative colitis (UC) after total resection of the large intestine, which is the site of inflammation, and that ileal pouchitis develops after total colectomy. These findings provided clues that led us to the recent discovery that colitogenic CD4+memory T cells, which are capable of reproducing colitis, continuously circulate throughout the body, not just the intestine, and that they are retained in organs outside the intestine and are involved in the intractability and persistence of UC.Additionally, CD29as a memory cell markerin many previous studies, and CD4+CD29+T cells are belived as a subtype of memory CD4+T cells.All of that reflected that CD4+CD29+T cells may be associated with replased UCclosely.Thus, CD4+CD29+T cells play an important role in the immune response of UC, but rarely study focus on it and no reasch explore the relationship between CD4+CD29+T cell and CD4+T cell subsets. It is still not clear whether CD4+CD29+T cells is an independent CD4+T cell subset or mainly consistent with one of Th1, Th2, Th17and Treg.Nowadays, a wide arsenal of drugs such as5-aminosalicylates, corticosteroids, immunosuppressives and tumor necrosis factor alpha (TNF-a) antibodies are available for UC therapy applied in a multi-tiered approach. Still, at one point of their life25%-40%of UC patients have to undergo abdominal surgeries (in most cases resections) due to failure of other therapeutic options. Furthermore, severe adverse effects of long-term medication negatively affect the quality of life of UC patients demonstrating the need for better targeted formulations.Due to the changing of the global disease spectrum and medical model, as well as the extensive adverse reactions by use of synthetic drugs, calling for the development and utilization of traditional Chinese herbal and natural medicine is growing, the natural medicines for maintaining health or disease prevention is full of expectations. Baicalin, a major active ingredient of traditional Chinese hebal radix scutellariae, has anti-inflammatory and immune regulating effects, studies have shown that baicalin can inhibit Th17cell expansion and limit their migration to the inflammation sites of kidneys, and can inhibit VCAM1expression on HUVEC. It was demonstated that baicalin can inhibit a certain type of CD4+T cellsand limit their migration to inflamed tissues. However, no study report whether baicalin can effect the function and differentiationof CD4+T cell subsets of UC patients. Therefore, further studies are needed to find a new way for treatment UC.In summary, the adhesion and memoryfeatures of CD29, meet the confined lesions andreplase clinicialpathoiogy of UC.Research on the unctional of CD4+CD29+T cells and its relationships with Thl, Th2,Th17and Treg, will benefit to clarify the mechanism of UC and prevent its reccurrent. In addition, baicalin has anti-inflammatory and immunomodulatory effects, discuss the effect of baicalin on T cells in peripheral blood of UCpatients is expected to develop a safe and effective natural medicine to treatment UC.In this study, we investigated the the number of cells, the expressing of major transcription factor mRNA and cytokine of CD4+T cells subsets in peripheral blood of UC patients, while the number of CD4+CD29+T cells and its relationship with CD4+T cell subsets was also conducted, whichfurther improvment the UC immunological mechanisms. We also explored the effect of baicalin on T cells in peripheral blood of UC patientsin vitro, and the possible mechanisms were discussed. It will provide valuableinsight for development of traditional Chinese medicine and the treatment of UC.Purpose1. To study the expression and significance of CD4+T lymphocyte subsets (ie,Th1、Th2、Th17and Treg) cells in peripheral blood of patientswith UC by flow cytometry and Rt-PCR.2To study the expression of CD4+CD29+T cell in peripheral blood of patients with UC and to explore its correlation with disease severity; The relationship between CD4+CD29+T cells with the level of CD4+T cell subsets was also investigated by flow cytometry gating techniques.3. To explore the changing of CD4+T cell associated cytokines and transcription factors of peripheral blood mononuclear cells interved by baicalin in vitro, and expected the effect of baicalin on the role of CD4+T cells in patients with UC and its mechanism.METHOD1.Expression of CD4+T cell subsets and related cytokines in patients with UC1.1Inclusion criteria and groupingAll patients are from Southern Medical University nanfang hospital from2012September to2013April.(n=26,14male,and12female,ages32.2±11.0years).Inclusion criteria refer to 《Consensus on the diagnosis and treatment of inflammatory bowel disease》 which was approved by inflammatory bowel disease group of Digestive disease branch of Chinese medical associationin2012. While, healthy volunteers were enrolled as control group.