【作者】 孙胜君；

【导师】 南征；

【作者基本信息】 长春中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：糖尿病肾病（DN）是糖尿病常见而严重的微血管并发症，以蛋白尿，肾功能进行性恶化为主要特征，病理变化主要为肾小球滤过率改变，基底膜增厚，系膜细胞增宽，直至肾小球硬化，发展至肾功衰竭。其发病率之高，进展速度之快，严重威胁着人类的健康。糖尿病肾病的发病机制多而复杂，近几年，从蛋白尿产生机制角度入手，对足细胞的研究成为热点。各种原因导致足细胞的损伤参与了蛋白尿的产生，而改善足细胞功能的药物成为治疗糖尿病肾病的关注点和创新点。目前对于糖尿病肾病蛋白尿的治疗，西医多以降糖药、ACEI/ARB类药、改善循环为主，发展至中晚期则直接采用透析疗法维持。而中药在治疗蛋白尿方面取得良好效果，具有西药无法比拟的优势。榛花消肾胶囊是导师常用的治疗糖尿病肾病蛋白尿的方剂，在临床患者中取得满意疗效，本次实验是应用榛花消肾胶囊干预实验性糖尿病大鼠，观察该中药对足细胞相关分子及转分化wnt/β-catenin通路的影响，进一步证明榛花消肾胶囊治疗糖尿病肾病蛋白尿的有效性。方法：1将Wistar大鼠高脂饲料喂养一个月后予链脲佐菌素（STZ）腹腔注射，复制糖尿病大鼠模型，将造模成功大鼠随机分成DM模型组，中药实验组，西药对照组，同时设立正常对照组。中药实验组予榛花消肾胶囊灌胃，西药对照组予罗格列酮合苯那普利做阳性对照。观察大鼠一般状态，检测血糖、血脂、肾功、尿量、尿微量白蛋白等变化。2应用光学显微镜和透视电镜观察肾组织超微结构，观察榛花消肾胶囊对糖尿病大鼠肾脏病理组织结构的改变。3采用免疫组化方法观察足细胞标志性分子nephrin、desmin、zo-1的蛋白表达；应用荧光定量PCR技术检测nephrin、desmin m RNA表达。4采用免疫组化方法观察足细胞转分化wnt/β-catenin通路相关分子wnt-1、β-catenin、sail1蛋白表达；应用荧光定量PCR技术检测wnt-1、sail1m RNA表达。结果：1榛花消肾胶囊能够改善实验性DM大鼠的一般状态以及多饮、多食、多尿、消瘦的临床症状。2榛花消肾胶囊对实验性DM大鼠具有一定的降血糖、降低甘油三酯和胆固醇的作用，改善糖脂代谢。3榛花消肾胶囊能够抑制实验性DM大鼠早期肾脏肥大，减少尿微量白蛋白的排出，降低血肌酐和尿素氮，改善肾功能。4榛花消肾胶囊具有改善实验性DM大鼠肾脏组织的病理形态，减轻肾小球基底膜增厚，细胞外基质的积聚，系膜增生，改善足细胞形态，从而减轻DM性肾小球硬化。5榛花消肾胶囊具有抑制实验性DM大鼠肾组织nephrin、zo-1蛋白及m RNA的表达，增加desmin蛋白及m RNA的表达。6榛花消肾胶囊具有抑制实验性DM大鼠肾组织转分化wnt/β-catenin通路中wnt-1、β-catenin、sail1蛋白及m RNA的表达。结论：榛花消肾胶囊能够改善实验性DM大鼠一般状态以及多饮、多食、多尿、消瘦的症状；抑制早期肾脏肥大，减轻肾组织病理结构；具有降血糖、降低甘油三酯和胆固醇、减少尿微量白蛋白，降低血肌酐和尿素氮的作用。同时，榛花消肾胶囊亦能改善足细胞相关分子nephrin、zo-1、desmin表达，抑制足细胞转分化wnt/β-catenin信号转导通路分子wnt-1、β-catenin、sail1表达。说明榛花消肾胶囊是通过干预足细胞转分化wnt/β-catenin通路而发挥改善糖、脂代谢，减少尿蛋白，改善肾功的作用。导师南征教授认为消渴肾病的病机关键是毒损肾络，邪伏膜原。创造性地将中医学毒损肾络、邪伏膜原理论与西医学糖尿病肾病发病机制，肾小球滤过膜-足细胞结构及功能联系在一起。榛花消肾胶囊在解毒通络、疏利膜原、益肾导邪的治疗大法指导下，通过干预足细胞转分化wnt/β-catenin通路而发挥治疗糖尿病肾病的作用。更多还原

