【作者】 刘蓉；

【导师】 唐方；

【作者基本信息】 天津医科大学 ， 中西医结合临床， 2007， 博士

【摘要】 目的胃肠运动功能是消化系统最主要的生理功能之一。正常的胃肠运动不仅传送食糜，而且推动肠道内细菌及其产物的排出，对胃肠道具有非特异免疫防御作用。已知，创伤、手术、放疗、化疗、严重感染等应激状态下，患者经常会出现腹胀、腹痛、恶心、呕吐等一系列胃肠动力障碍表现，早期使用药物恢复胃肠道功能对疾病的恢复和预防并发症是至关重要的。目前，研究发现造成胃肠动力障碍的因素是多方面的，促进胃肠动力的西药效果不佳，中药治疗取得了较好的临床疗效，具有广阔的应用前景。芳香类中药富含挥发油，能疏通气机，宣化湿浊，消胀除痞，醒脾开胃。药理、药效学研究证明，本类药物的挥发油与水溶液两种组分均能不同程度刺激嗅觉、味觉及胃黏膜，增加胃粘膜血流、提高血清胃泌素水平和增强胃粘膜组织抗自由基损伤，保护应激状态下的肠粘膜屏障功能，并兴奋肠管蠕动，使胃肠推进运动加快。提示芳香类中药具有促进胃肠运动的功能。紫苏为芳香运脾法的常用药物，有芳香醒脾，和中开胃的功效。紫苏茎叶含挥发油0．3%-0．7%，主要成分为紫苏醛、柠檬烯、紫苏酮等。紫苏对于芳香运脾，疏通中州，恢复脾胃受纳运化的临床应用效果显著，但缺乏科学的实验研究依据。为进一步探讨芳香醒脾中药调整胃肠运动的作用，揭示芳香药物“运脾”作用机理，我们选用紫苏为代表，采用肢体缺血-再灌注模拟应激导致的结肠动力障碍模型，拟通过结肠动力障碍观察紫苏梗水提液和紫苏叶油对离体结肠平滑肌条收缩振幅、单个结肠平滑肌细胞长度、细胞内钙离子浓度、细胞膜电位、Ca²⁺-ATPase活性以及结肠平滑肌细胞膜流动性等的影响，探讨其促进结肠收缩运动的作用机制。方法1建立肢体缺血-再灌引发的胃肠动力障碍模型，设立不同浓度中药观察组。2制备大鼠离体结肠平滑肌环行肌条和离体单个结肠平滑肌细胞，通过观察大鼠结肠平滑肌条收缩波平均振幅、结肠平滑肌细胞平均长度的变化，探讨紫苏结肠收缩运动的作用和机制。3通过采用激光扫描共聚焦显微镜技术，测定结肠平滑肌细胞内钙离子浓度和细胞膜电位，定磷法测定胞内Ca²⁺-ATPase活性，探讨紫苏对细胞内钙离子浓度的影响，以及钙动员的作用途径，揭示紫苏促进结肠收缩运动的作用机理。4测定结肠平滑肌细胞膜流动性，观察紫苏对细胞膜的保护作用。结果1肢体缺血-再灌注模型成功地模拟了应激状态下的结肠动力障碍。再灌注12h后，大鼠结肠收缩波平均振幅、张力明显下降（P＜0．05，P＜0．001）；并且大鼠结肠平滑肌细胞舒张，平均长度明显增加（P＜0．05），呈现结肠动力障碍。模型大鼠出现结肠平滑肌细胞内[Ca²⁺]_i显著下降，胞内Ca²⁺-ATPase活性增加，细胞膜流动性降低。2紫苏梗水提液和紫苏叶油均能升高模型大鼠结肠平滑肌环行肌条的收缩波平均振幅，促进结肠平滑肌的收缩。钙通道阻断剂盐酸维拉帕米能够抑制紫苏两种成分对结肠环行肌条收缩的增强效应，盐酸普萘洛尔仅抑制紫苏梗水提液的肌条收缩效应。胆碱能M受体阻断剂阿托品对紫苏两种成分的结肠环行肌条收缩增强效应无明显影响。3紫苏梗水提液和紫苏叶油均能减少模型大鼠结肠平滑肌细胞平均长度，增加平滑肌细胞的收缩率。钙鳌合剂EGTA和钙通道阻断剂盐酸维拉帕米对紫苏两种成分促进结肠平滑肌细胞收缩作用均由明显的抑制作用。4紫苏梗水提液和紫苏叶油均可增加模型大鼠结肠平滑肌细胞内[Ca²⁺]_i。IP3受体拮抗剂Heparin和ryanodine受体拮抗剂Ruthenium Red均可抑制紫苏梗水提液增加胞内[Ca²⁺]_i的作用。两种受体拮抗剂均不能抑制紫苏叶油升高胞内[Ca²⁺]_i的作用。5紫苏梗水提液和紫苏叶油均能降低模型大鼠结肠平滑肌细胞膜电位，减少胞内Ca²⁺-ATPase活性（P＜0．05，P=0．001）。6紫苏梗水提液和紫苏叶油均可增加模型大鼠结肠平滑肌细胞膜流动性（P＜0．001）。结论肢体缺血-再灌注模型成功模拟了应激状态下的结肠动力障碍。紫苏梗水提液和紫苏叶油均可通过增加结肠平滑肌条收缩振幅和平滑肌细胞收缩率，升高胞内[Ca²⁺]_i，达到促进结肠收缩运动的作用。紫苏两种成分均可通过增强平滑肌细胞膜的去极化，促进电压依赖型钙通道的开放，抑制Ca²⁺-ATPase活性，从而升高细胞内[Ca²⁺]_i，增强结肠运动功能。紫苏梗水提液促进结肠运动的机理还包括参与或调整肾上腺素能β受体途径，通过IICR和CICR两种胞内钙库释放钙途径，促进细胞内[Ca²⁺]_i的增加，以及参与第二信使IP₃介导的肌醇脂质信号系统对平滑肌收缩作用的调节。紫苏两种成分均能维持平滑肌细胞膜流动性，为平滑肌正常运动功能提供必要的物质基础。更多还原

【Abstract】 Objective:The gastrointestinal motility function is one of the most significant physiologicalfunctions of the digestive system. The normal gastrointestinal motion not onlytransmits chyme, but also promotes excreting the bacteria and its outcome in theintestinal tract. It also has the function of nonspecific immunity defense on thegastrointestinal tract. It is well known that under the stress conditions of trauma,operation, radiation, chemotherapy and severe infection, the symptoms ofgastrointestinal motion dysfunction will occur. The clinical symptoms includeabdominal distention, abdominal pain, nausea and vomiting, and so on. It is highlyimportant that the remedy should be used early for recovering gastrointestinalfunction, in order to come back to health and prevent complication. At present, it isdiscovered in study that there are many factors, which result in dysfunction ofgastrointestinal motility. The Western medicine