虎桂药对组分配伍治疗高尿酸血症及其机制的探究

发布时间：2018-05-08 09:52

本文选题：高尿酸血症 + 虎杖苷　；参考：《广东药科大学》2017年硕士论文

【摘要】：高尿酸血症是困扰人类的众多代谢疾病之一。大量的国内外研究显示,高尿酸血与许多疾病密切相关,如急性痛风性关节炎、尿酸性肾病、高血压、肥胖、糖尿病和心血管疾病等。伴随着我国经济水平的高速列车,人民的生活习惯,饮食规律发生了翻天覆地的变化。夜宵、快餐文化、啤酒、饮料(含果糖)等在我国的大肆泛滥,致使我国的高尿酸血症患者爆发式的增长,并呈现低龄化的趋势。严重影响着我国人民的身体健康以及生活质量。目前高尿酸血症的研究尚缺乏公认的合理的动物模型和安全有效的治疗药物,故本次实验我们希望尝试建立一种血尿酸值较为平缓、稳定、持久、对肾脏损害小的大鼠高尿酸血症模型,并在此基础上探究虎桂药对的组分——虎杖苷和桂皮醛相互配伍对高尿酸血症的治疗作用以及其作用机制。1.大鼠高尿酸血症模型实验目的:优化次黄嘌呤,氧嗪酸钾,乙胺丁醇三种药物联合制备大鼠高尿酸血症模型的使用剂量,以期得到更合适的大鼠高尿酸血症模型。方法:雄性SD大鼠32只,随机分为4组,每组8只。模型Ⅰ组:给以灌胃75mg/kg次黄嘌呤和250mg/kg盐酸乙胺丁醇,皮下注射200mg/kg氧嗪酸钾(1次/d)。模型Ⅱ组:给以灌胃75mg/kg次黄嘌呤和250mg/kg盐酸乙胺丁醇(1次/d),皮下注射100mg/kg氧嗪酸钾(1次/12h)。模型Ⅲ组:给以灌胃50mg/kg次黄嘌呤和250mg/kg盐酸乙胺丁醇,皮下注射200mg/kg氧嗪酸钾(1次/d)。空白组给予生理盐水灌胃及橄榄油皮下注射。连续造模24天。第4,11,18d皮下注射后6h,大鼠眼眶取血,测SUA含量。第24天皮下注射后,分别3、6、12、24h取血测SUA含量,并测6h血清中BUN、Cr的含量。大鼠处死取肾,测察其病理改变。结果:与空白组对比,三组造模方式均能引起大鼠的血尿酸含量明显上升(P0.01),可维持12h,尤其是模型Ⅱ组能达到24h;三组造模方式的大鼠肾脏病理切片表明,三种造模方式均能引起肾脏轻微损伤;模型Ⅰ组和模型Ⅱ组的血尿素氮含量与空白组对比明显的升高(P0.01,P0.05)。结论:三种造模方式均能成功制备大鼠高尿酸血症模型,并伴有轻微的肾脏器质和功能损伤(尤其是模型Ⅲ组损伤最轻),具有血尿酸值增长维持时间长的优点(尤其是模型Ⅱ组可持续24h)。2.虎桂药对组分配伍对高尿酸血症的治疗及机制的探究目的:探究虎桂药对的组分——虎杖苷(Polydatin)和桂皮醛(Cinnamaldehyde)互相配伍后,对大鼠高尿酸血症的降尿酸作用,并从尿酸的排泄和生成两个途径,初步探索其多靶点降尿酸的机理,为中医药治疗高尿酸血症提供新的思路和方法。方法:雄性SD大鼠70只随机分为7组。空白组、模型组、别嘌呤醇组、苯溴马隆组、虎杖苷和桂皮醛(PC)的低、中、高组,每组10只。皮下注射氧嗪酸钾(200mg/kg),7h后灌胃乙胺丁醇(250mg/kg)和次黄嘌呤(50mg/kg)制备高尿酸血症大鼠模型,连续26天(1次/d)。第4天,皮下注射后6h眼眶取血测大鼠的SUA值。第5天起,皮下注射前1h,灌胃治疗药,持续21天(1次/d)。第25天将大鼠放进代谢笼,收集尿液24h,备用。最后一天皮下注射6h后,大鼠腹主动脉取血,测定SUA、Cr、BUN和GPT的含量;取出大鼠肝,肾,小肠备用。ELISA试剂盒测定尿液中Kim-1、NGAL的含量;肾脏组织的病理学观察,统计评分;qPCR测定肝脏XOD、ADA和肾脏GLUT9、URAT1、OAT1、OCT1的基因表达;Western Blotting测定肝脏XOD和肾脏GLUT9、URAT1的蛋白表达;免疫组化测定小肠MRP4的蛋白表达,分析照片阳性的累积光密度值(IOD)。结果:造模后,大鼠SUA含量明显升高(P0.01)。给予治疗药后,PC中、高组SUA含量明显下降(P0.01,P0.05);PC中组BUN含量明显下降(P0.05);PC三组的NGAL含量明显下降(P0.01,P0.05);PC中、高组的Kim-1含量明显下降(P0.01);PC三组肝脏ADA,XOD mRNA表达明显下降,肾脏GLUT9,URAT1mRNA表达明显下降,OAT1、OCT1 mRNA表达明显上升(P0.01,P0.05);PC三组肝脏XOD蛋白表达明显下降,肾脏GLUT9、URAT1蛋白表达明显下降(P0.01,P0.05);PC中组小肠MRP4蛋白表达明显升高(P0.01);肾脏组织病理切片表明:PC中组的大多数肾皮质肾小管上皮细胞形态正常,胞浆丰富,肾髓质少数集合管管腔内见红细胞堆积。结论:虎桂药对成分——虎杖苷和桂皮醛相互配伍后,具有明显的抗高尿酸血症的药理作用。可通过抑制尿酸的生成和促进尿酸的排泄双重降低血尿酸含量,改善高尿酸血症对肾脏造成的器质及功能性损伤。其作用机制可能与抑制大鼠肝脏XOD、ADA和肾脏URAT1、GLUT9的表达;促进肾脏OCT1、OAT1和小肠MRP4的表达相关。
[Abstract]:Hyperuricemia is one of the many metabolic diseases that perplex human beings. A large number of domestic and foreign studies have shown that high uric acid blood is closely related to many diseases, such as acute gouty arthritis, uric acid kidney disease, hypertension, obesity, diabetes and cardiovascular diseases. There has been a great change. The night, the fast food culture, the beer, the drink (including fructose) and so on in our country, caused the outbreak of hyperuricemia in our country to increase, and present a low age trend. It seriously affects the health of our people and the quality of life. At present, the research of hyperuricemia is still lack of recognition. In this experiment, we hope to try to establish a model of hyperuricemia in rats with a relatively gentle, stable and persistent blood uric acid value, and to explore the therapeutic effect of the combination of Polygonum cuspidin and cinnamaldehyde