【作者】 周红光；

【导师】 徐肇敏； 于成功；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 溃疡性结肠炎（UC）是一种直肠和结肠粘膜的炎症性肠病（IBD）,病因尚不明确,是易感基因、环境和免疫系统等相互作用导致的复杂疾病,免疫系统的参与在UC的病机发展中起重要作用。随着免疫学、分子生物学等学科的迅速发展,以及近年来对细胞因子、粘附分子的研究,UC的发病机制正逐步得以揭示。对于UC的治疗,中药与西药发挥着相似的作用。但是,中药治疗UC极少发生西药治疗所引起的易复发、具有毒副作用等方面的问题。中医药治疗UC在临床发挥着不可轻视的作用。中医药在治疗UC方面具有广阔的研究空间,中医药治疗UC的实验研究尤其是作用机理的研究是目前中医药治疗UC的重点。溃克灵（KKL）是在南京市鼓楼医院消化科多年临床经验基础上总结出的用于治疗UC的有效中药复方。我们认为脾虚是UC发病之本,湿热邪毒为致病之标,气滞血瘀贯穿始终,脾肾阳虚是本病主要转归和病情迁延不愈的重要因素。KKL具有健脾益气、清热祛湿、活血化瘀的功效,临床应用显示较好的疗效,无明显毒副作用。KKL先期实验研究已经证实,中药KKL对TNBS法灌肠复制模型大鼠结肠炎的发生具有一定预防保护作用,能明显减少大鼠结肠溃疡个数和面积,改善结肠粘膜炎症和水肿等病理变化,可能与下调CD4⁺/CD8⁺比值、抑制促炎因子IL-1β分泌和促进抗炎因子IL-10分泌等免疫调节机制有关。为了进一步研究溃克灵治疗溃疡性结肠炎药效及免疫机理,我们选择了溃克灵治疗溃疡性结肠炎药效及免疫机理研究,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,并为开发高质量的抗UC中药新药打下坚实的基础,有重要的理论意义和广阔的应用前景。本论文分上、下两篇对KKL治疗UC进行了理论和实验研究。上篇理论研究:1.中医学对溃疡性结肠炎的研究现状对中医学关于UC的病名的认识、病因病机的认识及治疗现状进行了全面综述。并根据UC的主要临床表现,经严密剖析与推理,提出我们的观点:脾虚湿盛是UC发病的基本病机,贯穿UC的始终。2.白介素在溃疡性结肠炎发病机制中的研究进展白介素（IL）是细胞因子中最主要的具有多种生物活性的一组淋巴因子,并在免疫细胞的发育、分化、免疫应答及某些细胞的激活过程中有重要调节作用。目前研究表明IL主要通过影响机体整体和/或局部免疫系统的功能介导UC的产生,并与UC的迁延难愈和反复发作有关。本文分别对促炎细胞因子和抗炎细胞因子在UC发病机制中的研究进展及中药调控作用做以综述。3.ICAM-1在IBD炎症过程中的表达及作用粘附分子（AM）是一类介导细胞与细胞、细胞与细胞外基质间粘附作用的,具有多种生物功能的膜表面糖蛋白。其中内皮细胞黏附分子-1（ICAM-1）属于免疫球蛋白超家族,在IBD发病中的作用近年来受到关注。本文阐述了ICAM-1的生物学特性、在炎症中的作用、在IBD中的表达及作用、抗细胞粘附分子抗体在IBD治疗中的研究、中药通过干预ICAM-1表达治疗UC实验大鼠的研究等几个方面内容。下篇实验研究:1.研究内容1.1溃克灵治疗溃疡性结肠炎主要药效学研究①分别从溃克灵对TNBS法UC模型大鼠一般表现的影响,体重增长率的影响,结肠病理组织学的影响三方面观察溃克灵对实验性溃疡性结肠炎大鼠的影响;②分别从溃克灵对巴豆油混合致炎剂所致小鼠耳廓肿胀的影响,对腹腔注射醋酸所致小鼠腹腔毛细血管通透性增高的影响,对大鼠塑料环肉芽肿的影响三方面观察溃克灵的抗炎作用;③分别采用热板法测定溃克灵对小鼠痛阈值和对小鼠痛阈提高百分率的影响,醋酸扭体法测定溃克灵对小鼠扭体次数的影响观察溃克灵的镇痛作用。1.2溃克灵治疗溃疡性结肠炎免疫机理研究①分别从2.5二甲苯致耳肿胀,角叉菜胶致足肿胀,大鼠腹腔白细胞游走三方面观察溃克灵对迟发性超敏反应的影响;②分别从溃克灵对TNBS法UC模型大鼠血清IL-8含量、血液流变学、结肠黏膜NO含量、NOS活性、结肠组织ICAM-1基因表达、外周血和肠系膜淋巴结CD₄⁺CD₂₅⁺调节性T细胞数量等几方面观察溃克灵对实验性UC大鼠的免疫调节作用。2.主要研究方法将健康Wisater大白鼠随机分成六组,即正常对照组、模型组、溃克灵大剂量组、溃克灵中剂量组、溃克灵小剂量组、柳氮磺胺吡啶组。采用TNBS/乙醇溶液（100mg/kgTNBS+50%的乙醇0.25ml）灌肠法造模。按人与动物等效剂量,分别对各组大鼠进行中、西药及生理盐水灌胃,每日一次,连续三周。第3周末处死大鼠,采集血液与结肠组织标本,检测相关指标;急性炎症模型、慢性肉芽肿病理模型建立,检测相关指标;热板法和扭体法观察溃克灵镇痛作用。2.结果一般状态:正常组大鼠明显优于模型组大鼠,治疗组用药1周左右能明显改善模型组大鼠的状态。光镜观察:正常组结肠各层结构清晰,无炎性改变。模型组结肠粘膜炎症显著,重者有严重溃疡形成。各治疗组（包括KKL各组、SASP组）炎症程度均有改善（P＜0.01,P＜0.05）。抗炎作用:KKL各剂量组均能抑制巴豆油所致小鼠耳廓肿胀反应,能抑制HAc所引起小鼠腹腔毛细血管通透性的增高,降低大鼠塑料环肉芽肿湿重和干重,有抗肉芽组织形成作用（P＜0.01,P＜0.05）。镇痛作用:KKL各剂量组均能提高热板法大鼠痛阈值,尤其在给药后1h效果最明显,KKL高、中剂量能明显减少醋酸所致小鼠扭体次数,（P＜0.01,P＜0.05）。对迟发型变态反应的影响:KKL各剂量组均对二甲苯致小鼠耳廓肿胀有极显著的抑制作用,对角叉菜胶致足肿胀有极显著的抑制作用,对腹腔注射羧甲基纤维素钠引起的白细胞在腹腔游出、聚集有显著抑制作用（P＜0.01,P＜0.05）。血清IL-8含量:与正常组相比,模型组大鼠血清IL-8水平显著增高（P＜0.01）;与模型组相比,各治疗组血清IL-8水平显著降低（P＜0.01）。结肠黏膜NO含量、NOS活性:与正常组相比,模型组大鼠结肠黏膜NO含量、NOS活性显著增高（P＜0.01）;与模型组相比,各治疗组（包括KKL各组、SASP组）结肠黏膜NO含量、NOS活性显著降低（P均＜0.01）。结肠组织ICAM-1基因表达:与正常组相比,模型组大鼠结肠组织中ICAM-1基因表达显著增高（P＜0.01）,而溃克灵大剂量组、SASP组较模型组明显降低（P＜0.01）。血液流变学变化:与正常组相比,模型组大鼠全血粘度、血浆粘度和红细胞压积均明显增高（P＜0.01）。与模型组相比,溃克灵三个剂量组均能显著降低造模大鼠全血粘度、血浆粘度和红细胞压积（P＜0.01,P＜0.05）。CD₄⁺ CD₂₅⁺调节性T细胞:与正常组相比,模型组外周血和肠系膜淋巴结CD₄⁺ CD₂₅⁺调节性T细胞数量明显下降（P＜0.01）,而溃克灵高、中剂量组、SASP组较模型组明显增高（P＜0.01,P＜0.05）。3.结论采用TNBS/乙醇溶液（100mg/kgTNBS+50%的乙醇0.25ml）灌肠法复制UC大鼠模型成功,完全符合实验要求。以健脾祛湿为主要功用的KKL对实验性UC大鼠治疗效果肯定,不论在一般状态、病理状态、抗炎、镇痛等方面均显示出中医药治疗溃疡性结肠炎具有较大优势,进而说明脾虚湿盛是UC发病的基本病机,其疗效的产生与该方能抑制迟发型变态反应、降低结肠黏膜NO含量、NOS活性、下调炎性细胞因子IL-8、下调细胞间粘附分子ICAM-1的表达、改善造模大鼠血液流变学及提高CD₄⁺ CD₂₅⁺调节性T细胞数量有关,明确了KKL治疗UC的部分作用机制,开拓了中医药治疗UC的新思路。更多还原

