【作者】 王月华；

【导师】 陈志强；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的:糖尿病肾病(diabetic nephropathy,DN)是糖尿病(diabetic melliutus,DM)常见而严重的微血管并发症,是西方国家终末期肾病(end stage renal disease,ESRD)及进行肾替代治疗的最主要原因。在我国DN在ESRD中的比例也逐渐上升,预计在未来的20年中DN将成为终末期肾衰竭最重要的原因。因此探讨DN的发病机制,寻求延缓DN进展的治疗方法具有重要意义。由于众多生物活性物质参与了糖尿病肾病发病过程,具有多途径、多环节相互作用的复杂病理生理机制,其防治一直是临床难点。西医在治疗DN上主要是控制高血糖、控制高血压、低蛋白饮食及纠正脂代谢紊乱等对症治疗,至今尚没有一个能完全阻断DN进展的药物。而中药复方治病的特点就是通过多途径、多靶点实现治疗目的,故应用中药复方防治DN具有很大的潜力。在前期临床实践中,我们根据中医学病机理论,结合糖尿病肾病的临床特征、发病机制及其病理特点,逐步认识到该病的病情虽然复杂,但在早期和中期大多以气阴两虚、瘀血阻络、癥瘕积聚为其基本病机,治疗当以益气养阴、化瘀软坚、消癥通络为主,为此我们结合多年的临床用药经验,拟定了以益气养阴、消癥通络为主的中药复方制剂,主要药物有黄芪、玄参、鳖甲、地龙等。用该方在临床上治疗早中期的糖尿病肾病,取得了良好的临床疗效。为了进一步证实该方的治疗作用,探讨其可能的机制,进行了本项研究。即借助糖尿病肾病动物模型,观察其对糖尿病肾病大鼠干预作用及对TGFβ/Smad信号传导系统的影响。方法:1益气养阴消癥通络中药对糖尿病肾病大鼠干预作用健康雄性SD大鼠46只,适用性饲养一周。按体重随机分为正常组(Normal)10只,造模组36只。除正常组外,造模组大鼠一次性腹腔注射链脲佐菌素(STZ) 60 mg/kg,正常组注射相应量生理盐水,72小时后测血糖,以≥16.7mmol/L为DM模型成模标准。造模组在DM模型成模后,随机分为模型组(Model)、贝那普利组(Benazepril)、中药(ZY)组各12只。成模一周后开始给药,正常组、模型组每天每只大鼠2毫升生理盐水灌胃,治疗各组按成人剂量的20倍给药,即贝那普利组予贝那普利10mg/kg.d灌胃,中药组予中药煎剂26g/kg.d灌胃,每日一次,连续给药23周。于治疗开始后23周末收集24小时尿液,杀检动物,留取血、肾组织,测定实验动物的24小时尿蛋白定量、糖化血红蛋白、血脂、肾功能、观察肾组织超微结构及肾小球硬化指数的变化。2益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ及其受体的影响同上方法复制糖尿病肾病大鼠模型,于成功复制糖尿病肾病模型24周末留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠Ⅳ型胶原(Col-Ⅳ)、TGFβ1及TGFβΙ型受体(Transforming growth factor-beta TypeⅠReceptor, TβR-Ⅰ)、Ⅱ型受体(Transforming growth factor-beta TypeⅡReceptor, TβR-Ⅱ)的影响。3益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ/Smad信号传导系统的影响同上方法复制糖尿病肾病大鼠模型,于成功复制糖尿病肾病模型24周末留取肾组织标本,观察益气养阴消癥通络中药对DN大鼠smad2、smad3和smad7的影响,进一步探讨其对糖尿病肾病可能的作用机制,结果:1益气养阴消癥通络中药治疗糖尿病肾病大鼠的干预作用1.1各组大鼠24h尿蛋白定量的比较24h尿蛋白定量,模型组、贝那普利组、中药组与正常组比较均明显升高(P<0.01),贝那普利组、中药组与模型组比较明显降低(P<0.01),而贝那普利组与中药组之间无显著差异(P>0.05)。1.2各组大鼠糖化血红蛋白(HbA1C)的比较模型组、贝那普利组、中药组大鼠糖化血红蛋白与正常组相比明显升高(P<0.01),而模型组、贝那普利组、中药组之间无显著性差异(P>0.05)。1.3各组大鼠血脂的比较模型组、贝那普利组、中药组血清总胆固醇(TC)明显高于正常组(P<0.01),中药组则比模型组明显偏低(P<0.01),模型组与贝那普利组之间比较无显著性差异(P>0.05)。甘油三酯(TG),两治疗组与正常组均明显低于模型组(P<0.01),而两治疗组与正常组之间无显著性差异(P>0.05)。1.4各组大鼠肾功能的比较两个治疗组与正常组的血肌酐(Scr)明显低于模型组(P<0.01),贝那普利组与正常组相比明显升高(P<0.05),而中药组与正常组之间无显著性差异(P>0.05);尿素氮(BUN),两治疗组与正常组均明显低于模型组(P<0.01),而与正常组比较,贝那普利组和中药组的BUN则明显升高(P<0.05),两治疗组之间则无显著性差异(P>0.05)。1.5肾脏病理形态学观察光镜:正常组肾小球结构完整,未见肾小球肥大,肾小球毛细血管基底膜、系膜、基质未见明显异常改变;模型组可见肾小球肥大,肾小球毛细血管基底膜增厚,肾小球系膜细胞增生、系膜基质增多,系膜区增宽;部分肾小管发生空泡变性,肾小管萎缩,透明管型形成;贝那普利组与中药组也均有不同程度病理改变,但与模型组相比病变减轻。电镜:正常组肾脏组织结构正常,肾小球基底膜均匀无增厚,上皮足突分布均匀,模型组大鼠肾小球毛细血管基底膜高度增厚,大部分呈匀质样增厚,部分增生呈丘状隆起;足细胞足突广泛融合;内皮细胞高度融合,窗口大部分消失。贝那普利组与中药组大鼠肾小球毛细血管基底膜较正常呈匀质样增厚,足细胞局部融合,内皮细胞部分融合,窗口部分消失。各组肾小球硬化指数比较:与正常组相比,模型组和两个治疗组的肾小球硬化指数要明显升高(P<0.05),与模型组比较,贝那普利组和中药组要明显降低(P<0.05),两治疗组比较,贝那普利组高于中药组(P<0.05)。2益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ及其受体的影响2.1 Col-Ⅳ免疫组化检测结果正常组Col-Ⅳ在系膜基质及基底膜及包曼氏囊有基础表达。与正常组相比,模型组Col-Ⅳ表达的范围和强度明显扩大和增强(P<0.01);而中药组、贝那普利组与模型组相比,其表达显著下降(P<0.01)中药组与贝那普利组比较无显著性差异(P>0.05)。2.2对大鼠肾组织TGF-β1蛋白及其基因表达的影响免疫组化染色显示:TGF-β1在肾小球上皮细胞及系膜区,毛细血管袢,肾小囊壁层上皮细胞,肾小管上皮细胞胞浆以及小管间质均呈弱阳性表达,半定量分析结果显示:与正常组相比,模型组和两个治疗组的表达要明显加强(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显降低(P<0.05),两治疗组比较,贝那普利组高于中药组(P<0.05)。RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-β1表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。2.3对大鼠肾组织TGF-βΙ型受体蛋白表达及基因表达的影响免疫组化结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βΙ型受体表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βΙ型受体表达则明显降低(P<0.01);两治疗组比较,贝那普利组高于中药组(P<0.05)。2.4对大鼠肾组织TGF-βⅡ型受体蛋白表达及基因表达的影响免疫组化染色显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βⅡ型受体表达则明显降低(P<0.01);贝那普利组和中药组比较则无明显差异(P>0.05)。RT-PCR结果显示:与正常组相比,模型组和各治疗组表达明显升高(P<0.01),而与模型组比较,贝那普利组和中药组大鼠肾组织TGF-βⅡ型受体表达则明显降低(P<0.01);贝那普利组和中药组比较则无明显差异(P>0.05)。