on hyperuricemia on the basis of this model. And its mechanism of action mechanism.1. rats hyperuricemia model experiment to optimize the dosage of hypoxanthine, potassium oxazine and ethambutol to prepare the model of hyperuricemia in rats, in order to get a more suitable model of hyperuricemia in rats. Methods: 32 male rats were randomly divided into 4 groups, 8 rats in each group. Group I: group I: group I: group I: group I: group I: group I: group I: group I: group I: group I: group I: group I: group I 75mg/kg hypoxanthine and ethambutol 250mg/kg were injected subcutaneously with potassium 200mg/kg oxazine (1 /d). Model II group was given 75mg/kg hypoxanthine and 250mg/kg hydrochloric ethambutol (1 /d), and hypodermic potassium oxazine (1 /12h). Model III group: gavage of 50mg/kg hypoxanthine and 250mg/kg hydrochloric ethambutol, 200mg/kg potassium oxazine (1 times /d) was injected subcutaneously in the blank group, and the normal saline was given to the stomach and the olive oil was injected subcutaneously for 24 days. After the subcutaneous injection of 4,11,18d, the blood was taken from the orbit of the rat and the content of SUA was measured. After twenty-fourth days of subcutaneous injection, the content of SUA in the serum was measured by 3,6,12,24h, and the content of BUN and Cr in the serum of 6h was measured. Rats were executed and the kidneys were killed and detected by the rats. Pathological changes. Results: compared with the blank group, the blood uric acid content in the three groups could increase significantly (P0.01), and could maintain 12h, especially in the model II group. The pathological section of the kidney of the three model rats showed that the three modes of modeling could lead to slight renal injury; the hematuria in model I group and model II Group The content of vegetarian nitrogen was significantly higher than that in the blank group (P0.01, P0.05). Conclusion: the model of hyperuricemia in rats can be successfully prepared by the three models, with slight renal organic and functional damage (especially in model III group), which has the advantage of long duration of increasing blood uric acid value (especially in model II Group sustainable 24h).2. The purpose of the treatment and mechanism of group distribution of tiger and cinnamon to hyperuricemia is to explore the effect of Polydatin and Cinnamaldehyde on the components of the Chinese medicine, and to explore the effect of uric acid on hyperuricemia in rats, and the excretion and formation of uric acid in two ways. To provide new ideas and methods for the treatment of hyperuricemia in Chinese medicine. Methods: 70 male SD rats were randomly divided into 7 groups. The blank group, the model group, the allopurinol group, the benzene bromide Malone group, the Polygonum glycoside and the cinnamaldehyde (PC) were low, middle, high group, 10 in each group. The subcutaneous injection of potassium oxazine (200mg/kg), 7h after 7h and hypoxanthine (250mg/kg) and hypoxanthine (250mg/kg). 