【Abstract】 Ulcerative colitis（UC） is an inflammatory bowel disease of the rectal and colonic mucosa.The aetiology of UC remains unknown.UC seems to result from a complex series of interactions between susceptibility genes,the environment and the immune system.An involvement of the immune system plays an important role in the pathogenesis of UC.With the fast development of immunology,molecular biology and investigation of cytokines,adherence factor,the mechanism of UC has been revealed step by step.Therapeutic effect of traditional Chinese medicine（TCM） on UC was similar to than of Western medicine.But the defect of Western medicine such as easy recurrence,toxicity and side effects were seldom found in using TCM. The prospect of utilization and development of TCM is cheerful.The key point of investigation on treating UC with TCM was empirical studied on mechanism of action.Kuikeling decoction（KKL） was summarized for the treatment of UC effective Chinese herbal compound in Gastro enterology of Nanjing Drum Tower Hospital on the basis years of clinical experience.We consider spleen deficiency are the root of UC,and the fire,humid,food stasis are the tip of UC.The therapeutic principle of the decoction was clearing away heat,removing humid,invigorating the spleen and activating blood.It showed affirmative therapeutic effect on patients without obvious toxicity and side effects.In the past investigation,effect of KKL on UC model rats induced by TNBS was observed.It was observed that the number and area of ulcer model rats were significantly reduced with the administration of KKL.Pathological changes such as inflammatory cell infiltration and edema were improved.It showed affirmative curative effect of KKL on treating UC model rats induced by TNBS.In order to further study the treatment of ulcerative colitis KKL efficacy and immune mechanism,we have chosen the ulcer treatment of ulcerative colitis KKL efficacy and mechanism of immunity.This research will enable the Chinese medicine treatment of UC into the mechanism of gene regulation level,and for the development of high-quality anti-UC Chinese medicine and lay a solid foundation, there are important theoretical significance and wide application prospects.In this paper,upper and lower two pairs of ulcer treatment kling UC theoretical and experimental studies.The study in theory1.The status of UC in Traditional Chinese medicine（TCM）It is rounded up thoroughly about the knowledge of name of disease of UC, cause of disease and pathogenesis and the status of treatment in TCM.We put up with our viewpoint that is the weak spleen and stomach,hygrothermal intrinsic is the fundamental pathogenesis of UC and has during UC by the main clinical manifestation.2.Interleukin in the pathogenesis of ulcerative colitis Research ProgressInterleukin（IL） is the most important cytokine with multiple biological activity of a group of lymphokines and immune cells in the development,differentiation, immune response and certain cells in the activation process as an important regulatory role.The