基于P2X1、P2X3受体探讨加味五苓散对糖尿病膀胱功能障碍大鼠的作用机制

发布时间：2018-09-12 11:59

【摘要】：一、文献及理论研究糖尿病膀胱功能障碍(DBD)是糖尿病慢性高血糖的一个并发症。在糖尿病发展的过程中,部分患者逐渐出现排尿间隔延长、排尿感觉迟缓、排尿乏力、点滴而下与排尿不净。尿动力学检测提示有膀胱感觉受损、膀胱容量增加、逼尿肌收缩能力下降、残余尿量增多等特点。由于膀胱的生理活动受周围神经所支配,因此多年来研究糖尿病膀胱病的发病机制主要针对于神经源性病变。可是,在临床上也能观察到患病不久的糖尿病患者出现下泌尿系的症状,表现却为尿急、尿频、夜尿与急迫性尿失禁,而且尿动力学检测也提示不同程度的逼尿肌不稳定或逼尿肌反射亢进。这两种系列相反的症状体征被认为与疾病的时间推演有联系,是糖尿病高血糖引起膀胱经历两个阶段、两种病理变化的具体体现,即从早期膀胱过度活跃(OAB)的代偿期阶段到晚期膀胱失张力(UAB)的失代偿期阶段。而且,研究也发现膀胱的上皮细胞、神经元、逼尿肌与尿道任何一方面出现异常都可以引起膀胱发生储尿或排尿的功能障碍。非肾上腺素能非胆碱能(NANC)通道对膀胱的生理病理有重要的作用。既往认为,膀胱的收缩主要是因为副交感神经释放的ACh与M受体结合而成。其后发现,嘌呤受体P2X与ATP结合后也能达到相似的作用。而且,P2X受体的表达异常往往与膀胱病变有密切关系,因此在膀胱病变中探索不同P2X亚型的角色也变得重要起来。P2X1受体主要分布于逼尿平滑肌细胞边缘,而P2X3受体主要分布在膀胱上皮细胞下的神经束。在病理状态下,两者的表达量变化可能提示膀胱在感觉或收缩活动上出现异常。中医方面,以往把糖尿病膀胱病(DC)或糖尿病神经源性膀胱(DNB)归属于中医“癃闭”的范畴。这只是按糖尿病后期常见的局部下泌尿道症状来命名,并不能体现与糖尿病有整体相关性,而且也未能体现膀胱从功能活跃到功能低下状态的一个演变过程。糖尿病以多饮、多食、多尿与体重下降为症状特点,属于中医“消渴病”的范畴。所以,本文提出DBD早期出现OAB状态仍然属于中医“消渴病”的范畴,而后期出现DAB状态则应该称为“消渴—癃闭”,以明确区分单纯的“癃闭”。糖尿病患者自身的阳气亏虚是消渴病的一个重要发病因素,并发的膀胱功能障碍的病机也因此离不开阳虚的一面。从六经辨证来说,糖尿病涉及六经,而糖尿病膀胱功能障碍的基本病位主要在太阳。太阳走体表属阳。阳气虚弱,邪犯太阳,从经入腑,可以导致太阳腑病。太阳腑属膀胱,膀胱的正常气化功能可以调节津液的输布与尿液、汗液的排泄。膀胱气化失常,失于固摄或失于开合是直接导致膀胱发生储尿或排尿功能障碍的两个重要病机。五苓散是中医经典《伤寒论》的处方,对调节膀胱功能具有重要的影响。从药物组成来看,茯苓、猪苓、·泽泻、白术四味药物都有利尿作用,因此五苓散常常被视为利尿剂,多用于膀胱排尿障碍出现少尿的时候。然而,桂枝的温阳通阳作用可能对膀胱的气化起了决定性的影响,因此有学者认为五苓散不仅仅是利尿剂,也是发汗剂。它的温阳化气作用,有助于水液代谢得到正常调节。这可能也适用于尿频、尿急、尿量增多的膀胱储尿障碍阶段。本病的病程发展与症状变化往往涉及太阴与少阴。温补太阴、少阴阳气不仅是要顾护先后天之本,也意在加强五苓散对膀胱的温阳化气作用。因此,加味五苓散是在五苓散的基础上加用黄芪、熟附子与乌药,以黄芪补太阳、太阴,熟附子补少阴,并佐以乌药温肾理气。由于文献报道糖尿病膀胱功能障碍的发病率高,并且严重影响患者的生活质量,中医药的干预可能为患者提供一种治疗途径。已知本病的代偿期与失代偿期会有不同的下泌尿系表现,而且五苓散被认为有双向调节作用。然而,加味五苓散的作用靶点与机制仍然存疑。本研究假设加味五苓散是透过调节嘌呤受体的表达来发挥药效,从而达到与症状相反的治疗目的。以加味五苓散作为中药干预措施,预期嘌呤受体P2X1与P2X3的表达会降低,并且出现膀胱储尿功能改善的相关指征。研究结果可能有助于理解嘌呤受体在加味五苓散双向调节作用的角色。二、实验研究目的：以DBD大鼠模型探讨加味五苓散对大鼠膀胱P2X1与P2X3受体表达的作用机制。方法：110只SPF级雄性SD大鼠经普通饲料适应性喂养1周后,进行随机分组。正常组10只,其余纳入造模组,包括模型组、托特罗定组、中药高剂量组、中药中剂量组、中药低剂量组。其后,造模组大鼠改以高脂饲料喂养1个月后,按45mg/kg剂量腹腔内注射1%STZ。72hr后消毒剪尾取尾静脉血,检测血糖≥16.67mmol/L,并且伴见多饮、多食、多尿等情况,即考虑为糖尿病大鼠模型。每2周观察体重与血糖的变化,并且每4周以代谢笼收集大鼠的饮水量与排尿量。造模后第9周开始,对托特罗定组大鼠灌服2m1托特罗定,对中药高、中、低剂量组分别灌服加味五苓散水煎剂6ml、3ml、1.5ml。第12周所有大鼠腹腔注射25%乌拉坦以麻醉,剖腹暴露膀胱并且在膀胱顶部造瘘,以30ml/h速率灌注生理盐水,以检测糖尿病大鼠的尿动力学变化。其后,分离膀胱测量湿重。经常规脱水、石蜡包埋和切片,膀胱组织在HE染色后用光镜观察组织病理情况。最后,以免疫印迹、免疫细胞组化技术对嘌呤受体P2X1、 P2X3的表达进行检测。结果：造模组大鼠出现高血糖的成模率为94.74%。在实验结束时共有44只大鼠完成实验,占总体的40%。其中正常组10只,造模组34只(包括模型组7只,托特罗定组6只,中药高剂量组6只,中药中剂量组7只,中药高剂量组8只)。实验期间,正常组大鼠的整体情况良好,而造模组大鼠表现精神紧张,毛发稀疏枯黄。其余各项指标的结果如下：①体重：组间对比发现中药低剂量组大鼠比托特罗定组大鼠要重,但与其它组没有统计学差异。组内比较则发现只有模型组大鼠体重下降(p=0.009),其它组则没有统计学变化。②血糖：组间对比发现中药低剂量组大鼠血糖较模型组大鼠的低。但是,组内比较则没有发现药物干预后各组的血糖有统计学变化。③饮水量：组间比较发现托特罗定组、中药中剂量组、中药低剂量组的饮水量较模型组为低。组内比较则只有托特罗定组大鼠的饮水量下降(p=0.018)。④排尿量：组间比较并没有发现造模组大鼠的排尿量有差异,但是组内比较则发现只有模型组与托特罗定组大鼠的排尿量有减少(p=0.006与p=0.002)。⑤尿动力学：除中药高剂量组外,其余造模组大鼠的排尿时间较正常组大鼠为延长。模型组与托特罗定组大鼠的最大膀胱排尿压均较正常组大鼠的为低,以托特罗定组的差异较为显着(p0.005),但中药组各组大鼠与正常组和模型组大鼠并没有差异。除中药高剂量组外,其余造模组大鼠的膀胱顺应性较正常组大鼠的为高,以托特罗定组大鼠的差异最为显着(p0.005)。造模组大鼠之间并没有统计学差异。⑥膀胱湿重与膀胱湿重指数：造模组各组大鼠的膀胱湿重较正常组为重,其中以模型组、中药高剂量组与中药中剂量组的差异较为显着(p0.005)；中药中剂量组的膀胱湿重最重,而托特罗定组最轻,两者有统计学差异。而膀胱湿重指数方面,造模组大鼠均比正常组大鼠为高。⑦病理检测：光镜观察HE染色切片,发现造模组大鼠的膀胱逼尿肌层有增厚现象,肌细胞肥大,形态多样,肌束排列紊乱有断裂,结构松散,肌束间间隙明显增宽,胶原纤维减少。⑧免疫印迹：各组的P2X1、P2X3受体表达未见明显异常,但是发现模型组的P2X3比值下降,托特罗定组与中药低剂量组又下调,而中药中剂量组与高剂量组则上调。⑨免疫组化：P2X1受体主要表达在逼尿肌、上皮细胞和血管壁；P2X3受体表达在逼尿肌。结论：本研究有以下几点结果：1)本研究成功建立DBD大鼠模型。2)未发现加味五苓散能降低DBD大鼠的体重、血糖、饮水量与排尿量。3)未发现加味五苓散能改善第12周DBD大鼠的尿动力学排尿时间、最大膀胱排尿压与膀胱顺应性。4)未发现加味五苓散能减轻膀胱重量与修复膀胱组织结构。5)发现DBD大鼠的P2X1受体主要表达在逼尿肌、上皮细胞和血管壁；P2X3受体的表达也见于逼尿肌。6)发现加味五苓散未能影响膀胱P2X1受体的表达,但是对P2X3受体的表达有调控作用,惟暂未发现有明显差异。此外,本研究有几点发现值得注意：1)虽然加味五苓散并不能降低高血糖状态,但它可能避免体重病态性下降。它的利尿作用可能延缓膀胱从OAB阶段向UAB阶段发展的趋势。中、低剂量的加味五苓散可能有助于改善糖尿病口渴多饮的症状。对症而言,未发现加味五苓散具有抗利尿的作用。2)加味五苓散的利尿效果可能有助于延缓逼尿肌向UAB发展的进程。3)糖尿病使P2X3受体表达下调。加味五苓散低剂量与中高剂量对P2X3受体表达分别为下调与上调,提示可能对P2X3受体有双向调节作用。
