| BackgroundInflammatory bowel disease (IBD) is a chronic, relapsing disorder of the gastrointestinal tract that may manifest as either Crohn’s disease (CD) or ulcerative colitis (UC). The disease is characterized by unpredictable attacks of inflammation of the intestine and the clinical symptoms include weight loss, diarrhea accompanied by blood, and abdominal pain. The highest incidence rates and prevalence of UC and CD have been reported from northern Europe and North America, where the rates are beginning to stabilize. Rates continue to rise in low-incidence areas such as southern Europe, Asia, and most developing countries including China. The etiology of IBD is unknown, but the condition seems to be the result of a combination of environmental, genetic and immunologic factors in which an uncontrolled immune response within the intestinal lumen leads to inflammation in genetically predisposed individuals. There is strong evidence to support that deregulated immune response to commensal bacteria in a genetically susceptible individual drives IBD.CD4+T cells are important for combating infectious diseases and cancers. However, when aberrant, they are responsible for chronic inflammatory diseases including inflammatory bowel disease (IBD), because each of the different subsets plays a role in IBD. The various CD4+T cells subsets contain Thl cells, Th2cells, regulatory T cells (Treg) and newly reported Th17cells. Before Th17cells characterization, it had been considered that Thl/Th2imbalance plays an important role in the pathogenesis of IBD. The discovery of Th17and Treg cells has enabled the IBD field to rapidly expand what is known about the disease. Therapeutics targeting these aberrant Th responses is already under development and hold promise for treating IBD or other chronic inflammatory diseases.Treatment of IBD includes conservative measures as well as surgical approaches in those who are non-responders to medical treatment. Pharmaceutical treatment of IBD includes five major categories, namely anti-inflammatory drugs, immunosuppressant, biologic agents, antibiotics, and drugs for symptomatic relief. The primary therapeutic goals are related to improvement of patient quality of life by inducing and maintaining remission, predicting, preventing and treating complications, restoring nutritional deficits, providing appropriate psychosocial support, and modifying the course in those with aggressive disease. But current conventional treatment opportunities for IBD are limited, and may have severe side-effects. Combined with the limited efficacy and potential adverse effects of current treatments, new effective and safe treatment for IBD is urgently needed. Chinese/Oriental herbal medicine has long been used for treating IBD. Herbal cocktails take advantage of synergy and interactions among a myriad of phytochemicals present in the different herbs to achieve therapeutic efficacy targeting multiple biological and pathological processes while minimizing side effects. Huangqin-Tang is one of the most well-known Traditional Chinese Medicine formula. Although Huangqin-Tang has long been used to treat IBD, the underlying mechanisms by which these effects remains to be defined.To explore the mechanism of Huangqin-Tang in the treatment of IBD, we investigated whether the Th1/Th2and Th17/Treg imbalance could be reversed by Huangqin-Tang and whether the reversal was related to the improvement of clinical indications. Chapter I Huangqin-Tang confers a protective effect on TNBS-induced colitis in ratsObjectiveThe aim of the present study was to investigate whether Huangqin-Tang may protect and down-regulate the inflammatory response in2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats.Methods1. Induction of colitis and experimental design:34specific pathogen-free (SPF) rats were randomly assigned to two groups as follows:control group (Cntrl, n=9) and colitis group (n=25). Then30mg of TNBS dissolved in0.25ml of50%ethanol was administered through the catheter to the rats of colitis group. Rats in the control group underwent the same procedure but were administered with physiological saline instead. Two rats were randomly chosen from each group and then the colon tissue sections that stained with HE method by light microscope to confirm colitis rats replicated successfully. Then, the colitis group was randomly divided into three groups:TNBS-induced colitis group (TNBS, n=8), Huangqin-Tang group (HQT, n=8) and Mesalazine group (ML, n=8).2. Treatment:Drug administration to animals was started24h after induction of colitis and repeated twice a day for one week. HQT group that received the TNBS challenge and treatment with HQT (11.67%.0.12g/100g-d). ML group that received the TNBS challenge and treatment with ML (1%1ml/100g·d).3. Assessment of protective effect of HQT on