【作者】 谭莹；

【导师】 吴清和；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 中医认为,“肾主开阖”,具有主持和调节人体津液代谢的功能,尿的排泄,虽在膀胱,但要依赖肾阳的气化。若肾气充足,则能温煦膀胱,使之固摄有权、开阖有度,维持水液的正常代谢;若肾气不固,则致封藏失职,膀胱失约,出现多尿、遗尿、甚则小便失禁。机体的衰老状态被认为属于生理性肾虚的范畴,即老年人肾元亏虚,遇夜间阴盛阳衰,膀胱摄纳无权,易出现的夜尿、遗尿、尿急等症状。缩泉丸出自《校注妇人良方》,由乌药、益智仁、山药组成,具有温肾祛寒,缩尿止遗的功效,主治膀胱虚寒证,小便频数、遗尿不止等,现代临床被常用于夜尿、遗尿、尿频、尿急、尿失禁等老年下尿路功能症状等的治疗,其显著的临床疗效和治疗优势得到临床医师及病患的首肯。为了进一步阐明中医“温肾缩尿”理论,本课题组开展了一系列探索缩泉丸”缩尿”功效及关键作用靶点的研究,为中医经典理论的现代研究和缩泉丸的深层次研发奠定基础。夜尿、尿频、遗尿、尿失禁等下尿路功能症状（LTUS）在老年人群中的发生率日趋增高,成为继心脑血管系统疾病后的又一大严重影响老年人身心健康和生活质量的重要因素。针对老年LTUS虽有包括行为矫正、神经调节、外科手术、针炙等疗法,但效果不甚理想,存在副作用大,复发率高,患者依从性较差等问题。近年来,关于老年LTUS的发病机制研究取得了较大进展,已证实引发老年LTUS的因素除神经、血管系统病变和膀胱流出道梗阻等尿道因素外,老龄本身对膀胱功能改变的影响也是重要因素之一。衰老的膀胱逼尿肌发生肌源性和神经源性的改变,导致逼尿肌舒缩功能障碍,出现储尿及排尿功能异常是引发老年LTUS的主要原因。鉴于缩泉丸通过温补肾阳、温化肾气,达到“温肾缩尿”功效的治疗特色与现代医学所提及的通过调整膀胱逼尿肌功能状态失常治疗老年LTUS具有内在相关性,本研究以此为切入点,以中医传统古方缩泉丸为研究对象,并在成功复制衰老“多尿”动物模型并初步探讨其“多尿”机制的基础上,围绕缩泉丸干预调节尿液生成的相关因素和膀胱逼尿肌舒缩功能状态,对老年LTUS发病链上与尿液生成、排泄相关的各指标参数进行分析测定,并从对膀胱逼尿肌胶原蛋白、神经受体相关基因、蛋白表达调控的影响入手,从分子机制及“多尿”发生发展的重要环节探索缩泉丸“缩尿”作用机制和关键作用靶点,为进一步阐明中医“温肾缩尿”经典理论的现代医学内涵奠定基础。衰老“多尿”动物模型的研究结果表明:D-gal致亚急性衰老动物出现24h总尿量增加及水负荷后6h排尿潜伏期缩短、排尿次数增多,但总尿量没有明显改变的“多尿”症状。其多尿“机理”包括:（1）神经内分泌系统功能失调导致抗利尿激素（ADH）、醛固酮（ALD）合成与分泌减少,心钠素（ANP）分泌增加,尿液中水通道蛋白-2（AQP-2）浓度降低;肾脏病理形态学改变,包括肾单位数量减少,肾小管萎缩,近曲小管玻璃样变性等可能是模型动物24h总尿量增加的主要原因。（2）逼尿肌功能、组织酶学及病理形态学异常可能是模型动物排尿次数增多的主要原因。在组织酶学方面:逼尿肌丙二醛（MDA）含量增加,超氧化物歧化酶（SOD）、ATP酶（ATPase）、线粒体琥珀酸脱氢酶（SDH）活性降低;在功能改变方面:逼尿肌兴奋性增强,膀胱顺应性、弹性降低;对ATP敏感性增强的同时对ISO敏感性降低;在病理形态学方面:膀胱肌层肌纤维排列紊乱,血管壁纤维化,胶原纤维占肌层比例增加等。缩泉丸“缩尿”功效的研究结果表明:缩泉丸可减少亚急性衰老动物24h总尿量及水负荷后6h内排尿次数,延长排尿潜伏期。缩泉丸在减少动物尿液Na⁺、Cl^-排出量的同时增加K⁺排出量,说明药物具有保钠排钾的作用。缩泉丸可能通过恢复神经内分泌系统功能增加血中ALD、ADH,尿中AQP-2浓度及减少ANP的分泌及改善模型动物肾小球萎缩、硬化,肾小球基底膜增厚,入球血管硬化及肾小管萎缩等达到减少24h尿量的作用。缩泉丸可改善逼尿肌的衰老状态,使MDA含量降低,SOD活性增强,药物可能通过降低逼尿肌兴奋性及对ATP敏感性;增强Na⁺-k⁺-ATPase、Ca²⁺-Mg²⁺-ATPase,线粒体SDH活性,膀胱顺应性、弹性,对ISO敏感性及减轻逼尿肌肌层胶原纤维的增生,改善肌层肌纤维排列紊乱、增粗及肌纤维断裂等发挥延长水负荷后排尿潜伏期、减少排尿次数的作用。缩泉丸对逼尿肌胶原蛋白及神经受体的影响研究结果表明:缩泉丸可以从肌源性和神经源性两个角度调节逼尿肌胶原蛋白及神经受体的表达,恢复逼尿肌舒缩功能。从肌源性角度出发,缩泉丸可使衰老“多尿”大鼠膀胱逼尿肌collagenⅠ、collagenⅢmRNA及蛋白的表达显著降低,使动物由于衰老而异常沉积的胶原蛋白减少,增加了膀胱的顺应性及弹性,减轻衰老逼尿肌纤维化趋势;从神经源性角度,缩泉丸在降低介导逼尿肌收缩的P2X₂受体mRNA及蛋白的同时显著提高介导其舒张的β₃-AR mRNA及蛋白的表达,使两者保持动态平衡,恢复逼尿肌的正常舒缩功能。亚急性衰老“多尿”动物模型的成功复制为本课题的研究提供了可靠的动物模型。缩泉丸通过影响尿液生成和排泄的多个环节发挥“缩尿”功效。药物可使老化逼尿肌舒缩功能恢复正常,这种作用与上调逼尿肌β₃-AR mRNA及蛋白的表达和下调collagenⅠ、collagenⅢ、P2X₂受体mRNA及蛋白的表达有关。缩泉丸”缩尿”功效的发挥与改善衰老“多尿”动物机体状态,调节神经-内分泌-免疫网络系统功能有关,充分体现中药复方多成分、多靶点、整体调节的作用特点及优势。更多还原

【Abstract】 The low-tract urinary symptoms（LTUS） like nocturia、urinary frequency、enuresis、urinary incontinence,etc have a highly incidence rate in the elder people.Substituted heart and cerebrovascular system,it become the critical reason to effect physical and mental health and life quality of the aged people. According study,we find out the cause of the LTUS in the aged people not only the reason of urethra,like nerve system dysfunction,blood-vascular system affection and bladder outflow obstruction,but