current study shows that IL mainly through the impact of the whole organism and/or local immune system mediated by the emergence of UC and UC’s persistent and repeated attack on the refractory.In this paper,respectively, pro-inflammatory cytokines and anti-inflammatory cytokines in the pathogenesis of UC’s research progress and the role of traditional Chinese medicine to control synthesis.3.ICAM-1 in IBD inflammation in the process of expression and the roleAdhesion molecule（AM） is a class of cells and cell-mediated,cells and extracellular matrix in the role of intercellular adhesion,with a variety of biological functions of the membrane surface glycoprotein.Which endothelial cell adhesion molecule -1（ICAM-1） belong to immunoglobulin superfamily,in the IBD pathogenesis of attention in recent years.In this paper,there are Several aspects, ICAM-1 of the biological characteristics,In the role of inflammation,Expression in IBD and the role,Anti-cell adhesion molecule antibody therapy in IBD Research, Chinese medicine through the intervention treatment of ICAM-1 expression in rats UC Research. The study in experiment1.ContentPharmacodynamic study:①Separately from the UC rat model of TNBS in general on the performance of the growth rate of the impact of body weight,colon histopathology observed the effects of three KKLon experimental ulcerative colitis in rats.②To observe anti-inflammatory and analgesic effects of KKL.Anti-inflammatory effect of KKL was investigated by mixed-croton oil-induced aruical swelling in mice and Plastic loop-induced granuloma.Analgesic effect of KKL was investigated by hot plate test and acetic acid writhing test in mice. Mechanism of immune:①Xylene-induced ear swelling and carrageenan-induced paw swelling,rat peritoneal leukocyte migration observed the impact of delayed-type hypersensitivity of KKL.②From serum IL-8 content,hemorheology,colonic mucosal NO content,NOS activity in the colon of ICAM-1 gene expression and the number of mesenteric lymph nodes and peripheral blood CD₄⁺ CD₂₅⁺ regulatory T cells,observed KKL the immune regulation role in experimental rats UC.2.MethodsThe healthy male Wister rats is divided 6 groups randomly,which is individually normal group,model group,3 Kuikeling treatment group included large dose group,medium and little dose group,SASP treatment group.The UC rats model is performed by TNBS/Alcohol Solution（100mg/kgTNBS+50%Alcohol Solution 0.25ml） enema.The all groups are given by pouring into stomach individually Chinese medicine,western medicine and 0.9%NaCl solution according to the equivalent of person to rats.It performs 1 times/day for 3 weeks.After 3 weeks,we collect blood and the tissue of colon of the rats.Then observe relevant target.Constituting acute inflammation model and pathological model of chronic granulomatous,then observe relevant target.Analgesic effect of KKL was investigated by hot plate test and acetic acid writhing test in mice. 3.ResultsGeneral conditions:The rats’ general conditions of normal group and all treatment groups after 1 week’s treatment are better than that of model group.Observation by optical