【作者】 李叶；

【导师】 罗云坚；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 研究目的:本研究实验部分以溃疡性结肠炎大鼠为研究对象,观察肠涤清合锡类散保留灌肠对溃疡性结肠炎的治疗效果和量效关系,及其对C反应蛋白(C reaction protein,CRP)、细胞因子表达的影响,探讨其作用机理。临床部分观察肠涤清灌肠组、肠涤清合锡类散灌肠组治疗溃疡性结肠炎的临床疗效,比较两组间的有效率,以便寻找一种疗效好、副作用少、价格便宜、使用方便的治疗方法。研究方法与内容:采取随机、对照研究。实验部分取清洁级SD大鼠80只,雄雌各半,体重200-220g。将大鼠编号,随机取出空白组10只(第1组),其余均予复合法造模(2,4,6-三硝基苯磺酸+乙醇)。造模后麻醉意外死亡14只,剩余56只随机分为4组:模型组(第2组),肠涤清合锡类散低剂量灌肠组(第3组),肠涤清合锡类散高剂量灌肠组(第4组),对照组灌肠组(第5组),每组14只。空白组常规饲料和蒸馏水喂养。造模后第三天,各组开始灌肠给药,每日一次,连续2周。空白组及模型组均给予生理盐水1.0ml/100g灌肠;肠涤清合锡类散低剂量灌肠组予肠涤清0.5ml/100g+锡类散0.05g/d灌肠;肠涤清合锡类散高剂量灌肠组予肠涤清1.0ml/100g+锡类散0.1g/d灌肠;对照组予生理盐水1ml/100g+柳氮磺吡啶片(SASP)0.1g/d灌肠。各组大鼠在实验结束后称体质量后全部处死,取全段结肠。取病变最明显处结肠段0.5cm,予病理学分级。取结肠标本,采用双抗体夹心ABC-ELISA法检测IL-4水平。并采血约8ml注入干燥管,分离血清-20摄氏度保存,测CRP、IL-6水平。所有标本均同批检测。临床部分,选择符合溃疡性结肠炎西医诊断标准轻、中度且属活动期以及中医辨证大肠湿热证患者,年龄在18～75岁。纳入病例共65例,随机分为治疗组(肠涤清合锡类散灌肠组)31例,其中男17例,女14例,平均年龄47.35±12.73岁。对照组(肠涤清灌肠组)34例,其中男18例,女16例,平均年龄50.45±17.20岁。两组均无并发症和肠外症状,两组患者性别、年龄均无显著性差异(p＞0.05)。治疗组予肠涤清150ml+锡类散1g,保留灌肠,每天一次;对照组予肠涤清液150ml,保留灌肠,每天一次。1个月为1个疗程。观察项目有一般项目、安全性观测项目、疗效性观察项目。最后评定临床综合疗效,包括完全缓解、有效和无效。计算有效率,为完全缓解和有效例数与单组治疗总人数的百分比,比较两组间有无差异性。研究结果:造模后大鼠均精神不振,食欲下降,排稀烂便夹少量鲜血,予药物灌肠治疗过程中,上述症状得到了不同程度的改善。相关结果如下:1.肠涤清合锡类散对UC大鼠肠粘膜病变的影响。光镜下,模型组镜下可见溃疡,及大量炎性细胞浸润,以中性粒细胞为主,病理损害分级为Ⅳ、Ⅴ级;肠涤清合锡类散低剂量组和对照组少见溃疡,但见大量炎性细胞浸润,病理损害分级以Ⅱ、Ⅲ级为主;肠涤清合锡类散高剂量组炎性细胞浸润明显减少,病理损害分级以Ⅰ、Ⅲ级为主。各组与模型组比较,病理损害分级有差异性(P＜0.05),而空白组、肠涤清合锡类散低剂量组、肠涤清合锡类散高剂量组和对照组之间病理损害分级无差异性(P＞0.05)。2.肠涤清合锡类散对UC大鼠血清CRP的影响。与空白组比较,模型组CRP含量升高,差异有非常显著性意义(P＜0.01);与模型组比较,肠涤清合锡类散低、高剂量组和对照组CRP含量降低,差异有非常显著性意义(P＜0.01);与肠涤清合锡类散低剂量组、对照组比较,肠涤清合锡类散高剂量组CRP含量降低,差异有显著性意义(P＜0.05);与对照组比较,肠涤清合锡类散低剂量组CRP含量则无差异性(P＞0.05)。3.肠涤清和锡类散对UC大鼠血清IL-6的影响。与空白组比较,模型组IL-6含量升高,差异具有非常显著性意义(P＜0.01);与模型组比较,肠涤清和锡类散低、高剂量灌肠组和对照组血清中IL-6含量降低,差异具有非常显著性意义(P＜0.01)。与肠涤清和锡类散低剂量灌肠组、对照组比较,肠涤清和锡类散高剂量灌肠组IL-6含量无显著差异(P＞0.05)。4.肠涤清合锡类散对UC大鼠结肠组织IL-4的影响。与空白组比较,模型组结肠组织中IL-4含量降低,差异有显著性意义(P＜0.01);与模型组比较,肠涤清和锡类散低、高剂量灌肠组和对照组结肠组织中IL-4含量升高,差异有显著性(P＜0.05);与肠涤清和锡类散低剂量灌肠组、对照组比较,肠涤清和锡类散高剂量灌肠组IL-4含量升高,差异有显著性意义(P＜0.05);与对照组比较,肠涤清和锡类散低剂量灌肠组IL-4含量差异无显著性意义(P＞0.05)。临床试验结束后,两组均无严重的不良反应,故病人顺应性可,两组均无脱落病例。将临床症状消失情况和结肠镜复查结果作为疗效判定标准。经治疗后,两组临床疗效结果为:治疗组治疗31例,完全缓解11例,有效17例,无效3例,总有效率为90.32%。对照组治疗34例,完全缓解6例,有效18例,无效10例,总有效率为70.59%。两组差异显著(P=0.0470,P＜0.05),说明治疗组对溃疡性结肠炎临床疗效明显优于对照组。结论:本研究采用复合法(2,4,6-三硝基苯磺酸+乙醇)复制UC动物模型,模型组病变组织镜下可见溃疡,及大量炎性细胞浸润,以中性粒细胞为主,病理损害分级为Ⅳ、Ⅴ级;且血清CRP和IL-6的含量高于正常组,IL-4的含量低于正常组(P＜0.01),表明复合法所致UC大鼠的发生与CRP和IL-6的高表达有关。其余各组均给予中药肠涤清、不同剂量的肠涤清合锡类散联合灌肠、柳氮磺吡啶灌肠,观察它们的药理作用,发现它们均具有一定的抗溃疡作用,可以改善UC动物模型的症状,减轻结肠溃疡及炎性病变。其中尤以肠涤清合锡类散高剂量灌肠组疗效最好,能明显改善UC大鼠的病理分级、减轻血清中炎症因子CRP和IL-6的含量;并能明显提高血清中抗炎症因子IL-4的含量,这些可能是肠涤清合锡类散发挥其药理作用的机制之一。临床上,溃疡性结肠炎病变始于直肠,直肠、乙状结肠患病率占70%。而且,我国溃疡性结肠炎病程均表现为反复发作,病情相对较轻,轻中度多见,病变范围以左半结肠为主。据此,中药灌肠治疗溃疡性结肠炎有相当多的优点。在前期工作的基础上,我们拟用肠涤清合锡类散联合灌肠,与单独肠涤清灌肠组比较。结果显示,两组均无严重的不良反应,病人顺应性可。肠涤清合锡类散联合灌肠组具有更好的临床疗效,且因为副作用少,顺应性强,价格便宜,易被我国广大溃疡性结肠炎患者所接受,解决他们的疾苦。其远期疗效和确切机制尚有待进一步研究。更多还原

【Abstract】 Purpose:This research took rats that suffered from ulcerative colitis as investigative objects to observe the therapeutic efficacy and dose-effect relationship of ulcerative colitis by using Changdiqing and Tin-like powder to do the retention enema and influences of the two kinds of drugs to C-reaction protein(CRP) and expression of cytokines as well as the mechanism of action.As to the clinical part,we observed the therapeutic effects of coloclysis group using Changdiqing and the other group using combined Changdiqing and Tin-like powder in treating of ulcerative colitis and intended to find a therapeutic approach with benefits such as satisfactory curative effect, minor side effect,reasonable price as well as convenient direction.Method and contents:Adopted randomized control study.Experimental