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养血活血方治疗血虚型便秘临床研究及对便秘模型小鼠结肠ICC的影响
【作者】 李国峰；
【导师】 李国栋；
【作者基本信息】 中国中医科学院 ， 中医外科学， 2008， 博士
【摘要】 便秘的危害是渐进的,不易引起医生及患者重视,但长期顽固性便秘对人体的危害是不容忽视的。人们普遍的惯性思维认为便秘仅仅是大便干硬难出,事实上便秘与许多疾病的发生发展密切相关。随着饮食结构的改变及精神心理和社会因素的影响,便秘已严重影响了现代人的生活质量;且在结肠癌、肝性脑病、乳腺疾病、早老性痴呆等疾病的发生中有重要作用;在急性心肌梗死、脑血管意外等症时便秘可导致生命意外;便秘和部分肛肠疾病有密切的关系,如痔、肛裂等。因此,早期预防和合理治疗便秘将会大大减轻便秘带来的严重后果和社会负担。随着现代生活水平的提高,人们对生活质量的期望越来越高,对于既往常被忽视的一些功能性疾病更加重视。生活方式的改变、饮食结构的变化使得便秘发病率逐渐升高,尤以老年及经产女性居多。流行病学资料显示,从美国的几个全国性调查数据中得出美国便秘的发病率为2%,即大约有4百万美国人患有便秘,每年有2—3百万人应用泻剂辅助排便,且便秘患病率随年龄的增大而增加。我国的流行病学资料显示各地区发病率有所差异,但都高于2%水平。治疗便秘的药物较多,但大多难收持久疗效,且长期应用易导致药物依赖及副作用发生机率增加。祖国医学对于便秘的治疗积累了丰富的经验,认为慢性功能性便秘有虚实之分,临床发现顽固性便秘以虚秘为主,虚秘中以血虚便秘居多,尤以老年及多产女性更为明显。血虚便秘多病程较长,临床症状顽固,治疗效果欠佳。导师李国栋教授经多年实践,参详古今,认为血虚便秘治疗疗效不佳的原因是血瘀病理常常被忽视,中医有“久病多瘀”、“久病入络”之说,慢性血虚便秘因病程迁延,导致肠壁络脉瘀阻,肠道微循环障碍,影响肠壁营养,出现肠道动力紊乱。洞察医理,导师治疗慢性血虚便秘在养血润肠的基础上投以活血化瘀之品,创养血活血方。顽固性便秘之所以疗效不尽如人意,因为现代医学对便秘的病因、发病机制尚未完全清楚。研究者们在不断进行着有益的探索,认为功能性便秘患者存在结肠动力紊乱,结肠平滑肌病变,结肠神经丛及神经递质异常,结肠粘膜水通道蛋白表达异常。尽管如此,仍不能完全阐释便秘的发病机制。随着对ICC研究的深入,其在胃肠动力障碍性疾病中作用越来越受到重视。便秘是临床常见疾病,也是多种疾病常见的症状之一,可分为原发性便秘和继发性便秘。本研究的内容即指原发性功能性便秘。原发性顽固性功能性便秘治疗困难,需长期反复用药,长期反复应用泻剂,可加重肠壁损害,导致便秘症状更加顽固。促动力药物因可导致恶性心血管事件,亦为临床少用。李国栋教授总结多年治疗顽固性便秘的经验创养血活血方治疗血虚型顽固性便秘,临床疗效满意。李师勤求古训,汇通中西,并结合自已多年临床实践,对便秘一症有独到认识。养血活血方是李教授多年临床经验的结晶。为进一步验证养血活血方治疗血虚便秘的临床疗效,探讨治疗机理并结合当前国内外研究进展特设计本课题。本课题主要从以下2个方面进行研究:第一部分临床研究目的:验证养血活血方治疗血虚型便秘的临床疗效。方法:临床研究共收集入选病例80例,随机分成2组,治疗组50例,对照组30例。分别给与养血活血方、麻仁软胶囊口服。疗程2周,观察治疗前后症状积分变化。结果:经临床观察进一步证实养血活血方治疗血虚型便秘,疗效确切,效果优于对照药物麻仁软胶囊。2组药物治疗便秘均有一定效果,但养血活血方治疗血虚型便秘其痊愈与显效率明显高于对照组,养血活血方治疗后患者排便情况积分及中医证候积分下降程度均优于对照组,且随访发现服用养血活血方患者,一般感觉良好,无大便溏泻、腹部不适等副作用。第二部分实验研究实验一:血虚型功能性便秘小鼠模型的制备目的:复制血虚型功能性便秘小鼠模型方法:综合国内外文献报道,结合前期实验研究,采用小鼠皮下注射盐酸吗啡的方法复制便秘模型小鼠,并结合小鼠断尾放血法制造血虚模型。皮下注射吗啡2.5mg/kg/d,共45天,对照组给予等体积等渗生理盐水。结果:模型组小鼠表现精神萎靡,倦卧少动嗜睡,毛耸无华,眼睛淡白无神,鼻唇淡白失泽;活动减少等一系列符合中医血虚证候的表现,且每日平均粪便量明显减少,便出粪球粘滞,便后不易脱落,部分排出粪块变大,基本符合便秘特点。实验二:养血活血方对血虚型便秘模型小鼠结肠cajal间质细胞的影响目的:探讨养血活血方治疗血虚型功能性便秘的机理方法:造模成功小鼠按照既定分组,分别给予灌胃养血活血方大、中、小剂量浓缩水煎剂,麻仁软胶囊,莫沙比利。空白组、模型组给予等体积生理盐水灌胃。共计2周。末次给药后,处死动物,打开腹腔,分离大肠,取距离肛门约1cm远端结肠肠段2段,于0.9%生理盐水中冲洗干净,分别置于福尔马林液中固定及液氮罐中冷冻保存待用。采用免疫组化技术及激光共聚焦显微镜扫描观察各组小鼠结肠Cajal间质细胞(Interstitial cells of Cajal ICC)的变化。结果:慢性功能性便秘模型小鼠存在结肠ICC的异常,便秘模型小鼠结肠ICC数量明显减少,并有形态异常,与空白组小鼠比较ICC连接网络欠完整。养血活血方灌胃14天后小鼠结肠ICC数量有明显升高。西药莫沙必利对便秘模型小鼠结肠ICC有一定影响,但未见统计学差异,麻仁软胶囊能够提高ICC数量,但与养血活血方大、中剂量比较存在明显差异。养血活血方中剂量能够明显增加便秘模型小鼠结肠ICC的数量,且对ICC数量的影响存在量效关系,大剂量养血活血方对ICC数量的改善更加明显,几乎接近空白组小鼠结肠ICC的数量。实验三:养血活血方对慢性便秘模型小鼠结肠c-kit蛋白表达的影响目的:从分子水平探讨养血活血方治疗血虚型功能性便秘的机理方法:取液氮冷冻保存的便秘模型小鼠结肠组织,利用western-blot技术检测养血活血方干预后的便秘模型小鼠结肠组织c-kit蛋白表达的变化。结果:实验结果表明便秘模型小鼠结肠组织中c-kit蛋白表达明显降低。养血活血方对c-kit蛋白表达有明显调节作用,且表现明显的量效关系,与实验二一致趋势。经组间比较,其对c-kit蛋白表达的影响养血活血方大剂量组优于养血活血方中、小剂量组,养血活血方中剂量组优于养血活血方小剂量组。
