【作者】 王镁；

【导师】 于世家；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2008， 博士

【摘要】 第一部分大黄酸对肥胖糖尿病大鼠的肾脏保护作用背景与目的糖尿病肾病(diabetic nephropathy,DN )是糖尿病(diabetes mellitus, DM)常见的慢性并发症之一,在糖尿病患者中20–40%会发生DN,在欧美,DN是引发终末期肾病(end stage renal disease,ESRD)的首要原因。其发病机制虽未完全阐明,但糖代谢紊乱引起多元醇通路、蛋白激酶C、晚期糖基化产物、己糖胺4种途径的激活、肾脏血流动力学改变、氧化应激、多种细胞因子以及遗传背景均起重要作用。近年研究认为,氧化应激在DN的发生、发展中起着重要作用,是DM微血管和大血管并发症病变发生的共同病理生理通路。中药大黄及其复方制剂在控制DN症状及延缓病情发展方面显示了很好的疗效,大黄酸(4,5dihydroxyanthraquinone-2-carboxylicacid,Rhein)为大黄的有效成分,在治疗DN的研究中已有一定的进展。研究资料显示,大黄酸除改善糖脂代谢外,有减少尿白蛋白排泄量,预防血肌酐水平的升高,改善肾小球肥大、基质增生,使肾组织转化生长因子β(transforming growth factor-β,TGF-β)以及纤维粘连蛋白(fibronectin,FN)等在肾脏的表达下调,抑制肾间质成纤维细胞的增殖等作用。但大黄酸是否具有抗氧化作用迄今未见报道。本实验旨在进一步验证大黄酸对肥胖糖尿病大鼠糖脂代谢、尿蛋白排泄的影响,并观察其对大鼠肾脏氧化应激的作用,揭示大黄酸防治DN的机制。方法肥胖糖尿病大鼠用高糖高脂饮食加链脲佐菌素(Streptozotocin,STZ)诱导,造模大鼠予高糖高脂饲料喂养8周后单次腹腔内注射2% STZ 25mg/kg,72小时后,取尾静脉血用快速血糖仪测定血糖,血糖﹥16.7 mmol/L,体重>395g者列入观察对象。将大鼠随机分5组,雷米普利组(DM-RA)予雷米普利1mg·kg-1·d-1灌胃;大黄酸组(DM-RH)予大黄酸100mg·kg-1·d-1灌胃;吡格列酮组(DM-PI)予盐酸吡格列酮10mg·kg-1·d-1灌胃;正常对照组(NC)和糖尿病模型组(DM)予生理盐水10ml·kg-1·d-1灌胃。各组大鼠均每天给药一次至实验结束,8周时留取24小时尿液测定24 h尿蛋白定量,用20%乌拉坦溶液腹腔内注射麻醉大鼠,留取血标本及肾组织,检测血糖、血脂、肾皮质超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(malondialdehyde, MDA)含量等的变化。结果采用高糖高脂加STZ饮食诱导的糖尿病大鼠血糖、体重、总胆固醇(cholesterol,CHOL)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol ,LDL-C)、24小时尿蛋白定量均明显高于正常对照组(P <0.01),治疗8周后大黄酸组、吡格列酮组血糖低于糖尿病模型组(P <0.01),且比治疗前明显下降(P <0.01),说明大黄酸与吡格列酮均有一定的控制血糖作用;大黄酸组与治疗前比较体重明显下降(P <0.05), CHOL、TG、LDL低于糖尿病模型组(P <0.01);大黄酸组、雷米普利组、吡格列酮组24 h尿蛋白量均显著低于糖尿病模型组(P <0.01);与正常对照组比较,糖尿病大鼠MDA含量明显增加,SOD活性明显降低(P <0.01);大黄酸治疗后肾组织MDA含量明显降低,SOD活性明显增加(P <0.05)。结论大黄酸具有改善糖脂代谢异常,减少尿蛋白排泄、抗氧化应激的作用,这可能是其预防和延缓DN进程的机制之一。第二部分大黄酸对肥胖糖尿病大鼠肾皮质过氧化物酶体增殖体激活受体γ表达的影响背景与目的过氧化物酶体增殖体激活受体(peroxisome proliferators activated-receptors, PPARs)是一类配体依赖性转录因子,属于核受体超家族成员,是调节脂肪活化因子转录、脂质代谢、炎症反应、细胞增殖和分化的重要因子,PPARs有三种亚型,即PPARα、PPARβ/δ和PPARγ,PPARγ及其配体在DN发病和治疗中的作用日益受到重视。大量的实验研究表明,激活PPARγ能减少蛋白尿及减轻肾组织的损伤,显著抑制系膜细胞的增殖反应、细胞外基质(extra cellular matrix, ECM)的分泌表达以及多种炎性细胞因子的分泌。临床研究也证明应用PPARγ激动剂可明显降低糖尿病患者尿白蛋白的排出,使尿白蛋白/肌酐比值(ACR)显著下降; TGF-β是DN复杂的细胞因子网络中的核心因子,是肾脏损伤的关键致病因子。具有促使肾脏固有细胞肥大、刺激ECM分子合成的作用,在DN发病机制的各个环节起着重要作用。有研究表明,激活PPARγ可下调TGF-β1、FN、Ⅳ型胶原、纤溶酶原激活物抑制因子-1的表达,达到肾脏保护作用。DM时肾脏PPARγ的表达报道不一,且大黄酸对肾脏PPARγ表达的影响也未有报道。本实验通过观察肥胖糖尿病大鼠肾脏PPARγ的mRNA和蛋白表达,以及TGF-β1的mRNA表达及大黄酸对二者表达的影响,试图进一步揭示大黄酸防治DN的机制。方法造模、分组、给药、处死方法等同第一部分实验,取血完毕后,经生理盐水反复灌洗至肾脏转为苍白色,迅速取肾,保存于-70℃冰箱中。采用免疫组化(immunohistochemistry,ICH)、逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)、蛋白印迹(western blotting)等方法,观察各组大鼠肾皮质PPARγ基因与蛋白表达和TGF—β1基因表达的影响。结果正常对照组大鼠肾皮质中PPARγmRNA有一定的基础表达,高糖高脂加STZ诱导的糖尿病大鼠肾皮质中PPARγmRNA表达和蛋白表达与正常对照组相比明显增加(P <0.01),经大黄酸和吡格列酮治疗8周后,PPARγ的表达量进一步升高(P <0.05,P <0.01)。免疫组化结果与RT-PCR和Western blotting结果相似。正常对照组大鼠肾皮质TGF-β1呈低水平表达,糖尿病模型组大鼠TGF-β1呈高表达(P <0.01),而大黄酸组、雷米普利组、吡格列酮组TGF-β1表达水平均低于糖尿病模型组(P <0.01)。结论肥胖糖尿病大鼠肾皮质PPARγ和TGF-β1的表达增加,大黄酸治疗可明显提高PPARγ的表达,下调TGF-β1的表达,大黄酸通过激活PPARγ从而下调TGF—β1可能是大黄酸防治DN的另一机制。更多还原

