【作者】 杜群；

【导师】 王建华； 王汝俊；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2006， 博士

【摘要】 免疫因素尤其是细胞因子在IBD的发生发展中起重要作用,对这些细胞因子的研究使UC的发病及治疗研究有了一定的进展,治疗上取得了一定的效果,但尚未能认识UC发病的关键环节。随着对细胞因子、粘附分子的研究,尤其近年对核因子-κB(NF-κB)及Toll样受体(TLRs)的研究,正逐步揭示着IBD的发病机制,TLRs/NF-κB通路的部分阐明也为寻找IBD的治疗提供了新的契机,通过抑制此通路的关键分子进而阻断IBD过激的炎症反应已成为众多学者研究的方向。中医药在UC的治疗上有较好的疗效,很多达到与西药相近的治疗效果,且可弥补西药较大毒副作用及停药后易复发的不足,有着广阔的应用和开发前景。中医药治疗IBD的实验研究尤其是作用机理的研究是目前中医药治疗IBD尤其是UC的重点,在这方面的研究取得进展,将会揭示中药作用的较确切的作用靶点。本研究对治疗UC疗效确切的中药复方溃结灵进行深入的作用机理研究,在前期临床、实验研究基础上更进一步从细胞因子表达的源头TLRs/NF-κB通路入手,观察溃结灵对此通路的一些关键环节TLR2、TLR4表达和NF-κB活化的干预作用,这一研究将使中医药治疗UC的机理研究深入到基因调控水平,并为高质量的抗UC中药新药的开发打下坚实的基础。 1 溃结灵对UC大鼠治疗作用的病理观察 我们研究所在多年临床经验的基础上,以清热健脾活血为治则,总结出治疗溃疡性结肠炎的中药复方溃结灵,临床取得了较好的疗效,无明显毒副作用。为验证其临床疗效,并进一步探讨其作用机理,我们采用三硝基苯磺酸法制作溃疡性结肠炎大鼠模型,并在此模型上观察了溃结灵的治疗效果,溃结灵采用三个剂量给药治疗,阳性组用SASP治疗,并设阴性(模型空白)及正常对照组。治疗结束进行病理观察,大体观察计数溃疡个数、测量溃疡总面积、并计算组织损伤积分,光镜下观察肠粘膜组织炎症、水肿和充血程度(分级法)。结果表明,模型组肠粘膜出现大小不等的溃疡,光镜下显示炎症和水肿程度较重;大体观察溃结灵高、中剂量组溃疡个数、溃疡面积、组织损伤积分均明显少于模型组(P<0.05或P<0.01);光镜下观察溃结灵高、中剂量更多还原