part:Took 80 clearing grade SD rats with equal sexual number and the weight of which between 200 to 220 grams.Numbered the rats and randomized took 10 of which out as blank group(the first group),others were built models according to combination method(2,4,6-trinitro-benzene-Sulfonic acid + alcohol).There were 14 rats died due to anesthesia accident post modeling and the rest 56 ones were randomized divided into 4 groups as follows: model group(the second group),coloclysis group with low dose of Changdiqing and Tin-like powder(the third group),coloclysis group with high dose of Changdiqing and Tin-like powder(the forth group) and control coloclysis group (the fifth group).Each group had 14 rats.Used routine forage and distilled water to feed the blank group.The other ones were begun to administrate drugs through coloclysis three days post modeling with frequency of one time per day and duration of a continuous two weeks.The blank group and modeling groups were adopting normal saline(NS) 1.0ml/100g to do the coloclysis.Coloclysis group with low dose of Changdiqing and Tin-like powder added Changdiqing 0.5ml/100g and Xileisan 0.05g/d additionally to do the colocysis.Coloclysis group with high dose of Changdiqing and Tin-like powder added Changdiqing 1.0ml/100g and Tin-like powder 0.1g/d additionally to do the colocysis.The control group chose normal saline 1ml/100g + Sulfasalazine(SASP) 0.1g/d to do the coloclysis.All the rats in each group were put to death after weighed when the research ended and got the whole colons and took 0.5cm with manifest pathological changes to do pathological classification.Meanwhile,did double antibody sandwich ABC-ELISA to detect the IL-4 level of the colon specimen. To measure CRP and IL-6 level to the separated serum from 8ml blood under storage of minus 20 degree centigrade as well.All the specimens were detected in the same batch.Clinical part:To select patients with light and midrange active stage ulcerative colitis in west medicine diagnosis and damp-heat in large intestine in traditional Chinese medicine(TCM) diagnosis as investigation objects with ages ranging from 18 to 75 years old.All the included 65 cases were randomized divided into treating group(Changdiqing combined Tin-like powder coloclysis group),which had 31cases and control group(Changdiqing coloclysis group), which had 34 cases.The former group had 17 male cases and 14 female ones, with an average age of 47.35±12.73 years old:meanwhile,the later had 18 male cases and 16 female ones,with an average age of 50.45±17.20 years old. All the cases in the two groups had no complications and symptoms beyond bowels. There were no significant differences on sex and age of the patients in the two groups(p＞0.05).To adopt 150ml Changdiqing + 1g Tin-like powder to do retention enema to the treating group with frequency of one time per day, meanwhile,to adopt 150ml Changdiqing to do retention enema to the control group with frequency of one time per day and to take one month duration as a course of treatment to the two groups.The observation items were including general data,safety surveying data and curative effect surveying data.After that,to evaluate the clinical comprehensive curative effects which could be divided as complete remission,utility and invalid.The effective powers were calculated from the percentages of the cases achieved complete remission and utility divided by the case number of each group and finally to compare whether the effective powers in the two groups had variability. Results:All the rats were suffered from symptoms and signs such as low spirit, poor