microscope:The structure of colon in normal group is clear and no inflammation.The structure of colon in model group show obvious inflammation and even forms severe ulcer.Treatment groups（including the KKL in each group,SASP group） there was some improvement in the degree of inflammation（P＜0.01,P＜0.05）.Anti-inflammatory effects:KKL can inhibit croton oil-induced ear swelling reaction in mice,inhibit murine peritoneal increased capillary permeability caused by Hac,reduce wet weight and dry weight of plastic ring granuloma in rats（P＜0.01, P＜0.05）.Analgesic effect:KKL can improve hot-plate pain threshold of rats,reduce the number of writhing of mice induced by acetic acid（P＜0.01,P＜0.05）.The impact of the delayed type hypersensitivity:KKL has a significant inhibitory effect for xylene-induced ear edema in mice,Carrageenan-induced paw swelling and intraperitoneal injection of sodium carboxymethyl cellulose in the abdominal cavity caused by travel of leukocyte accumulation（P＜0.01,P＜0.05）.Content of IL-8:Compared with normal group,model group,serum IL-8 level was significantly higher（P＜0.01）;Compared with model group,the treatment groups,serum IL-8 levels decreased significantly（P＜0.01）.Content of NO and NOS:Compared with normal group,model group rats colonic mucosal NO content,NOS activity was significantly higher（P＜0.01）; Compared with model group,the treatment group（including the KKL in each group, SASP group） colonic mucosal NO content,NOS activity significantly lower （P＜0.01）.Content of ICAM-1:Compared with normal group,model group,rat colon tissue ICAM-1 gene expression was significantly higher（P＜0.01）,while the collapse of high-dose group KKL,SASP group than in model group decreased significantly （P＜0.01）. Hemorrheology Change:Compared with normal group,model rats blood viscosity,plasma viscosity and hematocrit were significantly higher（P＜0.01）; Compared with model group,KKL can significantly reduce the rat whole blood viscosity,plasma viscosity and hematocrit（P＜0.01,P＜0.05）.CD₄⁺ CD₂₅⁺regulatory T cells:compared with normal group,the number of mesenteric lymph nodes and peripheral blood CD₄⁺ CD₂₅⁺regulatory T cells of model group significantly decreased（P＜0.01）,while the KKL high,medium dose group, SASP group than in model group increased significantly（P＜0.01,P＜0.05）.3.ConclusionsThe UC of rat model is succeeded by TNBS/alcohol solution（100mg/kgTNBS +50%Alcohol Solution 0.25ml） enema.The KKL,whose main effects is invigorating the spleen and removing humid,has a good therapeutic results to UC, which means weak spleen and stomach,hygrothermal intrinsic is one of fundamental pathogenesis of UC.KKL in the general status,pathological status,anti-inflammatory, analgesic,etc.show the advantages.The therapeutic results of KKL are associated with its effects which inhibit delayed type hypersensitivity,decrease the content of NO、NOS,the content of IL-8 and the expression of ICAM-1,improve the hemorheology and increase the number of CD₄⁺ CD₂₅⁺ regulatory T cells in rat model.So we make clear the partial mechanism of KKL to treat UC and exploit a new thinking of treating UC in TCM.更多还原