【Abstract】 PARTⅠ: To investigate the protective effect of Rhein on kidneys in obese diabetic rats.Background and objective Diabetic nephropathy(DN) is one of the most common complications of diabetes mellitus(DM), its incidence in DM is 20–40%.It is the leading cause of end-stage renal disease in the west world. The pathophysiological mechanism responsible for DN has not been fully understood yet. There are some important factors such as renal polyalcohol pathway, advanced glycation end products(AGEs), activation of Protein Kinase C,oxidative stress, hexosamine , renal hemodynamic change , many kinds of cytokines and genetic background and so on. Recent studies show that oxidative stress plays a role in the occurrence and development, it was the common physiological and pathological pathway of the diabetic complication of the great vessels and micrangium. Rhubarb and its compound preparation are effective on controlling the symptom of DN and delaying the progress of disease. Rhein as the effective ingredient of Rhubarb has major progress in the research of the treatment of DN. Reseach data demonstrates that Rhein can reduce the urinary albumin, prevent the elevation of SCr, relieve the glomerular sclerosis and improve the glucolipid metabolism. But up to now there is no report whether Rhein has antioxidant function. This experiment aims to confirm the effect of Rhein on glucolipid metabolism and urinary albumin excretion in Obese Diabetic Rats, to observe antioxidation of Rhein on renal, and to investigate the possible mechanism of Rhein preventing and delaying diabetic nephrophy.Methods Male Wistar rats were fed with the diets enriched with sucrose (20%) and lard (10%) and cholesterol (2.5%) and cholic acid (1%).Hyperglycemia was developed by intraperitoneal injection in these rats with 25 mg/kg streptozotocin (STZ) after 8 weeks on the diets.All rats were devided into 5 groups randomly: normal control group (NC),diabetic group(DM), diabetic rats receiving Rhein(DM-RH), diabetic rats receiving Ramipril(DM-RA) and diabetic rats receiving pioglitazone(DM-PI). After administration by gavage for 8 weeks,blood glucose, body weight, blood fat, 24h urinary protein excretion,the content of malondialdehyde (MDA) and superoxide dismutase(SOD) of the cortex renis were measured.Results After modeling the following indexes including blood glucose, body weight, Cholesterol(CHOL),Triglyceride(TG),High density lipoprotein-cholesterol (HDL-C),Low density Lipoprotein Cholesterol (LDL-C) and 24h urinary protein excretion were increased distinctly. After 8 weeks, compared with DM, the glucose of DM-RH and DM-PI was decreased markedly(P < 0.01), the glucose was decreased obviously after the treatment(P <0.01), and the group comparison was no significant difference(P >0.05);CHOL,TG,LDL of DM+RH was lower than DM(P <0.01), but DM-RA and DM-PI not(P >0.05); The 24h urinary protein excretion of DM-RA, DM-PI and DM-RH was obviously lower than DM(P <0.01), DM-RA was superior to DM-PI(P <0.05),but the group comparison between DM-RA and DM-RH was no significant difference(P >0.05).Compared with NC, the level of MDA of DM,DM-RA, DM-PI and DM-RH was increased significantly(P <0.01), the activity of T-SOD was decreased(P <0.01). After the treatment, The level of MDA of DM-RA, DM-PI and DM-RH was increased significantly(P <0.05), the activity of T-SOD was decreased(P <0.05,P <0.01).Conclusion Rhein can control glucose, adjust blood fat, reduce the urinary protein excretion. The antioxidation may be one of the possible mechanisms that Rhein can protect and remitte diabetic nephropathy.PARTⅡ: To investigate the effect of Rhein on the expression of peroxisome proliferators-activated receptors-γ(PPARγ) and transforming