【Abstract】 Doctors and patients often ignore the disease of chronic constipation because its harm is gradual.Even that we shouldn’t disregard of the jeopardise caused by chronic constipation.Many of patients think that the constipation is only difficult to defecate,in fact chronic constipation can result in so many kinds of diseases.With the change of drink and food,effect on mind and society,chronic constipation severely impact on quanlity of life in modern society.Chronic constipation is tightly relative to colon carcinoma,hepatocerebral disease(HCD),breast disease,presenile dementia and so on,Chronic constipation can result in emergency of life if someone got sick of acute myocardial infarction or cerebral accident.Some of patients with chronic constipation are prone to hemorrhoids and anal fissure.Accordingly,earlier prevention and rational therapy will greatly cut down the severe consequence and society burden resulted by constipation.With the elevation of life level,people hope to live more comfortable life and think highly of treatment on functional disease.Incidence of constipation gradually goes up,especially among the aged people and multiparous women because of the transform of life style and the change of diet composition.Data of epidemiology means that incidence of chronic constipation is 2%in USA and increases by age.The data told us that there are 4 millions people to get sick of chronic constipation every year and 2--3 millions patients with chronic constipation to defecate by luxative.Data of epidemiology in China is more than 2%,even that is different from city to city.There are so many kinds of drugs to treat on chronic constipation,but its therapeutic effect is difficult to last for long time and long-term application of medicine will result in drug addiction and side effect.Experts of Traditional Chinese medicine(TCM) accumulate a lot of experiences in treatment on chronic constipation and devided it into 2 types of deficiency and excess through differentiation of symptoms and signs.Most of patients with refractory constipation are the blood deficiency,especially among older or multiparous women patients through differentiation of symptoms and signs.Professor Li who is a famous expert for diseases of colon and rectum discovered that main reason of blood deficiency type refractory constipation is stagnation of blood.Professor Li conclude that stagnation of blood aggravates its deficiency.Professor Li researched and created herbs of yang xue huo xue fang(YXHXF) based on his experience in treatment on refractory constipation.Clinical observation found that herbs of YXHXF is effective for chronic constipation of blood-deficiency type.In order to study the mechanism of