[Abstract]:1. Literature and theoretical studies have shown that diabetic bladder dysfunction (DBD) is a complication of chronic hyperglycemia in diabetes mellitus. During the development of diabetes mellitus, some patients gradually appear to have prolonged voiding interval, slow voiding feeling, weak voiding, dripping down and unclean voiding. Because the physiological activity of the bladder is dominated by peripheral nerves, the pathogenesis of diabetic cystopathy has been studied for many years mainly for neurogenic lesions. However, symptoms of the lower urinary system can also be observed in patients with diabetes mellitus who have been ill for a short time. Urinary urgency, frequent urination, nocturia, and urgent urinary incontinence, and urodynamic tests also suggest varying degrees of detrusor instability or detrusor hyperreflexia. These two series of opposite symptoms and signs are thought to be associated with the timing of the disease, diabetic hyperglycemia causes the bladder to undergo two stages, two specific pathological changes. In addition, abnormalities in bladder epithelial cells, neurons, detrusors and urethra in any aspect of the bladder can cause dysfunction of urine storage or micturition. Non-adrenergic non-cholinergic NANC channels play an important role in the physiology and pathology of the bladder. It was previously believed that the contraction of the bladder was mainly due to the binding of ACh released by the parasympathetic nerve to M receptor. It was found that the binding of purine receptor P2X to ATP could also achieve a similar effect. It is also important to explore different P2X subtypes in cystic lesions. P2X1 receptors are mainly located at the edge of detrusor smooth muscle cells, while P2X3 receptors are mainly located in the nerve tracts under the bladder epithelial cells. In pathological conditions, the changes in the expression of the two may indicate abnormal sensory or contractile activities in the bladder. Diabetic cystopathy (DC) or diabetic neurogenic bladder (DNB) is classified as "amenorrhea" in traditional Chinese medicine. It is only named according to the common local lower urinary tract symptoms in the late stage of diabetes mellitus. It does not reflect the overall correlation with diabetes mellitus, nor does it reflect the evolution of bladder from active to dysfunctional state. Diabetes mellitus is characterized by polydipsia, polyphagia, polyuria and weight loss, which belongs to the category of "diabetes" in traditional Chinese medicine. Therefore, this paper suggests that the early onset of OAB in DBD still belongs to the category of "diabetes" in traditional Chinese medicine, while the late onset of DAB should be called "diabetes-amenorrhea" to clearly distinguish between simple "amenorrhea". Deficiency of Yang Qi is an important pathogenic factor of diabetes, and the pathogenesis of bladder dysfunction can not be separated from Yang deficiency. Diabetes mellitus is related to the Six Meridians, while the basic location of diabetic bladder dysfunction is mainly in the sun. The normal vaporization of the bladder can regulate the delivery of fluid and urine, sweat excretion. Bladder vaporization disorders, loss of solid uptake or loss of opening and