growth factor-β1 (TGF-β1) in the cortex renis of the obese diabetic rats.Background and objective Peroxisome proliferators-activated receptors (PPARs) are member of the nuclear receptor superfamily of ligands-dependent transcription factors. These receptors are important to regulate lipid-activated gene transcription, lipid metabolism, inflammation and cell proliferation and differentiation of the cells. and its ligand have been attracted enormous attention due to the key role these receptors play in treatment of DN. Growing laboratorial evidence points to activated PPARγcan reduce the urinary protein and the injury of the nephridial tissue, and inhibit the proliferation of the mesangial cells, the expression of extracellularmatrix secretion and secretion of the inflammatory cytokines. Clinical research demonstrates that PPARγactivator can reduce obviously diabetic urinary protein excretion, urinary protein excretion/creatinine(ACR). TGF-β1 is the kernel factor of the complicate DN cytokine network, and pivotal causative agent due to the renal injury. TGF-β1 has important effects in the pathogenesis of DN such as inducing the proliferation and hypertrophy of the renal cells and stimulating the molecular synthesizing of the ECM. Some research show that activated PPARγcan protect kidney due to reducing the genic expression of TGF-β1, plasminogen activator inhibitor-1 (PAI-1), fibronectin(FN) and typeⅣcollagen. There are different reports about the expression of PPARγin diabetic nephropathy. And there is no report about the effect of Rhein on the PPARγexpression of kidney. In this experiment, we observe the expression of PPARγand TGF-β1 in the cortex renis of the obese diabetic rats, and the effect of Rhein , to reveal the multiplicitas mechanisms that Rhein can protect and remitte diabetic nephropathy.Methods The methods of modeling, diving group, administration, and so on are the same to Part I. We observed that the effect of the Rhein on the expression of PPARγand TGF-β1 in the cortex renis of the obese diabetic rats induced by streptozotocin , high glucos and fat diet with the method of immune histochemistry, RT-PCR and western blot, and investigate the multiple mechanism of the prevention to DN of the Rhein.Results Compared with normal control group, the expression of PPARγin the rats of diabetic group(DM), diabetic rats receiving Rhein(DM-RH) and diabetic rats receiving pioglitazone(DM-PI) were increased markedly(P <0.01,P <0.05).The expression of PPARγin the rats of DM receiving Rhein(DM-RH) and diabetic rats receiving pioglitazone(DM-PI) were increased markedly than those in the rats of diabetic group(DM)(P <0.01,P <0.05).There was no significant difference between DM and diabetic rats receiving Ramipril(DM-RA) (P >0.05). The expression of TGF-β1mRNA of the cortex renis in diabetic group(DM) was increased than those in normal control group(NC)(P <0.01).The expression of TGF-β1mRNA of the cortex renis in diabetic group(DM) was decresed markedly than those in DM+RH, DM+RA and DM+PI(P <0.05).Conclusion Rhein can increase the mRNA and protein expression of PPARγ, and reduce the expression of TGF-β1, Rhein can activate PPARγand reduce the function of TGF-β1. It may be another possible mechanism that Rhein can protect and remitte diabetic nephropathy.更多还原