TNBS-induced colitis:Development and progress of colitis was assessed clinically. Throughout the experimental period multiple clinical parameters were measured. The body weight, stool consistency, and the occult blood in stool or at the anus were recorded daily. The disease activity index (DAI) was a mean score of the three parameters. Colon tissue was analyzed macroscopically and histologically. The indexes of spleen and thymus were measured. Colon was collected for myeloperoxidase (MPO) activity as well as cytokine levers measurement. The cytokine levers tumor necrosis factor-a (TNF-a) and interleukin-1β (IL-1β) were determined by the use of Enzyme-linked immunosorbent assay (ELISA).Statistical AnalysisAll statistical procedures were performed using SPSS software version16.0(SPSS for windows). All data are expressed as mean±S.D. Comparisons between two groups were analyzed with the unpaired Student’s-test. Comparisons between three or more groups were done with a one-way ANOVA. If the variance between groups were homogenous (levene’s test), groups were subjected the multiple comparison least significant differences (LSD) test. In case of no homogeneity variance, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett’s T3test. Repeated measured parameters in different time point was analyzed by repetitive measurement and analysis of variance. Macroscopic and histological scores were statistically analyzed using by Nonparametric Test Kruskal-Wallis H Test followed by the Mann-Whitney U test to compare the results of the different groups. P<0.05was considered statistically significant.Results1. Model verification:Two days after establishing the model of colitis, all rats had loose stools, followed by mucous stool and bloody purulent stool. Rats in TNBS group companied with control group showed slow response, lack of movement, tiredness, messy hair, hollow back, poor appetite, little drinking-water and loose weight. The macroscopic feature showed extensive mucosal injury throughout the distal part of colon; the mucosa appeared ulcerated, edematous, hyperemic, and hemorrhagic. The microscopic evaluation of colonic segments of healthy control group rats showed a histological normal structure. However severe mucosal damage was observed in rats treated with TNBS, characterized by increased neutrophil numbers, massive bowel edema, dense infiltration of the superficial mucosal layers, epithelial cell disruption, and the architecture of the crypts was distorted.2. By day7, the healthy control group had gained weight, however, the instillation of TNBS led to severe colitis rapidly with the typical signs including dramatic body weight loss (P<0.001) and shortened colon length (t=6.662, P<0.001), accompanied with obvious marked bloody diarrhea, which ultimately resulted in a sharp increase of the DAI from day3onwards compared with control group (P<0.001), and was maintained during the experimental period. The distal colon showed high macroscopic score (Z=-3.566, P<0.001) and microscopic score (Z=-3.578, P<0.001). Similarly, the MPO activity was significantly higher in TNBS rats than in control rats (t=-11.870, P<0.001).The index of spleen was higher compared with control group rats (t=-7.497, P<0.001), however, there was no difference in index of thymus between the two groups (t=1.009, P=0.330). The levels of TNF-a and IL-1β were higher in TNBS groups rats than that in the control group rats (t=-26.750, P<0.001.t=-25.228, P<0.001).3. HQT and ML ameliorated these parameters effectively compared with TNBS group rats. The administration of HQT or ML effectively reduced the clinical and histopathologic severity of TNBS colitis as well as markedly alleviated the macroscopic score (x2=16.334, P<0.001) and microscopic score (x2=15.205, P<0.001) of colitis. Furthermore, the beneficial effect was associated with a marked inhibition on the expression of MPO and inflammatory cytokines TNF-a as well as IL-1β. The difference of MPO activity (F=76.889, P<0.001), TNF-a (F=213.973, P<0.001) and IL-1β (F=191.073, P<0.001) in three groups was significant. The MPO activity, TNF-a and IL-1β were significantly decreased in the groups of HQT or ML compared with TNBS groups rats (each P<0.001). There was no statistical significance in MPO activity, TNF-a and IL-ip between HQT and ML group (P=0.364, P=0.595. P=0.163).Conclusions1. The results suggest that the instillation of30mg TNBS in50%ethanol led to a substantial wasting disease caused by severe diarrhea. So30mg TNBS is the best dose of enemas in the experiment.2. Our studies demonstrated that treatment with HQT down-regulates inflammatory response and ameliorates acute TNBS-induced colitis represented by weight gain, as well as improvement of clinical, macroscopic, microscopic and immunological parameters of colitis. Chapter II Huangqin-Tang attenuates