【作者】 王司军；

【导师】 金讯波；

【作者基本信息】 山东大学 ， 外科学， 2012， 博士

【摘要】 目的：泌尿系结石是泌尿系统最常见、最痛苦的疾病之一。在世界各地,它的发病率有所不同,在亚洲发病率为1-5%,在欧洲为5-9%,北美洲为13%,,沙特阿拉伯为20%,世界上只有极少的地区泌尿系结石的发病率较低,如格陵兰、日本沿海地区。同其他疾病不同,泌尿系结石可以追溯到人类的远古时期。大约80%的泌尿系结石由草酸钙组成。泌尿系结石可以引起尿路梗阻、感染、血尿、肾积水和肾功能不全。随着腔内泌尿外科技术的发展和体外震波碎石技术的引入,在泌尿系结石治疗方面取得了很大进展。目前,泌尿系结石病人很少接受开放手术治疗。接受ESWL治疗的病人可以出现出血、高血压、肾小管坏死、肾脏纤维化、急性肾脏功能损伤、肾功能下降和导致结石复发率升高。而且,泌尿系结石的一个非常显著的特点是其高复发率。据报道,在没有采取任何预防措施的情况下,泌尿系结石1年复发率接近10%,5年复发率33%,10年复发率50%,15年复发率高达75%。至今没有有效的方式预防泌尿系结石的复发。肾结石的形成是一个复杂的过程,由过饱和、晶核形成、结晶生长、结晶聚集、结晶肾小管滞留等几个阶段构成。从广泛上来讲,肾结石可以分为两大类：组织附着型结石和组织非附着型结石。肾上皮细胞暴露于草酸／草酸钙结晶时可以导致活性氧产量增加,氧化应激增强,继发细胞损伤和炎症。到目前为止,肾上皮细胞损伤在肾结石的发展中起重要作用这一观点已经得到大多数专家的认同。据报道,在人类、动物实验、体外实验的研究中已经得出结论：泌尿系结石存在时氧化应激升高。草酸盐可以升高氧化应激,抗氧化可以降低氧化应激。自由基、和拥有非成对电子的分子和原子统称为活性氧。活性氧具有很强的活性,同时在很多信号旁路中起重要作用。另外,它们可以对很多细胞成分产生损害和化学修饰,如核苷酸、脂质、蛋白质、碳水化合物。活性氧主要包括氧化亚氮自由基、羟自由基、过氧化氢、和超氧阴离子。氧化亚氮自由基主要由内皮细胞氧化亚氮自由基合成酶介导的L-精氨酸的氧化时生成。超氧阴离子主要是黄嘌呤氧化酶、还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶、血红素氧化酶、脂氧化酶、环氧合酶等酶的产物、和作为呼吸链的副产物生成。脂自由基同样可以产生超氧阴离子。内皮细胞氧化亚氮自由基合成酶产生超氧阴离子的能力超过产生氧化亚氮自由基的能力。氧化亚氮自由基和超氧阴离子之间反应可以产生具有高反应性的过氧化亚硝酸盐ON0O。细胞自身同时配备有清除活性氧的系统,如清除超氧阴离子的超氧化物歧化酶、清除过氧化氢的谷胱甘肽过氧化物酶和过氧化氢酶。在正常状态下,细胞通过相应的酶系严格控制活性氧的产生,并参与相应的调控系统和信号旁路。但是,活性氧的过量生产和/或细胞抗氧化能力的降低可以导致氧化应激的发展。因为活性氧存在时间短和运动能力低,氧化应激认为是活性氧和碳水化合物、蛋白质、核苷酸、脂质之间反应的副产物所致。丙二醛、氧化脂质是活性氧诱导的氧化应激的主要副产物。氧化应激可以损伤细胞的所有组成成分,并可被抗氧化剂拟制。植物药物自从人类历史出现记载后就已经开始应用,至今已有几千年的历史。目前,自然植物药物已在亚洲广泛应用,同时在欧洲、美国也获得越来越多的人认同。有很多植物药物自古就用于防治泌尿系结石并取得良好效果,但是这些植物药物防治泌尿系结石的具体作用机制仍未清楚。茯苓是最常见的传统中国植物药物之一,并在防治泌尿系结石方面得到广泛应用。茯苓水溶性多糖在茯苓中含量较少,是其在水中提取的主要活性物质。β-(1-3)-D-多糖是茯苓中的主要成分,但基本没有任何生物活性,但是其衍生物已被实验证实具有抗肿瘤等活性。在本实验中,我们想研究茯苓水溶性多糖预防大鼠草酸钙肾结石形成中的作用机制。方法：在雄性Wistar大鼠研究茯苓水溶性多糖预防大鼠肾结石形成的作用机制。应用0.8%乙二醇和1%氯化铵8天诱导大鼠成石。除了应用乙二醇/氯化铵外,另外给予茯苓水溶性多糖溶液灌胃(150mg/kg),阳性对照组给予乙二醇/氯化铵,阴性对照组大鼠正常饮水,同时给予自来水灌胃。每组大鼠包括6只大鼠。8天后,收集24小时尿并测量尿Ph值、钙、镁、草酸含量,左肾用于草酸钙结晶沉积评价,应用elisa法测量右肾MDA、SOD、CAT含量。结果：阴性对照组大鼠体重正常增长,阳性对照组大鼠体重明显减轻,茯苓水溶性多糖干预组大鼠体重减轻程度小于阳性对照组。阳性对照组大鼠每个大鼠肾脏切片均有结晶沉积,茯苓水溶性多糖干预组大鼠肾脏切片结晶沉积评分明显低于阳性对照组。阳性对照组大鼠尿量同阴性对照组大鼠无明显变化,茯苓水溶性多糖干预组尿量同阳性对照组比较明显增加。茯苓水溶性多糖干预组大鼠尿草酸含量少于阳性对照组,但无明显差异,但阳性对照组、茯苓水溶性多糖干预组大鼠尿草酸含量同阴性对照组具有明显差异。茯苓水溶性多糖干预组大鼠尿镁含量较阴性对照组、阳性对照组明显升高。阳性对照组大鼠肾脏SOD、CAT较阴性对照组含量明显降低,MDA含量明显升高。而茯苓水溶多糖干预组大鼠肾脏SOD、 CAT含量较阳性对照组明显升高,MDA含量明显降低。结论：对乙二醇/氯化铵诱导的大鼠应用茯苓水溶性多糖溶液灌胃,可以有效的预防泌尿系结石的形成。其预防泌尿系结石的机制有可能与茯苓水溶性多糖的利尿作用、升高尿镁含量和拟制氧化应激作用有关。意义：通过对茯苓水溶性多糖预防大鼠肾草酸钙结石模型的作用机制研究,不仅在一定程度上明确了茯苓水溶性多糖预防泌尿系结石的机制,而且对下一步研究茯苓β-(1-3)-D-多糖衍生物在泌尿系结石中的作用机制、充分利用茯苓、节约自然药物、及研究其他单剂中药防治泌尿系结石的作用机制并研制新的预防泌尿系结石的方剂奠定了基础。更多还原

【Abstract】 Objective:Urolithiasis, one of the most painful ailments of the urinary tract disorder, is one of the most common diseases in the urinary system. The prevalence differs in various parts of the world,1-5%in Asia,5-9%in Europe,13%in North America, and20%in Saudi Arabia, and there are a few geographical areas in which stone disease is rare, e.g. in Greenland and in the coastal areas of Japan. Unlike any other human disease, the occurrence of urinary calculi was documented far back into antiquity. Approximately of80%of the stone are composed of calcium oxalate (CaOx). Urinary calculi may cause obstruction, infection, hemorrhage, hydronephrosis in the urinary tract, and renal function insufficiency. Great improvements have been made in the treatment of