(n=21,13male,and8female, ages30.55±10.44years).1.2Disease lesion was measured by colonoscope,biopsy was gotten for HE staining to observed the histopathological changes,Disease activity index for UC patients (DAI) score was also performed.1.3The proportion of CD4+T cells subsets(Th1,Th2,Th17,Treg)in PBMCs were detected by the flow cytometry.We defined the CD4+IFN-γ+T cells as Thl cells,CD4+IL-4+T cells as Th2cells,CD4+IL-17+as Th17cells and defined CD4+CD25+Foxp3+as Classic Treg cells.1.4The level of CD4+T Cell subsets related transcription factor mRNA in PBMCs were detectedby Rt-PCR.1.5Screening the CD4+T cell-associated cytokine in Serum use the human Th1/Th2/Th17antibody chips (QAH-TH17-1) which was produced by the American RayBiotech company,and semi-quantitative analysis was also performed.1.6Detect the variation of IL-17,IL-23,IL-10mRNA in mucosal biopsies by Rt-PCR. 2.The Relations between CD4+CD29+T cells and helper T cells in the peripheral blood of UC patients.2.1Inclusion criteria and grouping:All patients are from Southern Medical University nanfang hospital from2012September to2013April.(n=16,10male,and6female,ages28.9±10.2years).Inclusion criteria refer to 《Consensus on the diagnosis and treatment of inflammatory bowel disease》 which was approved by inflammatory bowel disease group of Digestive disease branch of Chinese medical association in2012. While, healthy volunteers were enrolled as control group.(n=16,10male,and6female,ages28.8±9..9years).2.2Disease lesion was measured by colonoscope,biopsy was gotten for HE staining to observed the histopathological changes, Disease activity index for UC patients (DAI) score was also performed.2.3The levels of CD4+CD29+T cells in peripheral blood between the control group and the UC patientswere compared by flow cytometry. And analyze its correlation with the DAI.2.4Gating with CD4+CD29+by flow cytometry,and compare the level of CD4+IFN-γ+(Thl),CD4+IL-4+(Th2) and CD4+IL-17+(Th17) between control group and the UC patients.3. The impact on IL-23R/Jak2/Stat3pathway with baicalin in vitro stimulation of peripheral blood mononuclear cells of UC patients.Peripheral blood mononuclear cell (PMBCs) of patients with moderate to severe UC (n=52) andnormal healthy person (n=12). The diagnostic criteria for UC and normal control groups refer to the part1.3.1Use Western Blotting detection the relative expression level of IL23R, JAk-2, Stat-3and Smad proteins in the isolated PBMCs from UC patients (n=12) and all the normal control group (n=12).3.2PBMCs were isolated fromUC patients (n=44), and stimulated cultures with different concentrations of baicalin.Cell culture groups:①control group:DMSO+an ti-CD3and anti-CD28stimulation group;②Low concentrations of baicalin group(BL group):5u mol baicalin+DMSO+anti-CD3+anti-CD28stimulation;③moderate baicalin concentration group (BM group):20μmol baicalin+DMSO+anti-CD3+anti-CD28stimulation;④high baicalin concentrations group(BH Group):40μml baicalin+DMSO+anti-CD3+anti-CD28stimulation. DMSO, dimethyl sulfoxide, as a solvent for baicalin.3.3Observe the effect of different concentrations of baicalin on the expression the expression of RORC mRNA and IL-17mRNABy Rt-PCR.3.4Using Western Blotting technique to compare the effects of different concentrations of baicalin on the expression of IL-23Rand JAK2.3.5Using Western Blotting technique to compare the effects of different concentrations of baicalin on the phosphorylation of STAT3and Smad.StatisticalAnalysisAll statisticalprocedureswereperformed using SPSS software version16.0(SPSSfor windows). All data areexpressedas mean±SD. Comparisons betweenthree or moregroupswere done with a one-way ANOVA. If the variance between groupswere homogenous,groups weresubjectedthemultiple comparisonleast signi ficant differences(LSD)test. In case of no homogeneity variance, differences were evaluatedby Welch and thegroupsweresubjectedto themultiple comparisons Dunnett’S T3test. Ccorrelation analysis fornormal distribution data were conducted by Pear son correlational analyses. P<0.05was considered statistically significant. Results1.Expression of CD4+T cell subsets and cytokines in UC patients.1.1Colonoscopy andhistopathological features of UC patients included in the experiment meet the diagnostic criteria for UC;1.2The Thl (CD4+IFN-γ+), Th2(CD4+IL-4+) cells proportions of UC patients were increased compared with the control group, but the difference was not significantly(t=1.791P=0.095and1=1.174, P=0.260). The proportion of Th17cells in the peripheral blood of patients with UC were significantly higher, the difference was significantly (t=3.421,P=0.008). The proportion of Treg cells in peripheral blood of patients with UC was lower than the control group,the difference was significantly (t=2.954, P=0.01).1.3There is no significant difference between T-bet and GATA-3mRNA expression in PBMCs of UC patient and the control group (t=1.195, P=0.246; t=0.310, P=0.760). RORc mRNA expression was higher than in control group,the difference was significantly (t=2.721, P=0.013). The Foxp3mRNA expression was lower than in the