养荣润肠舒合剂对慢传输型便秘的治疗作用及机理研究

【作者】 张虹玺；

【导师】 田振国；

【作者基本信息】 辽宁中医药大学 ， 中医外科学， 2009， 博士

【摘要】 便秘是一种临床常见疾病,同时也是多种疾病的一种临床症状,其病因复杂,跨越多个学科。由于饮食结构改变和精神心理社会因素影响,便秘的发病率逐年增高。有调查显示我国慢性便秘的发病率为6.07%,在老年人群甚至可达到15%～20%。长期的慢性便秘不仅给患者带来许多苦恼,而且还在结肠癌、肝性脑病、乳腺疾病、早老性痴呆等病的发病中起着推波助澜的作用。便秘甚至可以因诱发急性心梗或脑血管意外而直接危及患者生命。因此如何有效治疗便秘,减少长期便秘患者的苦恼,降低由便秘引发的恶性事件的发生率成为广大医务工作者的重要任务。在各型便秘中,慢传输型便秘占有近一半的发生率(约45%)。近年来,慢传输型便秘的诊疗有了一定的突破,但较之西医药,中医药在防治慢传输型便秘方面更显示出疗效确切、副作用小等突出优势,而且关于慢传输型便秘的中医药研究取得了可喜的成绩。中药复方养荣润肠舒是导师田振国教授立足于中医中药,博览古今文献,经多年潜心研究,在大量的临床实践基础上总结出的经验方,主要用于治疗慢传输型便秘。对中医辨证属于气血津液亏虚的虚性便秘患者,具有滋阴养血,补气助气,润肠通便之功效,可通过调肝理脾、补肺强肾、通腑润肠来达到以补治秘的目的。临床研究显示,总有效率为92.50%。为了更加科学阐述其疗效机理,本实验在前期临床研究的基础上对药效和作用机理进行深入研究,将观察养荣润肠舒对慢传输型便秘小鼠的通便作用,并同时从分子水平入手,应用蛋白免疫印迹方法和免疫组化的方法研究本复方对这种便秘模型小鼠结肠Cajial间质细胞、结肠肌间神经丛血管活性肠肽(VIP)和P物质(SP)含量的影响,进而为揭示便秘发病机理和治疗新药研发提供实验依据。第一部分药效学研究实验一:慢传输型便秘模型的制备目的:复制小鼠慢传输型便秘的模型材料与方法:将96只健康的普通级昆明种小鼠按性别、体重分层随机分成正常组、模型组、麻子仁丸组、养荣润肠舒低剂量组、养荣润肠舒中剂量组和养荣润肠舒高剂量组6组,每组16只,雌雄各半。除正常组外,其余各组均建立模型。参照文献报道,将造模小鼠按照2.5m/kg的剂量每日一次皮下注射盐酸吗啡,正常组则注射等量等渗生理盐水,连续注射45天,即可造成慢传输型便秘的小鼠动物模型。结果:模型组小鼠大便明显干结,呈圆珠状或串珠状,且排便粒数及重量均明显下降,活动减少,符合便秘的临床表现,故认为造模成功。结论:应用盐酸吗啡按照2.5mg/kg的剂量,给小鼠每天一次皮下注射,连续注射45天,此方法可以成功复制出小鼠慢传输型便秘的模型,且该模型安全性好。实验二:养荣润肠舒对小鼠排便功能的影响目的:通过观察养荣润肠舒对小鼠排便功能的影响,判断养荣润肠舒治疗慢传输型便秘的疗效。材料与方法:造模成功后各组小鼠日一次灌胃给与不同药液。其中正常组和模型组每日按0.4 ml/10g体重灌蒸馏水:麻仁软胶囊组用蒸馏水将麻仁软胶囊配制成1.5%的混悬液,按0.4ml/10g体重日一次灌胃给药,生药量为0.6g/kg;养荣润肠舒低剂量组将养荣润肠舒合剂稀释一倍,按0.4 ml/10g体重日一次灌胃给药,生药量为11.4g/kg;养荣润肠舒中剂量组将养荣润肠舒合剂原液按0.4 ml/10g体重日一次灌胃给药,生药量为22.8g/kg;养荣润肠舒高剂量组将养荣润肠舒合剂浓缩一倍,按0.4 ml/10g体重日一次灌胃给药,生药量为45.6g/kg。各组均连续灌胃给药10天。在给药期间,所有小鼠均自由摄食与饮水,每2天称重一次,并根据体重变化调整给药剂量。第九天给药前禁食不禁水12h,给药后将滤纸铺于饲养笼中进行观察,记录给药24h内各组小鼠排便的总粒数及粪便重量。结果:模型组小鼠大便明显干结,24小时排便粒数及重量均明显下降,与正常组差异显著(P＜0.01)。灌胃治疗后,各治疗组小鼠的大便均有不同程度的恢复,尤其是养荣润肠舒高剂量组,在排便数量及重量上均明显高于模型组(P＜0.01),且大便外观上与正常组已无差异。结论:1盐酸吗啡皮下注射复制出小鼠慢传输型便秘的模型稳定性好。2吗啡造模和养荣润肠舒合剂治疗均对慢传输型便秘小鼠的体重无明显影响。3养荣润肠舒可以增加慢传输型便秘小鼠的24小时排便粒数和排便重量,并呈现出显著的量效关系。实验三:养荣润肠舒对小鼠小肠墨汁推进率的影响目的:通过观察养荣润肠舒对小鼠小肠墨汁推进率的影响,判断养荣润肠舒治疗慢传输型便秘的疗效。材料与方法:各组小鼠在第10天给药前禁食不禁水12h,灌胃给药,所有药液中均含有2%的炭末。30min后脱颈椎处死小鼠,立即打开腹腔分离肠系膜,并分离小肠与大肠。剪取上端自幽门,下端至回盲部的小肠管,至于托盘上,轻轻将小肠拉成直线,测量肠管长度为“小肠总长度”,从幽门至墨汁前沿为“墨汁推进长度”,计算炭末在肠管中的推进率。结果:正常组小肠的墨汁推进率是78.20%,模型组下降至65.08%,两者有显著性差异(P＜0.05),而治疗后各组的墨汁推进率均明显增加,高剂量组达到93.12%,不仅明显高于模型组,且高于正常对照组(P＜0.01)。结论:养荣润肠舒可以显著提高慢传输型便秘小鼠的小肠推进率,进而达到增强胃肠动力、治疗慢传输型便秘的作用。第二部分作用机制研究实验一:养荣润肠舒对ICC、SP及VIP的分布和表达的影响目的:应用免疫组化的方法探讨养荣润肠舒治疗慢传输型便秘的作用机制。材料与方法:实验小鼠末次给药后2小时,用25%乌拉坦按0.4 ml/100g的剂量腹腔麻醉,灌流固定后迅速打开腹腔,分离大肠,切取小鼠的近端结肠段组织约15mm,放入预先备好的装有固定液的小瓶中二次固定,保存备用。采用免疫组化的方法进行染色处理,先在光镜下观察组织结构的变化及结肠Cajial间质细胞、结肠肌间神经丛血管活性肠肽(VIP)和P物质(SP)的分布和含量的变化。免疫组化结果判定方法:细胞浆出现褐色片状或颗粒状物为c-Kit阳性,出现黄色或棕黄色物质为SP、VIP阳性。结果:1.正常组小鼠结肠的ICC细胞分布于整个结肠肌层,且以环肌层和肌间丛区域分布最丰富。ICC细胞呈梭形、卵圆形或不规则形,并以胞体为中心向四周发出两个或两个以上的突起。细胞核大而明显,胞质相对较少,细胞膜较完整。与正常组比,模型组ICC阳性细胞数量明显减少,尤以环肌层和粘膜下环肌层表面区域减少地最为明显,部分模型此区域ICC几乎消失。残存的ICC其形态也出现异常,如细胞膜部分溶解,突起变短、变钝等。治疗后ICC细胞数增加,ICC细胞分布密度也明显增大,病理改变得到改善。