herbs of YXHXF,we designed the research program which include 2 sections.Section one:clinical trialObjective:to observe therapeutic effect of herbs of YXHXF for chronic constipation Method:80 patients with chronic constipation of blood-deficiency type were divided randomizedly 2 groups.There are 50 patients to take medicine of herbs yang xue huo xue fang(YXHXF) in observation group,Control group of 30 patients take medicine of maren soft capsule.2 weeks for course of treatment,we recorded the change of symptom scores.Result:Through clinical trial,we concluded that effect of YXHXF to treat on chronic constipation of blood-deficiency type is precise and surpass control group.Section two:empirical studyExperiment 1:Duplicate mouse model with chronic constipation of blood-deficiency typeObjective:make mouse model with chronic constipation of blood-deficiency type Method:Combination with literature reports and experience of experiment,we apply with hypo-dermic injection of hydrochloric acid morphine 2.5mg/kg/d for 45 days to make mouse model of constipation.While hypo-dermic injection of hydrochloric acid morphine,we cut off tails of mouse to loss blood for 7 times(one time for every 6 days) to make model of deficiency of blood.Result:successful mouse model of blood-deficiency type constipation showed that tool-weight decreased obviously.Mouse stool turned so sticking and stagnant that it’s difficult to fall from anal.In brief,symptoms of mouse model of constipation is consistent with clinical patients with constipation.Experiment 2:the influence of YXHXF on interstitial cells of Cajal(ICC) of mouse model with chronic constipationObjective:to study the mechanism of treatment on chronic constipation of blood-deficiency type by YXHXF.Method:mouse model took medicine of YXHXF,maren soft capsule and mosapride by intragastric administration for 2 weeks and then executed mouse to excise colon specimen.We applied with immunohistochemistry technology to analyze the amount of ICC.Result:The amount of ICC decreased obviously in colon of mouse model with chronic constipation.After treatment by YXHXF,the amount increased more than control group.Experiment 3:the influence of YXHXF on c-kit of colon tissue for mouse model with chronic constipationObjective:to study the mechanism of YXHXF treatment on chronic constipation of blood-deficiency type in molecular levelMethod:we analyzed the diversity of c-kit in mouse colon tissue with chronic constipation by western-blot technology.Result:The density of c-kit decreased obviously in colon of mouse model with chronic constipation.YXHXF can increased the amount of c-kit and the effect is precise,a contrast to control group.