3益气养阴消癥通络中药对糖尿病肾病大鼠TGFβ/Smad信号传导系统的影响3.1免疫组化检测大鼠肾组织p-Smad2/3蛋白表达的结果与正常组相比,模型组和各治疗组阳性增强的核占小管细胞的百分比显著增加(P<0.05),与模型组比较,贝那普利组和中药组的表达明显降低(P<0.05),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。3.2 Western印迹检测大鼠肾组织Smad7蛋白表达的结果:与正常组相比,模型组和各治疗组的表达要明显降低(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显上升(P<0.01),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。3.3 RT-PCR检测肾组织Smad2mRNA和Smad7mRNA表达结果3.3.1Smad2mRNA表达结果:与正常组相比,模型组和各治疗组Smad2mRNA显著增加(P<0.05),与模型组比较,贝那普利组和中药组的表达明显降低(P<0.05),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。3.3.2Smad7mRNA表达结果:与正常组相比,模型组和各治疗组的表达要明显降低(P<0.05),与模型组比较,贝那普利组和中药组的表达要明显上升(P<0.01),贝那普利组和中药组之间的比较则无明显差异(P>0.05)。结论:1.益气养阴消癥通络中药能改善DN大鼠一般状况,减少尿蛋白的排泄,降低血脂,改善大鼠的肾功能。2.益气养阴消癥通络中药可减轻减轻肾组织病理损害,从而延缓DN的进展。3.益气养阴消癥通络中药可降低糖尿病肾病大鼠IV型胶原合成,从而阻止肾小球硬化的发生和发展。4.益气养阴消癥通络中药能明显降低DN大鼠肾组织中TGF-β1及TGF-βⅠ、Ⅱ型受体蛋白及基因的表达,说明该药可能通过降低肾组织TGF-β1及TGF-βⅠ、Ⅱ型受体蛋白及基因的表达而达到抗纤维化、保护肾功能的作用。5.益气养阴消癥通络中药能够抑制受体活化性蛋白smad2和smad3的表达,促进抑制性蛋白Smad7的表达,从而抑制DN大鼠肾脏中TGF-β/smad信号传导通路的激活,减少大鼠肾脏ECM沉积,从而延缓DN的进程。更多还原

【Abstract】 Objectives: Diabetic nephropathy(DN)is a part of micovasclor complication and the most important reason of end stage renal disease. The researchers keep working on the reason and treatment of DN. It is difficult to prevent and cure DN because numerous biotic activator participate in the process of DN. The medical doctor give the DN patients only with symptomatic treatment such as control hyperglycosemia and hypertension, low-protein diet and so on. The traditional Chinese herbs play an important role in preventing DN because of its advantage. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of herbs of supplement qi and nourish yin resolve masses and dredge meridians(ZY) and investigate the mechanism in TGFβ/Smad signal transduction system in DN rats. All the above that have been done was to provide the foundation in order to generalization and exploitation.Motheds:1 Effect of ZY on the treatment in DN rats:Forty-six male Sprague-Dauley rats were selected in this experiment. According to the weight, the animals were randomly divided into normal group(10 rats)and DM model group(36 rats). Besides the normal group, all the rats were injected with streptozotocin (STZ) in the dose of 60 mg/kg, the normal group were given the corresponding quantity’s physiological saline. After 72 hours, measured their blood glucose, took“≥16.7mmol/L”as the DM model standard. According to the blood glucose level, the rats of DM model were randomly divided into three groups: 12 in the model group, 12 in benazepril group and 12 in ZY group. After one week of DM models were made successfully, the drugs started be given to the rats according to adult dosage 20 times, once daily. The rats of ZY group were given the ZY by gavage, 26g/kg, the benazepril group were administrated with benazepril by gavage, 10mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 23 weeks. After 24 week of DM models made successfully, all rats were put into metabolism cage to obtain 24 hour urine. All animals were killed and the blood and the renal tissue were obtained. 24 hour urinary protein quantitative, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr), urea nitrogen (BUN) were assayed with the automaticbiochemistry analyzer. Pathomorphology change and the glomerulosclerosis index were observed.2 Effect of ZY on TGFβand receptor in DN rats:DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The Col-Ⅳ、transforming growth factor-beta , transforming growth factor-beta typeⅠreceptor, transforming growth factor-beta typeⅡreceptor were measured by immunohistochemical method and