所有切片肌层中均可见SP和VIP阳性表达的细胞,只不过与正常组比,模型组SP、VIP阳性表达的细胞较少,分布稀疏,而治疗后细胞分布相对较密集。阳性细胞多呈圆形或椭圆形,胞浆可见染成黄色或棕黄色的颗粒。2.模型组近端结肠组织c-Kit、SP、VIP阳性细胞均较正常对照组显著减少,而治疗后各治疗组的c-Kit、SP、VIP阳性细胞均较模型组增多,尤其是养荣高剂量组增加显著(P＜0.01)。结论:养荣润肠舒合剂是通过调节动物模型神经递质(SP、VIP)的含量及ICC的数量和功能来调节结肠蠕动,进而达到治疗慢传输型便秘的作用的。实验二养荣润肠舒对c-Kit蛋白含量的影响目的:应用蛋白免疫印迹法检测小鼠结肠c-Kit蛋白表达的变化,探讨养荣润肠舒治疗慢传输型便秘的作用机制。材料与方法:实验小鼠末次给药后2小时,脱颈处死,立即打开腹腔,分离大小肠,选取小鼠近端结肠组织约2cm,生理盐水冲洗后,置于液氮中冷冻,-70℃保存备用。采用western-blot的技术方法检测小鼠结肠c-Kit蛋白表达的变化情况,并应用ChemiImager 5500凝胶成像分析软件(America)分析,记录每条蛋白电泳带的灰度值,进行定量分析。结果:c-Kit蛋白在模型组中的表达较正常组明显降低(P＜0.01),治疗后c-Kit蛋白的表达显著升高,约为模型组的2.95倍(P＜0.01),但仍明显低于正常组,两者差异显著(P＜0.05)。结论:养荣润肠舒合剂能通过调节c-Kit蛋白表达来增加ICC的数量和功能,进而增强结肠蠕动,达到治疗慢传输型便秘的作用。更多还原

【Abstract】 Constipation is a common clinical symptom.Changes in dietary structures and effects of psychosocial factors have been attributed as the cause of increased rates of constipation.It has been reported that the morbidity of chronic constipation is 6.07%in China and it has reaches 15-20%in aged population.Chronic constipation is a major predisposing factor for a variety diseases,such as colon cancer,hepatic encephalopathy,breast diseases and Alzheimers Disease.Furthermore,it can induce some life threatening diseases such as acute myocardial infarction and stroke.In order to develop effective approaches in constipation treatment,relief patients from the constipation and to reduce the incidence of severe diseases induced by constipation,it is important to make research into constipation an area of great importance.Slow transit constipation accounts for nearly half(approximately 45%) of cases in all kinds of constipation.Compared with western medicine,the advantages of traditional Chinese medicine in the prevention and treatment of slow transit constipation have been highlighted in recent years.More efficient effects and less adverse effects have been show in traditional Chinese medicine in treating slow transit constipation.YangRongRunChangShu(YRRCS) is a combined Chinese medicine which has been invented by Professor Tian Zhengguo.Based on large amount of clinical practice,and ancient and modern literatures,Prof.Tian has summarised YRRCS in treating slow transit constipation.For the constipation patients in Qi,Blood and Body Fluids Deficiency Type according to traditional Chinese differentiation,YRRCS can tonify qi and replenish blood and moisten the intestines.It can improve the function of colon by soothing the liver and regulating the spleen,tonifing the lung and reinforcing kidney,moistening the intestines and relax the bowels.The previous clinical study has shown the total effective rate of YRRCS is 92.5%.To provide the scientific evidence for the development of new Chinese medicine,the pharmacodynamics of YRRCS and its related mechanism have been