urinary calculi thanks to the development of extracorporeal shock wave lithotripsy (ESWL) and the endoscopic surgery. Currently, open renal surgery for nephrolithiasis is unusual and uesd only rarely since the introduction of ESWL and endoscopic lithotripsy. The patients who exposure to ESWL may cause hemorhage, hypertention, tubular necrosis, kidney fibrosis, acute kidney injury (AKI), decrease in renal function, and increase in calculi recurrence. But it is important to point out that urolithiasis is characterized by high recurrence rate. It is reported that the recurrence rate of urolithiasis without preventive treatment is approximately10%at one year, 33%at five year,50%at ten years and75%at15years. There has been no effective measures to prevent the occurrence of urinary calculi. Kidney stone formation is a complex process that results from a succession of several events including supersaturation, nucleation, growth, aggregation, and retention within renal tubules. Renal stone can be broadly classified into two large groups:tissue attached and tissue unattached. Renal cellular exposure to oxalate and/or CaOx crystals leads to the production of reactive oxygen species (ROS), development of oxidative stress (OS) followed by injury and inflammation. At present, it seems clear that renal epithelial cell injuries play a decisive role in renal calculi development. It has been reported that raised OS is often present in urolithiasis in human, animal and in vitro studies. Oxalate promotes oxidative stress, which is retarded by antioxidants. Free radicals, molecules or atoms with unpaired electrons, and their metabolites, collectively called ROS which are high reactive. ROS play a important role in a variety of signaling pathways. Furthermore, they can produce damage to, and modifications of nucleotides, lipids, protein and carbohydrates. Major cellular reactive oxygen species include nitric oxide radical, hydroxyl radical, hydrogen peroxide, and superoxide anion. ROS are produced from many sources. Nitric oxide radical are produced by the endothelial nitric oxide synthase (eNOS) mediated oxidation of L-arginine. Superoxide anions are produced by xanthine oxidase, NADPH oxidase, hemeoxygenase, lipooxigenase, cyclooxygenase, and as a byproduct of the mitochondrial respiratory chain. Lipid radicals can also produce superoxide anions. eNOS can also produce superoxide anions rather than nitric oxide radical. Reactions between nitric oxide and superoxide can produce the highly reactive peroxynitrite ONOO-.Cells are equipped with several scavenging systems to limit ROS, such as superoxide dismutase (SOD) which has the ability to eliminate superoxide anions, catalase and glutathione peroxidase to detoxify hydrogen peroxide. Under normal conditions ROS are generated by tightly controlled enzymes and involved in various regulatory processes and signaling pathways, but an overproduction of ROS and/or a reduction in cellular antioxidant capacities leads to the development of OS. Because most ROS are short-lived and do not travel long distance, the presence of OS is recognized by a lots of byproducts of ROS interaction with carbohydrate, lipids, nucleic acids, amino