also the age itself.The function of capsular bladder is to urinate and store.This physiological function depend detrusor of bladder,a kind of main smooth muscle of bladder, to contract and relax.Because of physical and neurogenic changes,the senescent detrusor muscle occur functional impairment of contraction and relaxation.This is the main cause of elder LTUS.The formula of Suoquanwan（SQW） is recorded in a Chinese medicine book named Furenliangfang,which collected hundreds of formulas of Chinese medicine combinations.SQW consists of three herb medicines,Radix Linderae,Radix dioscoreae,and alpinia oxyphylla Miq. The function of SQW is to warm kidney,stop polyuria and hold excessive urination.This research duplicate the animal model of senile hyperdiuresis and observes the effect of arresting polyuria,collagen protein of musculus detrusor vesicae,neuroceptor related gene and regulatory protein related expression.This study from different Angles,like the molecular mechanism and the occurrence and development of polyuria,search for the mechanism of the formula SQW.Objective:On the basis of duplicating D-galactose mimetic aging animal model and exploring its mechanism of hyperdiuresis,observed the pharmacological action and molecular mechanism of SQW.Methods:Investigated the urination of D-galactose mimetic aging animal model, including total volume and ionic concentration of urine during 24h;We also observed the incubation period and frequency of urine within 6h after water load.The pharmacological mechanisms of SQW were investigated in following aspects:Investigated the hormones（including:ADH,ALD,ANP） and enzymes （including:ATPase,SDH） which influence the production and excretion of urine;the detrusor strips of each rat was suspended in a thermostatically controlled organ bath.And their spontaneous contraction frequency,reaction tension to stress and response intensity to electric stimuli were determined and statistically analyzed;The reactivity of urinary detrusor strips of each rat to different concentrations of ISO and ATP was measured respectively using an in vitro tissue bath technique;The pathological changes of kidney and bladder in rats were observed with HE,Masson,PAS dying method before and after SQW administration.The influence of SQW on expression of collagenⅠmRNA,collagenⅢmRNA,P2X₂ mRNA,β₃-AR mRNA in urinary detrusor strips of O-galactose mimetic aging animal model by RT-PCR;The influence of SQW on the in situ and protein expressions of collagenⅠ,collagenⅢ,P2X₂,β₃-AR in urinary detrusor strips of polyuria model by immunohistochemistry method.Results:1、The mechanism of polyuria for D-galactose mimetic aging modelThe animal models have characteristics of polyuria.Total volume of urine during 24h increased.The incubation periods shorten and the frequency of urine increased within 6h after water load.There are about some reasons that caused these symptoms.