RT-PCR.3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats:DM model was duplicated the same as the above.After 24 week of DM models made successfully, the renal tissue were obtained. The smad2、smad3 and smad7 were measured by immunohistochemical method and RT-PCR.Results:1 Effect of ZY on the treatment in DN rats:1.1 The 24 hour urinary protein quantitative of each groupThe 24 hour urinary protein quantitative of the rats in model group, benazepril group and ZY group was significantly higher than that of normal group (P <0.01), compared with model group, the 24 hour urinary protein quantitative of benazepril group and ZY group was obviously lower (P <0.01). There was no difference between benazepril group and ZY group (P >0.05).1.2 The HbA1C of each groupCompared with normal group, the HbA1C of the rats in model group, benazepril group and ZY group was significantly higher (P <0.01), but there was no difference among model group, benazepril group and ZY group (P >0.05).1.3 The TC and TG of each groupThe TC of the rats in model group, benazepril group and ZY group was significantly higher than that of normal group (P <0.01), compared with model group, the TC of ZY group was obviously lower (P <0.01), there was no difference between benazepril group and model group (P >0.05). The TG of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P <0.01), there was no difference among normal group, benazepril group and ZY group (P>0.05).1.4 The Scr and BUN of each groupCompared with model group, the Scr of the rats in normal group, benazepril group and ZY group was significantly lower (P<0.01), and benazepril group was obviously higher than that of normal group (P<0.05). There was no difference between normal group and ZY group (P>0.05). The BUN of the rats in normal group, benazepril group and ZY group was significantly lower than that of model group (P<0.01). Compared with normal group, the BUN of benazepril group and ZY group was significantly higher (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).1.5 The pathomorphology change of kidneyObservations under light microscope:In the normal group: the structure of renal glomerulus was integrated, glomerular capillary basement membrane, mesenterium and base material were normal. In the model group: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial cell proliferation, mesangial matrix increased, mesangial area broadening and tubulointerstitial fibrosis were seen. vacuolar degeneration, analosis and hyaline cast were seen in some renal tubule. The benazepril group and ZY group also showed different degree of pathological changes, but the pathological degree was obviously lighter than the changes of the model group.Observations under transmission electron microscopy: In normal group: the structure of glomerular capillary basement membrane was clear and complete, microcirculatary endothelial cell, foot processes was normal. In the model group: most of the glomerular capillary basement membrane showed significant homogeneous thicking, partial basement membrane showed elevation in the shape of hills; microcirculatary endothelial cell significantly confluence and the most of window structure disappeared; the fusion of the foot processes was extensive. The benazepril group and ZY group also showed the similar pathological changes, but the pathological degree was lighter than that in model group.The glomerulosclerosis index in each group: The glomerulosclerosis index in model and treatment groups were higher than that in normal group (P<0.05);Compared with model group, the glomerulosclerosis index in benazepril group and ZY group