50mg/kg) the rat model of hyperuricemia was prepared for 26 days (1 times /d). Fourth days after fourth days, the SUA value of rats was measured by 6h orbital injection after subcutaneous injection. From the fifth day, before subcutaneous injection, 1H, gavage for 21 days (1 times /d). The rats were put into the metabolic cage for twenty-fifth days, and the urine 24h was collected. After the last day subcutaneous injection of 6h, the abdominal aorta of rat was taken blood, measured blood, measured the abdominal aorta of rats, measured blood of rat abdominal aorta after the last day, measured blood of abdominal aorta of rats, measured blood of rat abdominal aorta, measured blood, measured abdominal aorta rats, measured blood, measured abdominal aorta rats, measured blood, measured abdominal aorta rats, measured blood, measured rat abdominal aorta, measured blood, measured rat abdominal aorta after blood, measured blood, measured rat abdominal aorta, measured blood after abdominal aorta, measured rat abdominal aorta after blood extraction, measured the rat aorta, test Determine the content of SUA, Cr, BUN and GPT; take out the.ELISA kit of rat liver, kidney and small intestine to determine the content of Kim-1 and NGAL in urine; the pathological observation of kidney tissue, statistical score; qPCR determination of liver XOD, ADA and kidney GLUT9, URAT1, protein expression; immunization The protein expression of MRP4 in the small intestine was determined by histochemistry and the cumulative photo density value (IOD) of the photo positive was analyzed. Results: after the model, the content of SUA in the rats increased significantly (P0.01). After the treatment, the content of SUA in the high group decreased significantly (P0.01, P0.05) in PC; the BUN content in the PC group was obviously lower (P0.05), and the content of the PC three groups decreased significantly. The expression of Kim-1 was significantly decreased (P0.01), the expression of ADA, XOD mRNA in PC three groups was significantly decreased, the expression of GLUT9 and URAT1mRNA in the kidneys was significantly decreased, OAT1 and OCT1 mRNA increased (P0.01,), and the expression of egg white in the three groups was obviously decreased. The histopathological sections of renal tissue showed that most of the renal tubular epithelial cells in group PC were normal, rich in cytoplasm and accumulated in a small collection of tubules in the renal medulla. Conclusion: after compatibility of Polygonum cuspidin and cinnamaldehyde, the pharmacology of tiger cinnamon and Polygonum cuspidin and cinnamaldehyde have obvious pharmacological effects on anti hyperuricemia. Inhibiting the formation of uric acid and promoting uric acid excretion to reduce the content of uric acid and improve the organic and functional damage caused by hyperuricemia on the kidney. The mechanism may be related to the inhibition of the expression of XOD, ADA, URAT1 and GLUT9 in the rat liver, and the expression of OCT1, OAT1 and the expression of MRP4 in the small intestine.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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