TNBS-induced colitis in rats by shifting Thl to Th2profileObjectiveThe aim of this study was to investigate the effect of Huangqin-Tang on the balance of Thl/Th2in TNBS-induced colitis in rats.Methods1. Induction of colitis and experimental design:34specific pathogen-free (SPF) rats were randomly assigned to two groups as follows:control group (Cntrl, n=9) and colitis group (n=25). Then30mg of TNBS dissolved in0.25ml of50%ethanol was administered through the catheter to the rats of colitis group. Rats in the control group underwent the same procedure but were administered with physiological saline instead. Two rats were randomly choose from each group and then the colon tissue sections that stained with HE method by light microscope to confirm colitis rats replicated successfully. Then, the colitis group was randomly divided into three groups:TNBS-induced colitis group (TNBS, n=8), Huangqin-Tang group (HQT, n=8) and Mesalazine group (ML, n=8).2. Treatment:Drug administration to animals was started24h after induction of colitis and repeated twice a day for one week. HQT group that received the TNBS challenge and treatment with HQT (11.67%.0.12g/100g·d). ML group that received the TNBS challenge and treatment with ML (1%1ml/100g-d).3. The cytokine production of Thl related parameters interferon-y (IFN-y) and IL-12, Th2marks IL-4and IL-13, IL-12p70in colon of TNBS-induced colitis in rats were examined by ELISA.4. The mRNA lever of Thl related parameters IFN-y and IL-12, Th2marks IL-4and IL-13in colon of TNBS induced colitis in rats were examined by Real-Time PCR.5. The protein expression of Thl master regulator T-box expressed in T cells (T-bet) and GATA family of transcription factors3(GATA-3), a Th2master regulator were examined by Western blot.Statistical AnalysisAll statistical procedures were performed using SPSS software version16.0(SPSS for windows). All data are expressed as mean±S.D. Comparisons between two groups were analyzed with the unpaired Student’s-test. Comparisons between three or more groups were done with a one-way ANOVA. If the variance between groups were homogenous (levene’s test), groups were subjected the multiple comparison least significant differences (LSD) test. In case of no homogeneity variance, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett’s T3test. P<0.05was considered statistically significant.Results1. It is well established that TNBS induced colitis is characterized by a high production of proinflammatory cytokines IFN-y and IL-12compared with the control group (t=-16.597, P<0.001. t=-15.966, P<0.001). There were no significant differences of the suppressor cytokines IL-4and IL-13between TNBS and control group (t=-1.610, P=0.130. t=-2.061, P=0.063). Administration of HQT or ML down-regulated the Thl-related parameters, whereas Th2markers were up-regulated compared with the TNBS group (each P<0.001). The differences of IFN-y, IL-12, IL-4and IL-13in all groups were significant (F=117.449, P<0.001. F=91.052, P<0.001. F=30.377, P<0.001. F=73.923, P<0.001), however, there was no difference between HQT and ML groups (P>0.05).2. The results of Real-Time PCR verified that the significantly increased expression of Thl cytokines IFN-y and IL-12mRNA compared with the control group rats (t=-14.062, P<0.001. t=-23.781, P<0.001). However, there were no significant differences of Th2cytokines IL-4and IL-13between TNBS and control group (t=0.582, P=0.570.t=0.475, P=0.642). HQT-treated and ML-treated groups suppressed the expression of IFN-y and IL-12mRNA efficiently (P<0.001), which was significantly high expression in the TNBS group. The expression of IL-4and IL-13increased after the treatment of HQT and ML compared with TNBS group (P=0.001, P<0.001). However, there was no difference between HQT and ML groups (P>0.05).3. Expression of IL-12p70was increased at protein level in TNBS group compared with control group rats (t=16.546, P<0.001). HQT and ML significantly reduced IL-12p70secretion compared with TNBS group (each P<0.001). However, there was no difference between HQT and ML groups (P>0.05).4. The administration of HQT and ML led to significant reduction of T-bet and increase of GATA-3protein expression compared with TNBS-treated rats (each P<0.001).The difference between HQT and ML group was significant (P=0.001).ConclusionTaken together, these results demonstrated that by shifting Thl to Th2profile in inflamed tissues, HQT can down-regulate inflammatory response of TNBS-induced colitis, thereby contributing to the Thl/Th2balance in IBD. Moreover, for the first time, our findings suggest that the complex immune modulating effects of HQT may result from a down-regulation of proinflammatory capacities of intestinal DC as assessed by analysis of IL-12p70. Chapter Ⅲ Huangqin-Tang attenuates TNBS-induced colitis in rats by modulating the