closing are two important pathogenesis that directly lead to urinary storage or urinary dysfunction in the bladder. Poria cocos, Poria cocos, Alisma orientalis and Atractylodes macrocephala have diuretic effects. Wuling powder is often considered as a diuretic and is used in the treatment of oliguria due to bladder dysuria. However, the warming yang and clearing Yang effects of Guizhi may play a decisive role in the gasification of bladder. Some scholars believe that Wuling Powder is not only a diuretic, but also a sweating agent. Its function of warming Yang and removing Qi helps to regulate the metabolism of water. This may also be applicable to the stage of bladder dysuria with frequent, urgent and increased urine volume. Therefore, Jiawei Wuling powder is based on Wuling powder plus astragalus, aconite and Wuyao, Astragalus to fill the sun, Taiyin, aconite to fill the shaoyin, and aconite to warm the kidney and regulate qi. It is known that the compensatory phase and decompensated phase of the disease have different lower urinary tract manifestations, and Wuling Powder is considered to have two-way regulatory effect. However, the target and mechanism of Jiawei Wuling Powder are still questionable. Wuling Powder exerts its effect by regulating the expression of purine receptor to achieve the opposite therapeutic purpose. Taking Weiwuling Powder as an intervention measure of traditional Chinese medicine, it is expected that the expression of purine receptor P2X1 and P2X3 will be decreased, and the related indications of improving bladder urinary storage function will appear. Objective: To explore the mechanism of Jiawei Wuling Powder on the expression of P2X1 and P2X3 receptors in bladder of rats with DBD. Methods: 110 SPF male SD rats were randomly divided into normal group (10 rats) and model group (10 rats). The rats in the model group were fed with high-fat diet for one month, then were injected with 45 mg/kg of 1% STZ.72 HR intraperitoneally. The tail vein blood was sterilized and cut off to detect blood glucose (> 16.67 mmol/L) and accompanied with excessive drinking, eating and urine, which was considered as diabetes mellitus. Rat models were established by observing the changes of body weight and blood glucose every 2 weeks, and collecting water intake and urination volume every 4 weeks with metabolic cage. Rats in the Tolterodine group were given 2 M1 tolterodine at the 9th week after modeling. Rats in the high, middle and low dosage groups were given 6 ml, 3 ml, 1.5 ml modified Wuling Powder Decoction respectively. After anesthesia, bladder was exposed by laparotomy and fistula was made at the top of the bladder. Urodynamic changes in diabetic rats were detected by infusion of saline at a rate of 30 ml/h. Wet weight was measured by separating the bladder and routine dehydration, paraffin embedding and biopsy were performed. The expression of purine receptor P2X1 and P2X3 was detected by cytochemistry. Results: The model rate of hyperglycemia was 94.74%. At the end of the experiment, 44 rats completed the experiment, accounting for 40% of the total. During the experiment, the rats in the normal group were in good condition, while the rats in the model group were nervous and their hair was thin and yellow. The results of other indexes were as follows: 1. Body weight: The rats in the low dose group were heavier than those in the tolterodine group, but there was no