investigated in this study.This study was divided into two parts.In the first part,effects of YRRCS on passing stools were examined in Experiment 1,2 and 3.In the second part,the mechanism of YRRCS on slow transit constipation treatment was investigated by using western blot method and immunohistochemistry method to examine the colon interstitial cells Cajal(ICC) and vasoactive intestinal peptide(VIP) and substance P(SP) in colon myenteric plexus of slow transit constipation mice model.Section one:The Study of Effects of YRRCSExperiment 1:Duplicate model of slow transit constipationObjective:Duplicate mouse model of slow transit constipationMethod:We chose 96 healthy mice and randomly divided them into six groups,i.e.normal group、model group、Mazirenwan group、slight-dose YRRCS group、middle-dose YRRCS group and high-dose YRRCS group.We made the model of slow transit constipation on all groups but the normal one.According to literature reports,we applied with hypo-dermic injection of hydrochloric acid morphine 2.5mg/kg/d for 45 days.Then the mouse model of constipation was established.Result:The mouse stools of model group turned so sticking and stagnant that it is difficult to fall from anus.That is to say symptoms of mouse of model group is very similar with the patients of constipation.Experiment 2:The influence of YRRCS on defecation function of mouseObjective:To observe the influence of YRRCS on defecation function of mouse,we can judge the efficient effects of YRRCS on slow transit constipation.Method:After establishing the model of slow transit constipation,we began to give the different medicines to the mice of different groups.Normal group and model group were intragastic infused with normal saline.Mazirenwan group was intragastic infused with Mazirenwan.Slight-dose YRRCS group、middle-dose YRRCS group and high-dose YRRCS group were intragastic infused with different dose of YRRCS which contented the medicine of 11.4 g/kg,22.8g/kg and 45.6g/kg.The period of administration was 9 days.Before the ninth day,the mice had been forbided to drink and eat for 12 hours.Then,the mice were intragastic infused as usual.After that,we began to record the quantity and weight of stools in 24 hours.Result:The mouse stools of model group had turned obvious dry and hard.At the same time,the quantity and weight of stools declined evidently,which had significant different with the normal group(P＜0.01).After treating with medicines,all mice stools had recoverd in different level,especially the high-dose group,whose stools had no different with normal group in outward appearance.Conclusions:YRRCS can increase the quanlity and weight of mouse stools of slow transit constipation in 24 hours.The efficient effects depend on the dose of YRRCS.Experiment 