Th17/Treg balanceObjectiveThe aim of this study was to investigate the effect of Huangqin-Tang on the balance of Th17/Treg in TNBS-induced colitis in rats.Methods1. Induction of colitis and experimental design:34specific pathogen-free (SPF) rats were randomly assigned to two groups as follows:control group (Cntrl, n=9) and colitis group (n=25). Then30mg of TNBS dissolved in0.25ml of50%ethanol was administered through the catheter to the rats of colitis group. Rats in the control group underwent the same procedure but were administered with physiological saline instead. Two rats were randomly chosen from each group and then the colon tissue sections that stained with HE method by light microscope to confirm colitis rats replicated successfully. Then, the colitis group was randomly divided into three groups:TNBS-induced colitis group (TNBS, n=8), Huangqin-Tang group (HQT, n=8) and Mesalazine group (ML, n=8).2. Treatment:Drug administration to animals was started24h after induction of colitis and repeated twice a day for one week. HQT group that received the TNBS challenge and treatment with HQT (11.67%.0.12g/100g-d). ML group that received the TNBS challenge and treatment with ML (1%lml/100g-d).3. The cytokine production of Th17related parameters IL-17and IL-6, Treg marks IL-10and TGF-β, IL-23p19in colon of TNBS-induced colitis in rats were examined by ELISA.4. The mRNA lever of Th17related parameters IL-17and IL-6, Treg marks IL-10and TGF-β1in colon of TNBS-induced colitis in rats were examined by Real-Time PCR.5. The protein expression of Th17master regulator orphan nuclear receptor yt (RORyt) and forkhead box P3(Foxp3), a Treg master regulator were examined by Western blot.Statistical AnalysisAll statistical procedures were performed using SPSS software version16.0(SPSS for windows). All data are expressed as mean±S.D. Comparisons between two groups were analyzed with the unpaired Student’s-test. Comparisons between three or more groups were done with a one-way ANOVA. If the variance between groups were homogenous (levene’s test), groups were subjected the multiple comparison least significant differences (LSD) test. In case of no homogeneity variance, differences were evaluated by Welch and the groups were subjected to the multiple comparisons Dunnett’s T3test. P<0.05was considered statistically significant.Results1. It is well established that TNBS induced colitis is characterized by a high production of proinflammatory cytokines IL-17and IL-6compared with the control group (t=-120.374, P<0.001. t=-32.119, P<0.001). There were no significant differences of the suppressor cytokines IL-10and TGF-β between TNBS and control group (t=1.929, P=0.740. t=-1.153, P=0.268). Administration of HQT and ML down-regulated the Th17-related parameters IL-17and IL-6(each P<0.001), whereas Treg markers IL-10and TGF-β were up-regulated compared with the TNBS group (each P<0.001). The expression of IL-17, IL-6and TGF-β in HQT and ML groups were significantly different (each P<0.001), however, the expression of IL-10was not significantly different (P=0.310).2. The results of Real-Time PCR verified that the significantly increased expression of Th17cytokines IL-17and IL-6mRNA in TNBS group compared with the control group rats (t=-14.130, P<0.001. t=-30.937, P<0.001). There were significant differences of the suppressor cytokines IL-10and TGF-β between TNBS and control group (t=-5.741, P<0.001.t=-5.121, P<0.001). The HQT-treated or ML-treated groups suppressed the expression of Thl7cytokines IL-17and IL-6mRNA efficiently (P<0.001), which was significantly high in the TNBS group. Treg cytokines IL-10and TGF-β increased after the treatment with HQT or ML (P<0.001). The lever of IL-17, IL-6, IL-10and TGF-β mRNA between HQT and ML groups were significantly different (P<0.001, P<0.001, P<0.001,P=0.033).3. The expression of IL-23p19increased in at protein level in TNBS compared with control group rats (t=-9.021, P<0.001). HQT and ML significantly reduced IL-23p19secretion compared with TNBS group (each P<0.001). However, there was no difference between HQT and ML groups (P>0.05).4. The administration of HQT or ML led to a significant reduction of RORyt and increase of Foxp3protein expression compared with TNBS-treated rats (each P<0.001). The difference between HQT and ML group was significant (P=0.033, P<0.001).ConclusionTaken together, our data indicate that Huangqin-Tang may modulate the production of corresponding cytokines and transcription factors of Th17and Treg in TNBS-induced colitis, thereby contributing to the Th17/Treg balance in IBD. Furthermore, here we show for the first time that HQT affects DC mediators responsible for a proinflammatory differentiation of T cells in TNBS-induced colitis as assessed by analysis of IL-23p19. |
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