appetite,diarrhea accompanied with minor blood post modeling and all the symptoms and signs were recovered in different degree during the drug coloclysis treatment.The associated results were elaborated as follows:1.Influence of Changdiqing and Tin-like powder to the intestinal mucosa pathological changes of rats with ulcerative colitis.The model group could see ulcers and plenty of inflammatory cell infiltrate with neutrophil dominance under light microscope,with a pathological lesion classification asⅣ,Ⅴdegree.Whereas,coloclysis group with low dose of Changdiqing and Tin-like powder and control group seldom could find ulcers but plenty of inflammatory cell infiltrate,with a pathological lesion classification asⅡ,Ⅲdegree and coloclysis group with high dose of Changdiqing and Tin-like powder had significantly decreased inflammatory cell infiltrate,with a pathological lesion classification asⅠ,Ⅱdegree.Each groups had statistical significance in pathological lesion classification compared to model group (P＜0.05).However,there was no statistical significance in pathological lesion classification among blank group,coloclysis group with low dose of Changdiqing and Tin-like powder,coloclysis group with high dose of Changdiqing and Tin-like powder and control group(P＞0.05).2.Influence to serum CRP(C-reaction protein) of Changdiqing and Tin-like powder to rats with ulcerative colitis.Compared to blank group,the content of CRP in model groups increased significantly with high statistical significance(P＜0.01).Comparison between model groups,the content of CRP in coloclysis group with low dose of Changdiqing and Tin-like powder, coloclysis group with high dose of Changdiqing and Tin-like powder and control group decreased significantly with high statistical significance(P＜0.01). Compared to group with low dose of Changdiqing and Tin-like powder and control group,the content of CRP in coloclysis group with high dose of Changdiqing and Tin-like powder decreased significantly with statistical significance(P＜0.05).Compared to control group,the difference of content of CRP in coloclysis group with low dose of Changdiqing and Tin-like powder had no statistical significance(P＞0.05).3.Influence to serum IL-6 of Changdiqing and Tin-like powder to rats with ulcerative colitis.Compared to blank group,the content of IL-6 in model groups increased significantly with high statistical significance(P＜0.01). Comparison between model groups,the content of IL-6 in coloclysis group with low dose of Changdiqing and Tin-like powder,coloclysis group with high dose of Changdiqing and Tin-like powder and control group decreased significantly with high statistical significance(P＜0.01) Content of IL-6 had no statistical significance among coloclysis group with low dose of Changdiqing and Tin-like powder,control group and coloclysis group with high dose of Changdiqing and Tin-like powder(P＞0.05).4.Influence to serum IL-4 of Changdiqing and Tin-like powder to rats with ulcerative colitis.Compared to blank group,the content of IL-4 in model groups decreased significantly with high statistical significance(P＜0.01). Comparison between model groups,the content of IL-4 in coloclysis group with low dose of Changdiqing and Tin-like powder,coloclysis group with high dose of Changdiqing and Tin-like powder and control group increased significantly with statistical significance(P＜0.05).Compared to group with low dose of Changdiqing and Tin-like powder and control group,the content of IL-4 in coloclysis group with high dose of Changdiqing and Tin-like powder increased significantly with statistical significance(P＜0.05).Compared