control group,the difference was significantly (t=3.610, P=0.002).1.4Using protein chips, four cytokines(IL-2, IL-10, IL-21and IL-23)in peripheral blood were found different between the normal control group and UC group. The levels of these cytokines were higher in the UC group than in control group, the difference was significantly (t=5.631,P<0.001; t=4.539, P<.001; t=4.767,P<0.001; t=4.639,P=0.001)1.5Expression of IL-17mRAN and IL-23mRNA in biopsies were different among inflammation site, non-inflammation site and the control group (F=21.329, P<0.001; F=11.565, P<0.001), wherein, inflammation sites were significantly higher than that from non-inflammatory sites (P<0.001,P<0.001) andthe control group (P=0.238, P=0.250).However,there were no differences onf IL-10mRNA among inflammation sites and non-inflammatory sites and the control group (F=0.058,P=0.944).2. The relationsh between CD4+CD29+T cells and helper T cells in peripheral blood serum of UC patients.2.1The proportion of CD4+CD29+T cells in peripheral blood of UC patients were higher compared with the control group,the difference was significantly (t=7.142, P<0.001)2.2CD4+CD29+T cell levels in peripheral blood of UC patients have positive correlation with their DAI score (r=0.973, P<0.001),The correlation coefficient was statistically significant.2.3IFN-y, IL-4expression levels tested by Flow cytometric analysis were not different in CD4+CD29+T cells between the UC patients and normal control group (t=1.619, P=0.119; t=0,526, P=0.640), whereas, the expression of IL-17in CD4+CD29+T cells were higher in UC group than the control group (t=3.876, P=0.001).3The change of IL-23R/Jak2/Stat3pathway of peripheral blood mononuclear cells of UC patientsstimulating with baicalin in vitro.3.1Compared with the control group, expression of IL-23R, Jak-2, Stat-3in UC patients was increased significantly (t=5.787, P<0.001; t=3.486, P<0.006).While Smad expression was descreased (t=0.639, P=0.537)3.2Expressionof IL-17and RORc mRNA in Control group, BL group, BM Group and BH group were different significantly (F=5.78, p<.01and F=2.27, p0.01). Expression levels of RORc and IL-17mRNA were no difference in BL group compared with the Control group (P=0722, P=0.143).Expression level of RORc mRNA is significantly lower in BM and BH group than in the control group(P=0.001, P<0.001), Expression level of IL-17mRNA is significantly lower in BM and BH group than in the control group(P=0.002, P<0.001).3.3. Expressionof IL-23R and JAK-2protein in Control group, BL group, BM Group and BH group were different (F=9.142, P=0.006and F=6.910, P=0.013) Expression of IL-23R, JAK-2were not different in BL group compared with the Control group (P=0.376, P=0.513).Expression of IL-23R is lower in BM and BH group than the control group(P=0.022, P<0.001), Expression of JAK-2is lower in BM and BH group than the control group(P=0.040, P=0.003).3.4. Expression level of p-stat3in Control group, BL group, BM Group and BH group were different significantly (F=21.047, P<0.001). Expression levels of p-stat3were no difference in BL group compared with the Control group (P=0.08). p-stat3in BM and BH group was lower than the control group (P=0.033, P<0.001). Expression ofP-smad were different among the four groups (F=8.516, P=0.007).There was no difference between BLgroup and control group (P=0.534). Expression of p-smad in BM and BH group washigher than the control group(P=0.043, P=0.002).Conclusions1. Expression of Thl7cells and its major transcription factor RORc mRNA in peripheral blood of UC patientswas significantly higher thanthe control group,but, Treg cells and their specific transcription factor Foxp3mRNA expression level was significantly lower. The levels of IL-21, IL-23that maintain the stability of Th17cells was found to be elevated, that suggested that Th17/Treg imbalance may contribut to the occur of UC.2.Expression of IL-17, IL-23mRNA in UC intestinal inflammation sites was significantly higher thannon-inflammationand the control group, suggesting IL-23/IL-17pathway is involved in the maintenance of pathogenic Th17. 3.The memory CD4+CD29+T cells in peripheral blood of UC patients increased significantly and positively correlated with DAI, suggesting that it is closely related to the pathogenesis of UC;4. Expression of IL-17on the surface of memory CD4+CD29+T cells in UC patients was significantly increased, suggesting that Th17(CD4+IL-17+T) cells may be major "memory pathogenic cell" result in the recurrent of UC.5. Expression of IL-23R, Jak-2and Stat-3in PBMCs of UC patient were increased, suggesting that IL-23R/Jak2/Stat3pathway is involved in the occurrence of UC.6. Baicalin can inhibit the expression RORc and IL-17A in PBMCs of UC patients, suggesting that baicalin can inhibit Thl7function through down-regulating IL-23R and Jak2, which could be via inhibiting the phosphorylation of Stat3, upregulating the level of phosphorylation of Smad. |
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