【Abstract】 Above all the immune factors, cytokines played an important role in the morbility and development of inflammatory bowel disease (IBD). The study on morbility and therapy of ulcerative colitis (UC) had been made considerable progress by investigating these cytokines. However, the key component element was unknown. The mechanism of IBD has been revealed step by step with the investigation of cytokines, adherence factor, especially the Nuclear Factor-κB (NF-kB) and Toll-like receptors (TLRs) for the past few years. Furthermore, It was helpful to the treatment of IBD to study the TLRs/NF-κB signal transduction pathway. Now more and more experts has paid close attention to inhibiting the key elements of TLRs/NF-kB pathway to obstruct the irritable inflammation of IBD. Therapeutic effect of traditional Chinese medicine(TCM) on UC was similar to that of Western medicine. But the defect of Western medicine such as recur easily, toxical and side effects were seldom found in using TCM. The prospect of utilization and development of traditional medicine is cheerful. The key point of investigation on treating IBD with TCM was empirical study on mechanism of action. The exact effect target could be revealed with such investigation. In this experiment, the action mechanism of Kuijieling decoction, which showed more affirmative effects on treating UC patients, would be researched deeply. The on . The key elements of TLRs/NF-kB pathway Such as the activation of NF-kB and expression of TLR2, 4 were observed in the experiment go evaluate the interventional effect of Kuijieling decoction. The research on mechanism of TCM on treating UC would be penetrate deeply to gene level ,which would be helpful to develop new TCM. 1 Observation of Kuijieling Decoction in Pathological Changes on UC Model RatsKuijieling decoction was frequently used in PiWei institute to treat UC patients. The therapeutic principle of the decoction was clearing away heat, invigorating the spleen and activating blood .It showed affirmative therapeutic effect on patients without obvious toxical and side effects. To validate the clinical curative effect and investigate the mechanism of action, effect of Kuijieling Decoction on UC model rats induced by TNBSwas observed. All the rats were divided into six groups as follows: normal control (NC) group, model control (MC) group ,Kuijieling low dose (KLD) group, Kuijieling medium dose (KMD) group, Kuijieling high dose (KHD) group and SASP group. The rats were killed to observe pathological changes after 10-days treatment. The number of ulcer were calculated, the area of ulcer were masured and the index of injury were culated with macroscopic observation. The degrees of inflammation, hyperemia and edema in colonic mucosa were graded with microscopic observation. The results showed: There were several ulcers in the colonic mucosa of MC group and the degrees of inflammation and edema were serious. The number of ulcer on the colonic mucosa of KMD group and KHD group were significantly decreased compared to the MC group (P<0.05, P<0.01). The index of injury of KMD group and KHD group were significantly decreased compared to the MC group (P <0.01) . The degrees of inflammation of KMD group and KHD group were significantly relieved compared to the model control group (P<0.05, P<0.01) . The degrees of hyperemia and edema of KHD group were significantly relieved compared to the model control group (P<0.05). The conclusion was: the number and area of ulcer of model rats were significantly reduced with the administration of Kuijieling Decoction. Pathological changes such as inflammatory cell infiltration and edema were improved. It showed affirmative curative effect of kuijieling on treating UC model rats induced by TNBS.2 Effect of Kuijieling Decoction on Proinflammatory Cytokines IL-lp, TNF-a in Serum of UC Model RatsIt was proposed that cytokines especially the proinflammatory cytokines such as the interleukin-ip( IL-ip) and the tumor necrosis factor- a (TNF- a ) played an important role in the morbility and development of UC .To confirm the curative effect and study the treatment mechanism of Kuijieling Decoction, the effect of IL-ip and TNF-a in serum of UC model rats was studied. The grouping and administration were same as before. The rats were killed after 10-days administration to get their serum. The contents of IL-10 and TNF-a in serum were measured with double antibody sandwich ABC-ELISA. The results showed that the contents of TNF-a and IL-ip in serum of the MC group were significantly increased compared to the normal control group (P<0.01) .The contents of TNF-a and IL-ip in serum of KMD group and KHD group were significantly decreased compared to the MC group (P<0.01). The conclusion was: By using Kuijieling Decoction, the contents of TNF-a and IL-ip in serum of UC model rats were reduced.3 Effect of Kuijieling Decoction on Immunosuppressive Cytokines IL-4 in Serum of UC Model RatsOf all the immunologic mechanisms of UC, the unbalance between proinflammatorycytokines and immunosuppressive cytokines was important. To investigate the therapy mechanism of Kuijieling Decoction, the effect of immunosuppressive cytokines—IL-4 in Serum of UC model rats was observed. The grouping and administration were same as before. The content of IL-4 in serum was measured with double antibody sandwich ABC-ELISA. The results showed that the content of IL-4 in serum of the MC group was significantly increased compared to the NC group (P<0.01) .The content of IL-4 in serum of KHD group was markedly decreased compared to the model control group (P< 0.01) .The changes of IL-4 in UC patients or UC rats were under dispute. Even some results were contradictory. By using Kuijieling Decoction, the content of IL-4 in serum was reduced. What kind of role it played in UC should be investigated further.4 Effect of Kuijieling Decoction on Expression of NF-kB P65 in Colonic Mucosa of UC Model RatsAt present the investigation of NF-kB, TLRs/ NF-kB pathway was becoming a focus point of IBD research, which could reveal the nosogenesis of IBD. To investigate the deepgoing therapy mechanism of Kuijieling Decoction on UC, the expression of NFKB/p65 in colonic mucosa was detected with immunohistochemical method. The grouping and administration were same as before. The tissue sections of colon were stained with immunohistochemical staining. The positive rate of p65 expression was analysis with statistics. The results showed that the positive rate of p65 was significantly higher in the MC group than that of NC group (P<0.01). The positive rate of p65 was significantly lower in the KMD and KHD group than that of MC group (P<0.01). The positive rate of NF-kB p65 in colonic mucosa of UC model rats was reduced after the administration of Kuijieling Decoction.5 Effect of Kuijieling Decoction on Gene Expression of TLR2,4 in Colonic Mucosa of UC Model RatsTLRs were important component elements of TLRs/ NF-kB pathway, which could be activated by NF-kB. If the gene expression of TLRs was inhibited, the activation of TLRs/ NF-kB could be inhibited. It was important to inhibite the activation of TLRs/NF-kB in the period of the treatment for UC patients. To investigate the deepgoing treatment mechanism of Kuijieling Decoction on UC, the genes expression of TLR2 and TLR4 were detected in colonic mucosa of UC model rats induced by TNBS. The grouping and administration were same as before. After 10-days treated period, the rats were killed to get their colonic mucosa. Total RNA was isolated from these rats’ colonic mucosa. RT-PCR was used to detect TLR2 and TLR4 expressions and P -actin was used as internal index. The products of PCR were separated with gel electrophoresis and stained .To compare the relativeexpression which was calculated from the gray scale ratio of TLR2, 4 and 3 - acting. The results showed that relative gene expression of TLR2, 4 in MC group were significantly higher than that in NC group (P<0.01). Relative gene expression of TLR2 in KMD and KHD group was significantly lower than that in MC group (P<0.05). Relative gene expression of TLR4 in KHD group was significantly lower than that in MC group (P< 0.05). The gene expression of TLR2, 4.in colonic mucosa of UC model rats induced by TNBS were inhibitted after using Kuijieling Decoction.TLRs/ NF-kB pathway played crucial roles in the occurrence and development of IBD. Since NF-kB could regulate "many key cytokines in the morbility of UC, it maybe participate in inflammation and immunity reaction of UC. Combined with upstream stimulating factors, TLRs transferred activated signals to the intracellular regions and activated transcription factors such as NF-kB, then the productions of inflammatory mediators that could result in injury in mucosa of IBD were regulated. Once provoked by activated NF-kB, the inflammatory reaction was enlarged and couldn’t terminate at once even if the initial etiopathogenisis were removed. NF-kB, TLRs and upstream (downstream) factors such as IL-ip, TNF-a were key components of TLRs/ NF-kB pathway. Proceeding of UC could be prevented efficiently by inhibiting these components. In short, Kuijieling Decoction could be effectually on treating UC model rats with clearing away heat, invigorating the spleen and activating blood therapy. By using Kuijieling Decoction, the contents of TNF-a, IL-ip and IL-4 in serum of model rats were reduced, the positive rate of NF-kB p65 in colonic mucosa of UC model rats was reduced, and the genes expression for TLR2 and TLR4 in colonic mucosa were inhibitted. The results showed many component elements of TLRs/ NF-kB pathway were regulated after using Kuijieling Decoction , which could be its partial mechanism of treating UC.更多还原