to control group,the difference of content of IL-4 in coloclysis group with low dose of Changdiqing and Tin-like powder had no statistical significance(P＞0.05).There was no severe adverse effect detected in the two groups after the ending of clinical trail,which had suggested the patients compliance was satisfactory and there was no amotic case as well.We took the clinic symptom recover and colonoscopy finding on rechecking as curative effect assessment standards and the results of clinical therapeutic effect of the two groups post treatment were as follows:among the 31 cases in the treating group, 11 cases complete remission,17 utility and 3 invalid,with an in total effective rate of 90.32%:among the 34 cases in the control group,6 cases complete remission,18 utility and 10 invalid,with an in total effective rate of 70.59%.The two sets had statistical significance(P=0.0470,P＜0.05) which had suggested that clinical curative effect in the treating group was superior than the control group in ulcerative colitis.Conclusions:In this research,we adopted combination method(2,4,6-trinitro-benzene-sulfonic acid + alcohol) to cope the ulcerative colitis animal model,and could find ulcers and plenty of inflammatory cell infiltrate with neutrophil dominance under light microscope,with a pathological lesion classification asⅣ,Ⅴdegree.Meanwhile,the content of CRP and IL-6 were more than common ones while IL-4 lower than common ones(P＜0.01),which had suggested the occurrence of ulcerative colitis built through combination method might have relationship with high expression of CRP and IL-6.In another groups,we adopted Changdiqing(a kind of traditional Chinese medicine),Changdiqing and Tin-like powder with different dosage to do coloclysis and Sulfasalazine to do coloclysis and to investigate their pharmacologic action.We found all the drugs had a certain anti-ulcer effect which could release symptoms of ulcerative colitis in the animal models and soften colonic ulcer and inflammatory pathological changes.Among the treating groups,coloclysis group with high dose of Changdiqing and Tin-like powder had best curative effect by significantly improving the pathogenic classification of the rats with ulcerative colitis,decreasing the serum content of CRF and IL-6 and increasing serum content of anti-inflammatory factor IL-4,which might be one of the pharmacologic mechanism of Changdiqing and Tin-like powder.In clinic practice,ulcerative colitis were mainly started in rectum and the prevalence of rectum and sigmoid colon reached as many as 70%.Meanwhile, course of disease of ulcerative colitis in our country were mainly appeared to be recurrent attacks with comparatively gently pathogenetic conditions of minor and midrange and the extent of diseases were mainly located in left hemicolon.For the reasons above,using traditional Chinese medicine to do retention enema had considerable advantages.On the basis of prophase work, we chose Changdiqing and Tin-like powder to do the coloclysis and compared the effect to group just using Changdiqing only.Results had suggested both the two treating method had no severe adverse effect and the patient had satisfactory compliance to this way as well.However,group using Changdiqing and Tin-like powder to do the coloclysis had better clinical curative effect had had other benefits such as minor side effects,strong compliance, reasonable price,which could be easily widely accepted by patients suffered from ulcerative colitis and solve their sufferings.The prostecdtive efficacy and precisely treating mechanism still needed to be investigated.更多还原