加味乌梅丸防治溃疡性结肠炎的免疫调控机制研究

【作者】 薛新丽；

【导师】 韩涛；

【作者基本信息】 北京中医药大学 ， 临床中药学， 2008， 硕士

【摘要】 溃疡性结肠炎(Ulcerative Colitis,UC)是炎症性肠病(inflammatory boweldisease,IBD)的一种,以结肠溃疡形成为病理特征。近年来,在我国发病率逐年增高。本病确切的发病机制至今仍不清楚,认为是由多种因素相互作用所致,主要包括免疫、遗传、感染、及精神心理因素;其发病既有自身免疫机制的参与,也有遗传因素作为背景,感染和精神因素只是诱发因素。治疗方面,现代医学常用氨基水杨酸类和糖皮质激素类药物,但存在易复发和一定程度的不良反应。中医将溃疡性结肠炎归属于“泄泻”、“痢疾”等范畴,中医认为溃疡性结肠炎患者病位在肠,多以脾胃虚弱为本,湿热毒邪为标,瘀血痰浊阻滞肠络的病理变化贯穿疾病始终,病程日久,累及脾肾,则可见脾肾虚弱。古代经典名方—乌梅丸主治病机与本证相符,临床多年用于治疗溃疡性结肠炎,疗效好、复发率低、不良反应少,具有良好的研究价值。加味乌梅丸是导师韩涛副教授依据临床经验,结合现代研究的科研成果,组成的科研处方。该中药复方经过多年临床验证,具有较好疗效,与单纯乌梅丸相比,紧扣病机,在功效上有较大的突破。本论文复制溃疡性结肠炎大鼠模型,并对模型进行评价;观察加味乌梅丸对溃疡性结肠炎大鼠保护作用;在以往研究的基础上,选取与溃疡性结肠炎发病机理密切相关的血清IFN-γ、结肠组织iNOS、P-选择素、ICAM-1作为指标,运用ELISA、免疫组化等检测技术,从整体、组织、细胞、分子水平,深入研究加味乌梅丸对溃疡性结肠炎大鼠的免疫作用机理。主要研究内容如下:1文献综述上篇通过查阅、整理国内外溃疡性结肠炎的相关研究资料,从中医、西医两方面对溃疡性结肠炎进行系统的综述。2研究内容2.1处方研究加味乌梅丸由《伤寒论》厥阴病篇中的乌梅丸化裁而成。根据中医理论对该处方进行分析,将其功效定位为涩肠止泻,健脾益气,调和寒热,活血止痛。该方可用于脾虚日久,寒热错杂,瘀血阻滞引起的腹痛,腹泻,里急后重,黏液脓血便。同时,通过研究处方中诸药的药理学进展,进一步全面分析该处方。2.2加味乌梅丸对TNBS型溃疡性结肠炎大鼠治疗作用的研究本研究复制TNBS/乙醇UC大鼠模型。随机分为正常组,模型组,柳氮磺胺吡啶组(SASP),加味乌梅丸低、中、高剂量组。从疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)、结肠黏膜镜下病理形态学等方面,对加味乌梅丸的疗效进行评价。结果发现,模型组DAI与正常组之间差异非常显著(P＜0.01);模型组和治疗组之间有显著性差异,SASP和加味乌梅丸均可使DAI降低(P＜0.05),尤以加味乌梅丸中剂量更明显(P＜0.01)。模型组的CMDI与正常组相比,差异非常显著(P＜0.01);模型组和治疗组之间有显著性差异(P＜0.01),其中SASP和加味乌梅丸可使CMDI明显降低(P＜0.01)。