【Abstract】 Propose: Diabetic nephropathy (DN) is a common and serious diabetic complication ofblood vascular. The main features of DN are proteinuria and renal function changes.The mainpathological changes are glomerular filtration change, thickening of the basement membrane,mesangial cells widened, glomerular sclerosis, until the renal failure. Its high incidence andfast progress is a serious threat to human health.The pathogenesis of diabetic nephropathy is more complex. In recent years, for the pointof view of the mechanism of proteinuria, podocyte has become a hot research. All factorsinjury podocyte will be involved in proteinuria, so the drug for improving podocyte functionbecomes hot spot and innovation in treatment of diabetic nephropathy. For the treatment ofdiabetic nephropathy proteinuria, Western medicine mainly adopt degraded blood glucosedrugs, ACEI/ARB drugs, improving circulatory drugs, until dialysis therapy whenintermediate stage or advanced stage. And traditional Chinese medicine in the treatment ofproteinuria has fine effective, which the western medicine can not be matched.ZhenHuaXiaoShen capsule my advisor commonly used is a prescription in the treatment ofdiabetic nephropathy proteinuria, which achieves satisfactory effect in clinical patients. Thisexperiment observes the influence of podocyte correlated molecules and podocyte EMT inwnt/β-catenin signal transduction pathway by ZhenHuaXiaoShen capsule interventionexperimental diabetic rats. And further evidence of ZhenHuaXiaoShen capsule for thetreatment of diabetic nephropathy proteinuria is effective.Method:1Wistar rats being feed with hypsi-lipoids feeder for a month has been intraperitonealinjected with streptozotocin (STZ) for making diabetic rat model. The successful DM rats arerandomly divided into DM model group, the experimental group with traditional Chinesemedicine, the control group with western medicine, and normal control group.ZhenHuaXiaoShen capsule has been used in the experimental group, while rosiglitazone andbenazepril has been used together in the control group. Observed rats the general state, testingthe blood glucose, blood lipids, renal function, urine output and urine micro albumin.2Observed renal ultrastructure by optical microscope and perspective electronmicroscopy, and analyzed the renal organism pathological structure changes for ZhenHuaXiaoShen capsule intervention experimental diabetic rats.3Observed the protein expression of podocyte signal molecule，like nephrin, desmin,zo-1by immunohistochemical method, and m RNA expression of nephrin, desmin byfluorescent quantitative PCR methods.4Observed the protein expression of podocyte EMT related molecules, like wnt-1、β-catenin、snail1in wnt/β-catenin signal transduction pathway by immunohistochemicalmethod, and m RNA expression of wnt-1, snail1by fluorescent quantitative PCR.Result:1ZhenHuaXiaoShen capsules can improve the experimental DM rats the general stateand clinical symptoms, such as polydipsia, polyphagia, polyuria and athrepsy.2ZhenHuaXiaoShen capsules can degrade the experimental DM rats blood glucose,triglyceride, cholesterol in a certain degree, and improve glycometabolism and lipidmetabolism.3ZhenHuaXiaoShen capsule can inhibit the experimental DM rats the early renalhypertrophy, reduce urinary albumin discharge, degrade serum creatinine and blood ureanitrogen, improve renal function.4ZhenHuaXiaoShen capsules improve the experimental DM rats nephridial tissuepathology, reduce the thickening of the glomerular basement membrane, extracellular matrixaccumulation, mesangial hyperplasy, improve podocyte morphous, thereby reducing DMglomerulosclerosis.5ZhenHuaXiaoShen capsule can inhibit the experimental DM rats nephridial tissueprotein and m RNA expression of nephrin and zo-1, increase protein and m RNA expressionof desmin.6ZhenHuaXiaoShen capsule can inhibit the experimental DM rats nephridial tissueprotein and m RNA expression of wnt-1, β-catenin and sail1in wnt/β-catenin signaltransduction pathway.Conclusion：ZhenHuaXiaoShen capsules can improve the experimental DM rats the general state andclinical symptoms of polydipsia, polyphagia, polyuria and athrepsy; inhibit the early renalhypertrophy and relieve the renal pathological structure; degrade blood glucose, triglyceride,cholesterol, reduce urinary albumin discharge, degrade serum creatinine and blood urea nitrogen. ZhenHuaXiaoShen capsules can also improve podocyte correlated proteinexpression of nephrin, zo-1and desmin, inhibit the expression of wnt-1, β-catenin and sail1in podocyte EMT in wnt/β-catenin signal transduction pathway. Therefore,ZhenHuaXiaoShen capsules can improve glycometabolism and lipid metabolism, reduceurine protein, and protect renal function by the way of intervention in podocyte EMT in wnt/β-catenin signal transduction pathway.My advisor NanZheng professor considers that the pathogenesis key of diabeticnephropathy is poison damaged renal meridian and hidden in pleurodiaphragmatic spaces.Creatively associate two theory of the traditional Chinese medicine theory of poison damagedrenal meridian and hidden in pleurodiaphragmatic spaces and western medicine diabeticnephropathy pathogenesis, glomerular filtration membrane-structure and function ofpodocytes together. ZhenHuaXiaoShen capsule could treat diabetic nephropathy in the way ofdetoxification-deoppilation meridian and pleurodiaphragmatic spaces-tonifying kidney-eliminate pathogen by intervention podocyte EMT in wnt/β-catenin signal transductionpathway.更多还原