was significantly lower (P<0.05), and the glomerulosclerosis index in benazepril group was higher than that in ZY group(P<0.05).2 Effect of ZY on TGFβ1 and receptor in DN rats2.1 Expression of CollegeⅣexamined by immunohistochemistry:In normal group, Col-Ⅳhas the basal expression. Compared with normal group, the expression of Col-Ⅳin model group was stronger(P<0.01). Compared with model group, the expression of Col-Ⅳin benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).2.2 Expression of TGFβ1 in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).The results detected by RT-PCR showed: The expression of TGFβ1 in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβ1 in benazepril group and ZY group was significantly decreased (P<0.01), and the TGFβ1 in benazepril group was higher than that in ZY group(P<0.05).2.3 Expression of TGFβtypeⅠreceptor in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).The results detected by RT-PCR showed: The expression of TGFβtypeⅠreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅠreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and the TGFβtypeⅠreceptor in benazepril group was higher than that in ZY group(P<0.05).2.4 Expression of TGFβtypeⅡreceptor in renal tissueThe results detected by immunohistochemistry showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P<0.01), and there was no difference between benazepril group and ZY group (P>0.05).The results detected by RT-PCR showed: The expression of TGFβtypeⅡreceptor in model and treatment groups were higher than that in normal group (P<0.01);Compared with model group, the expression of TGFβtypeⅡreceptor in benazepril group and ZY group was significantly decreased (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).3 Effect of ZY on TGFβ/Smad signal transduction system in DN rats3.1 Expression of p-Smad2/3 protein in renal tissueCompared with normal group, the expression of p-Smad2/3 in model group was stronge(rP<0.01). Compared with model group, the expression of p-Smad2/3 in benazepril group and ZY group was significantly lighter (P <0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.2 Expression of Smad7 protein in renal tissue by WesternblottingCompared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.3 Expression of Smad2 and Smad7mRNA by3.3.1 Smad2mRNACompared with normal group, the expression of Smad2 in model group was stronger(P<0.01). Compared with model group, the expression of Smad2 in benazepril group and ZY group was significantly lighter (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).3.3.2 Smad7mRNACompared with normal group, the expression of Smad7 in model group was lower(P<0.01). Compared with model group, the expression of Smad7 in benazepril group and ZY group was significantly stronger (P<0.01), and there was no difference between benazepril group and ZY group (P >0.05).Conclusions:1. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased the DN rat’s proteinuria, improve the lipid metabolism, decrease Scr and BUN2. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could lessen the damage of nephridial tissue.3. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decrease the ColIV deposition in order to slow down the progress of glomerulosclerosis.4. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could decreased TGF-β1and receptorⅠ、Ⅱwhich could resist renal fibrosis and protect renal founction.5. Herbs of supplement qi and nourish yin resolve masses and dredge meridians could inhibit the expression of smad2, smad3 and promote the expression of smad7which could delay the process of DN.更多还原