3:The influence of YRRCS on the length of ink progradation of the small intestine of mouseObjective:To observe the influence of YRRCS on the length of ink progradation of the small intestine of mouse,we can judge the efficient effects of YRRCS on slow transit constipation.Method:Before the tenth day,the mice of each group had been forbided to drink and eat for 12 hours.Then,all mice were intragastic infused with the medicines which contained the ink. After 30 minites,the mice were killed.Then,we opened the abdomen,cut the small intestine, measured the length of the ink and small intestine.At last,we caculated the rate of ink moving forward.Result:The rate of ink moving forward of small intestine of normal group is 78.20%,while the model group decrease into 65.08%.There are significant different between them(P< 0.05).After treating,the rate of ink moving forward of small intestine of all groups increased especially the high-dose one,who had got to 93.12%.It is not only higher than model group, but also higher than normal group.Conclusions:YRRCS can strengthen the force of stomack and intestine and treat the slow transit constipation.Section two:The Study of Mechanism of YRRCSExperiment 1:The Effects of YRRCS on ICC,SP and VIPObjective:To investigate the mechanism of YRRCS on slow transit constipation treatment by using immunohistochemistry method.Method:After intragastric administration for 2 hours,the mice were anesthetized and excised colon specimen。Then,we used immunohistochemistry method to examine the change of amount and form of ICC,VIP and SP in colon myenteric plexus of slow transit constipation mice model. Result:The amount of ICC,VIP and SP decreased obviously in colon of mouse model with chronic constipation.After treatment by YRRCS,the amount increased more than control group.There are significant different between control group and high-dose group(P＜0.01).Conclusions:YRRCS can strengthen colon peristalsis by increasing the amount of ICC,VIP and SP.So,it can cure slow transit constipation.Experiment 2:The Effects of YRRCS on c-KitObjective:To investigate the mechanism of YRRCS on slow transit constipation treatment by using western blot method.Method:After intragastric administration for 2 hours,the mice were killed and excised colon specimen。Then,we used western blot method to examine the change of amount of c-Kit in colon myenteric plexus of slow transit constipation mice model.Result:Comparing with normal group,the amount of c-Kit in model group decreased obviously(P＜0.01).After treatment by YRRCS,the amount increased 2.95 times than model group,but still lower than normal one(P＜0.05).Conclusions:YRRCS can strengthen colon peristalsis by increasing the amount of c-Kit.So, it can cure slow transit constipation.更多还原