（1） The synthesis and externalization of ADH and ALD decreased, at the same time,the level of ANP and AQP-2 increased.（2） The activities of SOD,Na⁺-k⁺-ATPase、Ca²⁺-Mg²⁺-ATPase and SDH in urinary detrusor strips are reduced.The frequency of spontaneous contraction was elevated significantly; The complaisance and elasticity of bladder were both attenuated.Compared with the young adult rats,significantly decreased relaxation response to high concentration of ISO and increased contractile response to ATP were found in aged model.Some histological changes were observed in aging model:the quantity of nephron was decreased,such as nephric tubule;glassy degeneration appeared in proximal convoluted tubule;the alinement of muscle fiber which in tunica muscularis vesicae urinariae was chaotic;fibrosis for vessel wall; the percentage of collagen fibers are increased.2、The pharmacologic action of SQWThe results show that SQW reduce significantly the urine and the concentration of sodium,chlorine in urine,and elevate the excretion of urine potassium in aging rats.It also can improve the frequency of urine and extend incubation period of urine.SQW can increase the releasing of ALD,ADH and the concentration of AQP-2 in urine,meanwhile,it can reduce the content of ANP.SQW can delay the proceeding of aging in detrusor muscle.The activity of SOD,Na⁺-k⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,SDH and complaisance and elasticity of bladder increased after SQW administration in aging rats.The frequency of spontaneous contraction was cut down significantly.The decreased relaxation response to ISO and increased contractile response to ATP were also changed after SQW administration.The pathological characteristics of kidney and bladder function were switched:nephric tubule analosis was improved;the hyperplasia of collagen fibers which in tunica muscularis vesicae urinariae and thickening,chaotic muscle fiber was decreased. 3、The molecular pharmacology mechanism of SQWThe results showed that both mRNA and protein expressions of collagenⅠ, collagenⅢ,P2X₂ were decreased and mRNA and protein expressions ofβ₃-AR was increased in D-galactose mimetic aging model with polyuria afer SQW administration.SQW can recover the normal physiologic function of detrusor of bladder by myogenic and neurogenic ways.Conclusion:Sucessful duplication of subacute aging animal model with polyuria provided a reliable and effective model for our study.SQW reduced significantly the urine through effecting the production and excretion of urine;it can recover the normal physiologic function of detrusor of bladder. This recovery might be through up-regulatingβ3-AR mRNA and its protein expression and down-regulating collagenⅠ,collagenⅢ,P2X2 mRNA and their protein expression.更多还原