连钱草抗泌尿系草酸钙结石的药理作用及其机制研究

发布时间：2018-05-14 21:19

本文选题：草酸钙 + 乙二醇　；参考：《兰州大学》2017年博士论文

【摘要】：目的泌尿系统中结石的形成主要包括了结石结晶的成核、生长、聚集及滞留。其全球发病率为1%-20%,平均约为10%。伴随微创外科新技术的不断进步,结石患者绝大多数得到了满意的疗效,但结石的高复发率,抗结石药物的不良反应,仍是一个困扰人类的大难题。虽然研究表明,噻嗪类利尿剂与柠檬酸盐等能在一定程度上预防泌尿系统结石的形成与生长,但发现长期使用该类药物会有较为明显的副作用及不良反应。因此,人们目前试图解决的一大难题便是重新开发研究一些较为安全有效、价格低廉、能够长期服用并且并发症相对较少的抗结石药物。近几年,以银杏叶、紫杉醇、青蒿素与喜树碱等一批植物为研究基础研发了一些新的药物,其抗结石作用得到了肯定,更多植物制剂的研究为人们的研究提供了新的思路。祖国医学中,连钱草常用来治疗肾结石,但其作用并不肯定。我们拟将中药多作用靶点的特点运用起来,通过蛋白组学的方法,探讨其预防泌尿系结石的可能作用机制。方法细胞实验中,通过草酸钙结晶(67μg/cm2)持续24小时来刺激人肾皮质近曲小管上皮细胞(HK-2)来建立草酸钙结石的细胞模型。试验时,细胞培养液中给予不同浓度的连钱草(0.5,1.0,2.0,or 4.0mg/mL)or 1.0mg/mL柠檬酸钾,当CaOx结晶作用HK-2细胞1天后,收集细胞及培养液,并检测氧化应激的指标:丙二醛(MDA)、超氧化物歧化酶(SOD)以及过氧化氢酶(CAT)。而后用免疫印迹(Western blotting)检测泌尿结石相关蛋白骨桥蛋白(OPN)和肾损伤分子-1(KIM-1)的表达情况。动物实验中,60只成年的雄性SD大鼠被随机分入6组(分别为正常组、实验组、阳性对照组、低中高浓度连钱草组)。当大鼠适应环境一周以后,予以实验组大鼠1%的乙二醇饲料喂养4周,同时以不同浓度水平的连钱草灌胃。于第28天将其置入代谢笼中,留取24h尿液标本。10%的水合氯醛行腹腔注射麻醉大鼠后,采集血标本和肾标本。而后检测尿液中的各项生化指标,在显微镜下观察结晶的沉淀情况及病理变化情况,同时测定OPN以及KIM-1的免疫组化、免疫印迹和氧化应激相关指标。结果体外和体内实验均证明,与阳性对照组相比,连钱草显著降低了草酸钙引起的OPN、KIM-1表达及氧化应激水平(p0.05)。和结石组大鼠相比,连钱草组的大鼠表现出显著减少的草酸钙沉积和病理改变(p0.05)。此外,尿液标本中草酸、肌酐和尿素的含量亦明显低于结石组的大鼠,而柠檬酸的含量则明显增高。结论连钱草提取物预防结石的作用比柠檬酸钾更好。
[Abstract]:Objective the formation of stone in urinary system mainly includes nucleation, growth, aggregation and retention of stone crystallization. Its global incidence is 1-20, with an average of about 10. With the development of the new technique of minimally invasive surgery, the majority of calculi patients get satisfactory curative effect. However, the high recurrence rate of stones and the adverse reaction of anti-stone drugs are still a big problem for human being. Although studies have shown that thiazide diuretics and citrate can prevent the formation and growth of urinary calculi to some extent, it is found that long-term use of these drugs has obvious side effects and adverse reactions. Therefore, one of the most difficult problems that people are trying to solve is to re-develop and study anti-stone drugs that are safe, effective, inexpensive, can be taken for a long time and have relatively few complications. In recent years, some new drugs were developed on the basis of ginkgo leaf, paclitaxel, artemisinin and camptothecin. In traditional Chinese medicine, even herbs are often used to treat kidney stones, but their role is not certain. We intend to apply the characteristics of multi-action targets of traditional Chinese medicine to explore the possible mechanism of preventing urinary calculi by proteomics. Methods the cell model of calcium oxalate calculi was established by stimulating the epithelial cells of proximal tubules of human renal cortex with calcium oxalate crystal 67 渭 g / cm 2 for 24 hours. In the experiment, the cells were treated with different concentrations of 4.0mg/mL)or 1.0mg/mL citric acid or 4.0mg/mL)or 1.0mg/mL in different concentrations. After CaOx crystallized for 1 day, the cells and the culture medium were collected, and the cells and the culture medium were collected. The indexes of oxidative stress were: malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT). Then Western blotting was used to detect the expression of osteopontin (OPN) and renal injury molecule-1 (KIM-1) in urolithiasis. In animal experiment, 60 adult male SD rats were randomly divided into 6 groups (normal group, experimental group, positive control group, low, medium and high concentration group). After one week of adaptation, rats in the experimental group were fed with 1% ethylene glycol forage for 4 weeks. On the 28th day, the rats were placed in a metabolic cage and 24 h urine samples were collected. 10% chloral hydrate was injected intraperitoneally into anesthetized rats. Blood and kidney samples were collected. Then the biochemical indexes in urine were detected, the precipitation and pathological changes of crystals were observed under microscope, and the immunohistochemical, immunoblotting and oxidative stress related indexes of OPN and KIM-1 were also determined. Results both in vitro and in vivo experiments showed that the expression of OPNN KIM-1 and the level of oxidative stress (P 0.05) were significantly decreased by Lianqiangcao compared with the positive control group. Compared with the lithiasis group, the rats in Lianchencao group showed significantly decreased calcium oxalate deposition and pathological changes (p 0.05). In addition, the contents of oxalic acid, creatinine and urea in urine samples were significantly lower than those in rats with calculi, while the content of citric acid was significantly increased. Conclusion the extract of Lianqiangcao is more effective than potassium citrate in preventing stone.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R691.4

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