for promoting gastrointestinalmotility have not better effect on it, while traditional Chinese medicine havesatisfying curative effect. So there are broad foregrounds of application of traditionalChinese medicine.It is full of volatile oil in the fragrant drugs, which can regulate the movement ofqi, eliminate the wetness-evil, remove distention and fullness, and invigorate thespleen and the stomach. The research of pharmacology and the effect of medicineprove that the volatile oil and aqueous components of the fragrant drugs both canexcite olfaction, gustation and gastric mucosa, increase gastric mucosa blood flow,promote the level of gastrin in plasma, strengthen gastric mucosa to resist the injuryof free radical, protect the function of the intestinal barrier in stress. They are alsoeffective to excite peristalsis of the intestinal tract and accelerate propulsion of thestomach and intestine. They suggest that the fragrant drugs have the function ofgastrointestinal movement regulation.Being one of the commonly used drugs in“fangxiang yunpi”therapy, Perillafrutescens have the effect on activating the function of spleen with fragrant scent,adjusting the middle jiao and invigorating the stomach. Both Caulis Perillae andFolium Perillae contain 0.3%-0.7% of volatile oil, whose main active ingredientsincludes perilladyhade, limonene, perillaketone, etc. Perilla frutescens has obviouscurative effect in clinic without sufficient scientific evidence, about restoring thefunction of the spleen, regulating qi movement of the middle jiao, and improving thecontaining, transporting and transforming function of the spleen and stomach. Thepurpose of the study is to explore the Perilla frutescens on adjusting gastrointestinalmotion function and demonstrating the fragrant drugs’“yunpi”mechanism. Lowerlimb ischemic-reperfusion （LIR） model was induced to simulate the dysfunction ofcolonic motility due to stress in wistar rats. To elucidate the promoting coloncontractile movement mechanism of Perilla frutescens, the average contractile amplitude of colonic muscle strips, the contraction response, cytoplasmic freecalcium concentration ([Ca²⁺]_i), cell membrane potential, Ca²⁺-ATPase activity, cellmembrane fluidity of the colonic smooth muscle cells are examined.Methods:1 The gastrointestinal motion dysfunction model is induced in LIR rats.2 The isolated circular muscle strips and single smooth muscle cells of colon areinduced in rats. Through testing the changes of the average contractile amplitude ofcolonic smooth muscle strips and the average length of the colonic smooth musclecells, we explore the action and mechanism of Perilla frutescens in increasing coloniccontractile movement.3After stained with Flou3/AM and DiBAC4_（3）, the calcium concentration and thecell membrane potential in colonic smooth muscle cells were detected with laserscanning confocal microscope. The Ca²⁺-ATPase