statistical difference between the two groups. Blood glucose: The blood glucose of rats in the low dose group was lower than that in the model group. However, there was no statistical change in blood glucose among the groups after drug intervention. It was found that the water intake of tolterodine group, middle-dose group and low-dose group was lower than that of model group (p=0.018). Urodynamics: Except for the high dose group of Chinese medicine, the urination time of the other model group was longer than that of the normal group. The maximum urinary pressure of the model group and the tolterodine group was lower than that of the normal group, and the difference between the tolterodine group and the tolterodine group was more significant (p0.005). There was no significant difference in bladder compliance between the two groups. Except for the high dose group, the bladder compliance of the other model groups was higher than that of the normal group, and the difference was most significant in the Tolterodine group (p0.005). There was no significant difference between the two groups. The wet weight of bladder in each group was heavier than that in the normal group, and the difference between the model group, the high dose group and the middle dose group was more significant (p0.005); the wet weight of bladder in the middle dose group was the heaviest, while that in the tolterodine group was the lightest, and the wet weight index of bladder in the model group was higher than that in the normal group. _Pathological examination: HE staining slices showed that the detrusor layer of bladder in the model group was thickened, the myocytes were hypertrophic, the shape was diverse, the muscle bundles were disorderly arranged, the structure was loose, the space between muscle bundles was significantly widened, and the collagen fibers were reduced. At present, the P2X3 ratio of model group was decreased, while that of tolterodine group and low-dose group was down-regulated, while that of middle-dose group and high-dose group was up-regulated. _Immunohistochemistry: P2X1 receptor was mainly expressed in detrusor, epithelial cells and vascular wall; P2X3 receptor was expressed in detrusor. BD rat model. 2) No modified Wuling powder was found to reduce the body weight, blood sugar, water intake and urination in DBD rats. 3) No modified Wuling powder was found to improve the urodynamic urination time, maximal bladder pressure and bladder compliance in 12-week DBD rats. 4) No modified Wuling powder was found to reduce bladder weight and repair bladder tissue structure. The expression of P2X1 receptor in detrusor, epithelial cells and vessel wall was found in DBD rats, and the expression of P2X3 receptor was also found in detrusor. Although Jiawei Wuling Powder does not reduce hyperglycemia, it may avoid morbid weight loss. Its diuretic effect may delay the development of bladder from OAB stage to UAB stage. 2) The diuretic effect of Jiawei Wuling Powder may be helpful to delay the development of detrusor to UAB. 3) The expression of P2X3 receptor is down-regulated by diabetes mellitus. The expression of P2X3 receptor is down-regulated and up-regulated by low dose and medium and high dose of Jiawei Wuling Powder respectively, suggesting that it may have two-way regulation on P2X3 receptor.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R285.5

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