以上结果表明,本实验的溃疡性结肠炎模型复制成功,加味乌梅丸对TNBS/乙醇所致的溃疡性结肠炎大鼠模型有良好的防治作用。2.3加味乌梅丸对TNBS型溃疡性结肠炎大鼠的免疫机制的研究2.3.1加味乌梅丸对溃疡性结肠炎大鼠模型结肠组织iNOS的影响模型组iNOS阳性率明显增高,与正常组比较有显著差异(P＜0.01);加味乌梅丸各组和SASP组iNOS阳性率下降,与模型组比较,有显著差异(P＜0.05),以加味乌梅丸中剂量组下降最为明显(P＜0.01)。2.3.2加味乌梅丸对溃疡性结肠炎大鼠模型结肠组织P-选择素的影响模型组P-选择素阳性率明显增高,与正常组比较有显著差异(P＜0.01);加味乌梅丸各组和SASP组P-选择素阳性率下降,与模型组比较有显著差异(P＜0.05),尤以加味乌梅丸中剂量组下降最为明显(P＜0.01)。2.3.3加味乌梅丸对溃疡性结肠炎大鼠模型结肠组织ICAM-1的影响模型组ICAM-1阳性率明显增高,与正常组比较有显著差异(P＜0.01);加味乌梅丸各组和SASP组ICAM-1阳性率下降,与模型组比较有显著差异(P＜0.05),尤以加味乌梅丸中剂量组下降最为明显(P＜0.01)。2.3.4加味乌梅丸对溃疡性结肠炎大鼠模型血清IFN-γ的影响模型组IFN-γ阳性率明显增高,与正常组比较有显著差异(P＜0.01);加味乌梅丸高、中、低剂量组血清IFN-γ的水平下降,与模型组比较有显著差异(P＜0.05),尤以加味乌梅丸中剂量组下降最为明显(P＜0.01)。2.3.5机理分析实验结果提示,加味乌梅丸具有明显的抗溃疡性结肠炎的效果;分析其机理,可能是通过下调iNOS的表达阻止机体免疫防御系统的启动,使T、B、NK细胞等淋巴细胞的分化减弱,下调促炎细胞因子IFN-γ的表达,从而使中性粒细胞、NK细胞和血管内皮细胞等炎性细胞的活化和浸润减少,血小板活化减少。那么在结肠组织中,P-选择素不能介导白细胞的复始黏附,不能介导ICAM-1黏附、趋化、淋巴细胞归巢等作用,参与炎症和免疫反应。最终,导致肠黏膜的损伤减少,起到治疗溃疡性结肠炎的作用。通过本实验,也证实免疫因素在溃疡性结肠炎发病中的重要作用。上述免疫分子可能是加味乌梅丸防治溃疡性结肠炎的作用靶点。3结论脾虚为本,湿热为标是溃疡性结肠炎的主要病机;以涩肠止泻,益气健脾,调和寒热,活血止痛立法的加味乌梅丸能有效的控制炎症的启动。通过本实验,我们得出结论,加味乌梅丸对TNBS型溃疡性结肠炎大鼠有着良好的保护作用,其治疗效果尤其在改善机体免疫系统方面,显示出中医药在提高机体的免疫防御功能方面具有的巨大优势。它通过益气健脾提高机体的免疫功能,调和寒热阻止炎症的发生,避免发生免疫系统级联反应,最终达到治疗溃疡性结肠炎的目的。我们相信随着对溃疡性结肠炎发病机制的深入研究,加味乌梅丸对溃疡性结肠炎的治疗作用机理将会得到更全面,更深刻的阐释。更多还原