activity was determined withquantitative determination of phosphorus. It was discussed that the action andapproaches of Perilla frutescens regulating the [Ca²⁺]_i.4 The membrane fluidity of colonic smooth muscle cells were examined, inorder to investigate the protecting cell membrane action of Perilla frutescens.Results:1 The dysfunction of colonic motion under stress was induced triumphantly byLIR model. After LIR rats were induced, the amplitude and tension of colonic musclestrips decreased markedly （P＜0.05, P＜0.001）, and the average length of colonicsmooth muscle cells increased （P＜0.05）. Of LIR model, the calcium concentration ofthe colonic smooth muscle cells decreased, Ca²⁺-ATPase activity was increased, andcell membrane fluidity was reduced.2 Both water solution of Caulis Perillae and volatile oil of Folium Perillaeincreased the amplitude of LIR rats colonic muscle strips. The contraction effectcaused by two components of Perilla frutescens was inhibited by verapamil, not byatropine. But propanolol only inhibited the contraction due to water solutioncomponent.3 Both water solution of Caulis Perillae and volatile oil of Folium Perillaeincreased contraction of LIR rats colonic smooth muscle cells. The contraction causedby two components of Perilla frutescens was inhibited by EGTA or verapamil.4 Both water solution of Caulis Perillae and volatile oil of Folium Perillaeincreased [Ca²⁺]_i of colonic smooth muscle cells in LIR rats. Not only IP（3）R blockerHeparin but also RyR blocker Ruthenium Red reduced the water solutioncomponent-evoked [Ca²⁺]_i increase, but not the volatile oil component-evoked[Ca²⁺]_i increase.5 Both water solution of Caulis Perillae and volatile oil of Folium Perillaedecreased membrane potential and Ca²⁺-ATPase activity of colonic smooth musclecells in LIR rats （P＜0.05,P=0.001）.6 Two components of Perilla frutescens all increased the membrane fluidity ofLIR rats colonic smooth muscle cells markedly （P＜0.001）. Conclusion:LIR model rats can induce the colonic motility disturbance under stress.Through increasing the amplitude of colonic muscle strips, contraction rate ofcolonic smooth muscle cells, and [Ca²⁺]_i, water solution of Caulis Perillae andvolatile oil of Folium Perillae promote colonic contractive motion. Two componentsof Perilla frutescens promote cell membrane depolarization and open the L-typecalcium channel, so as to increase ca²⁺ influx. Ca²⁺-ATPase activity can be inhibitedby the two components too. The adjusting action of water solution of Caulis Perillaeis mediated by adrenalβreceptor. It increases [Ca₂₊]_i in the process ofⅡCR andCICR, and regulates colonic dysfunction through IP₃-Ca²⁺ signal transductionpathway. Two components of Perilla frutescens can maintain the membrane fluidityof colonic smooth muscle cells in LIR rats. The results show that Perilla frutescenshas the regulation effect on colonic contractive motion.更多还原