acids, and protein. Malondialdehyde (MDA), oxidized lipids are the major byproducts of ROS induced OS. OS is injurious to all components of the renal epithelium which can be retard by antioxidants. Herbal medicine has been used for several thousands years and is as ancient as the history of mankind. Actually, natural herbal medicine has been used widely in Asia, and has gained popularity in Europe, and in United Stated as well. Various herbal medicines have been used in treating urolithiasis, but the exact mechanism of herbal medicine in treating urinary calculi has not been revealed. Poria cocos is one of the most popular traditional Chinese medicinal plant and has pharmacological importance in antilithic activities. Water soluble polysaccharides (WSP) is the main active material derived from poria cocos which is very little content in poria cocos, β-(1-3)-D-glucan is the major content of poria cocos, which shows little activity, but its derivatives can show anti-tumor activity.In this study, we want to evaluate the antilithic mechanism of WSP derived from poria cocos on ethylene glycol induced urolithiasis in rats. Methods:The effect of WSP derived from poria cocos on calcium oxalate urolithiasis was studied in male Wistar rats. Urolithiasis rat model were induced by0.8%ethylene glycol [v/v](EG) and1%ammonium chloride [w/v](AC) for eight days. In addition to EG/AC treatment rats were also treated solutions containing WSP derived from poria cocos (150mg/kg). Positive control rats were treated only with EG/AC. Negative control rats were provided with normal drinking water, and were administered normal water by gavage. Each group contained six rats. After eight days, twenty-four hour urine samples were collected for analysis, the left kidney was removed and assessed for crystal deposition using light microscopy, the right kidney was assessed for malondiadehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels by elisa method.Result:There was a significant loss weight after inducing crystal deposition in Group Ⅱ while a normal body weight increase in Group Ⅰ,and a less loss weight in Group Ⅲ. The rats treated with EG/AC alone had higher amounts of crystal deposition in the kidneys compared to negative control group of rats. This EG/AC-induced increase in kidney crystal deposition was inhibited by the administration of WSP derived from poria cocos. An increased level of SOD, CAT, magnesium excretion and decreased level of MDA were also found in polysaccharide-treated group, as compared with positive control group.Conclusion:We concluded that administration of WSP derived from poria cocos to rats with EG/AC induced rats, prevented the formation of urolithiasis. The mechanism underlying this effect is related to increased diuresis, urinary magnesium excretion and inhibition of OS activity.Significance:To study the efficacy of WSP derived from poria cocos on ethylene glycol induced urolithiasis in rats, not only understand the exact antilithic mechanism of WSP from poria cocos, but also make a basis of studying the mechanism of derivatives of β-(1-3)-D-glucan isolated poria cocos, making full use of poria cocos and sparing the natural medicinal herb.更多还原