【Abstract】 The ulcerative colitis is one of the inflammatory bowel disease,takes ulcer as pathology characteristic.In recent years,its incidence advanced year by year in our country. In spite of this sickness accurate pathogenesis is not clear until now,we thought that is interacted by many kinds of factor,mainly including immunity,heredity,infection,and stress disorder factor;Its morbidity already has own immunity mechanism participation,also has the hereditary factors to take the background.Obviously,infection and energetic factor only induces the factor.In treatment aspect,aminosalicylic acids and sugar cortical hormone medicine commonly were used in clinic,but exists easy to recur and untoward effect in the certain extent.UC is relegated in "the diarrhea","dysentery" and so on in Chinese medicine,it revealed that the UC patient shortcoming is in the intestines,many take the weakness of spleen and stomach as the base,but wet hot is the sign,the stasis bloody sputum hinders the pathological change.These changes penetration disease which muddy the intestines wind,and it will implicate the spleen kidney at last,then the obvious spleen kidney is weak.Ancient classical prescription-WMW mainly treated the pathogenesis with this card match case.It used to treat UC in the clinical many years,and the curative effect are good.the recurrence rate low,the untoward effect were few,so the good research value is obvious.According to the theory of TCM,Jiawei Wumei Wan(JWW)which bases of clinical experience,and unites modern research achievements in scientific research,composes scientific research prescription.We duplicates UC rat model,and carries on the appraisal to the model;observate JWW to UC rat protective function;select indexs which closely related UC pathogenesis:blood serum IFN-γ,the colon organizes iNOS,P-selection,ICAM-1,to take the target.Then we utilizes ELISA and Immunohistochemistry to deeply research JWW to immunity action mechanism of UC rat from the whole,the organization,the cell,the molecular level.The main research content is as follows:1 Literature reviewWe had reviewed literatures about the general situation of UC from TCM and western medicine in domestic and abroad.2 Research2.1 Prescription researchJWW stems from "Treatise on Febrile diseases" jueyin disease.The demonstration of JWW that guided by TCM theories analysis:antidarrhea with astringent,invigorate the spleen and profit pneuma,reconcile chills and fever,promote blood circulation to arrest pain.This will use in spleen weakness in the course of time,the cold-heat jumble,and ecchymosis will hinder the abdominal pain which will cause the diarrhea,the tenesmus,the mucus pus bloody stool.At the same time,through researching pharmacology progress in prescription,further overall is analysised this prescription.2.2 The research on treat UC/TNBS rat through JWWThis research duplicates TNBS/alcohol UC rat model.Divided into the normal group,the model group,sulfasalazine enteric-coated tablets(SASP)group,JWW high-dose, middle-dose,low-dose group stochastically.From disease activity index(DAI),colon mucous membrane damage index(CMDI),the colon mucous membrane the pathology morphology on light microscope aspect,we appraise JWW curative effect.The result discovered that DAI between model group and normal group the difference is remarkable(P＜0.01);Between the model group and the treated group has the significance difference,SASP and JWW both possibly cause DAI to redUCe(P＜0.05).Especially,JWW middle-dose group is more dramatic(P＜0.01).Compared with normal group,model group’s CMDI on light microscope elevates obviously(P＜0.01);Between the model group and the treated group,it has the significance difference(P＜0.01),SASP and JWW cause CMDI to reduce obviously (P＜0.01).The above result indicated that this experiment’s UC model duplicates successfully.JWW to TNBS/alcohol UC rat has obvious prevention function.2.3The research of immunologic mechanism about JWW for TNBS/UC rat2.3.1 The influence about JWW for UC rat colon tissue iNOSThe masc rate of colon tissue iNOS was clearly raised up in the model group,compared with the normal group(P＜0.01).While in the three-dose JWW groups and SASP group,the masc rate of colon tissue iNOS was dramaticly descended,compared with the model group(P＜0.05),especially the middle-dose JWW group(P＜0.01).2.3.2 The influence about JWW for UC rat colon tissue P-selectinThe masc rate of colon tissue P-selectin was clearly raised up in the model group,compared with the normal 1 group(P＜0.01).While in the three-dose JWW groups and SASP group,masc rate of the colon tissue P-selectin was dramaticly descended,compared with the model group(P＜0.05),especially the middle-dose JWW group(P＜0.01).2.3.3The influence about JWW for UC rat colon tissue ICAM-1The masc rate of colon tissue ICAM-1 was clearly raised up in the model group,compared with the normal group(P＜0.01).While in the three-dose JWW groups and SASP group,the masc rate of colon tissue ICAM-1 was dramaticly descended,compared with the model group(P＜0.05),especially the middle-dose JWW group(P＜0.01).2.3.4 The influence about JWW for UC rat serum IFN-γThe masc rate of serum IFN-γwas clearly raised up in the model group,compared with the normal group(P＜0.01).While the high,middle and low-dose JWW group,the level of serum IFN-γ,was dramaticly descended,compared with the model group(P＜0.05).2.3.5 Mechanism analysisThe experimental result indicates,JWW has obvious anti-UC effect;analyzing its mechanism:possibly first declining iNOS expression to prevent organism immune defense system,cause lymphocyte and T,B,NK cell differentiations to weaken,decline IFN-γexpression,thus causes the polymorphonuclear neutrophil,NK cell and the blood vessel endothelial cell’s activation and infiltration to reduce,blood platelet activation is reduced. Therefore,the P-selectin cannot mediate the white blood cell to adheres and accumulation; cannot mediate the ICAM-1 to adhere,hasten,and the lymphocyte to turn over nesting,participate inflammation and immune response.Finally,the intestines mucous membrane damage is reduced and ulcerous colitis is treated.Through the experiment, immunity factor was confirmed to influential role once more in the UC morbidity.The target which JWW could treat UC possibly relates with the above immune molecule.3 ConclusionUC main pathogenesis is based of spleen weakness and signed of hot and damp; Throught antidarrhea with astringent,invigorate the spleen and profit pneuma,reconcile chills and fever,promoting blood circulation to arrest pain,JWW can control inflammation starting effectly.According to this experimental study,we conclude that JWW could pretect TNBS/UC rat.Treatment is better in improvement organism immune system.It demonstrated TCM have huge superiority in enhancing organism immune defense function aspect, moreover,not poisonous side effect comparing with western medicine.JWW is good for the spleen through nourishing qi,enhances organism immunologic function;well distributed cold and heat could prevent inflammation’s occurrence and avoids having immune system cascade respond,thus achieves to treat UC.更多还原