【作者】 马瑞霞；

【导师】 关广聚；

【作者基本信息】 山东大学 ， 内科学肾脏病， 2009， 博士

【摘要】 第一部分高脂高糖喂养联合低剂量键脲佐菌素诱导的T2DM大鼠模型及其肾病特点研究背景和目的:糖尿病肾病（DN）是导致终末期肾脏疾病（ESRD）的主要原因,在这些糖尿病（DM）患者中,大部分是2型糖尿病（T2DM）,DM动物的研究目前多侧重于大剂量链脲佐菌素(STZ,50～65 mg.kg^-1)诱导的1型DM大鼠。因此,建立与人类T2DM发病特点类似的动物模型非常重要。但临床实验中T2DM模型建立方法不一,其特点也不尽相同,本研究以高脂高糖喂养联合小剂量链脲佐菌素诱导建立T2DM大鼠模型,旨在探讨其可行性及其肾病特点。方法:Wistar大鼠高脂高糖喂养8周后,腹腔注射STZ(30mg.kg^-1)诱导建立T2DM模型,观察体重（BW）、肾重（KW）、空腹血糖（FBG）、空腹胰岛素（INS）、收缩压（SBP）、血脂、白蛋白排泄量（UAL）、肾功能,并行光镜及电镜观察肾脏结构改变。Wistar大鼠喂养常规饲料,作为对照组,实验期间不注射胰岛素。结果:高脂高糖喂养联合小剂量STZ诱导的DM大鼠的BW、FBG、胆固醇（CH）、甘油三酯（TG）、低密度脂蛋白（LDL）、INS及SBP较对照组明显增高,高密度脂蛋白（HDL）及胰岛素敏感指数（ISI）下降。造模成功8周后,肾肥大指数（KW/BW）、UAL、尿素氮（BUN）、肌酐（Scr）、肾小球截面积、基质面积、基质面积比均较正常对照组升高,而毛细血管面积比下降,电镜下肾脏超微结构明显受损。结论:高脂高糖喂养联合小剂量STZ诱导建立的DM大鼠符合T2DM发病特点及DM肾病病理改变。第二部分雷公藤甲素联合厄贝沙坦对T2DM大鼠肾足细胞的影响及其机制探讨研究背景和目的:糖尿病肾病（DN）是糖尿病（DM）最常见的慢性并发症之一,也是导致终末期肾脏疾病（ESRD）的主要原因。新近研究表明,DN是一种代谢紊乱诱导的炎症性疾病,其中巨噬细胞浸润是DN炎症的特征性表现之一,也是DN发生、发展的中心环节。DN肾组织炎症与肾小球滤过屏障通透性改变及蛋白尿形成密切相关。足细胞位于滤过屏障的外层,其损伤参与了蛋白尿形成等DN早期病理进程。研究表明,免疫抑制剂—雷公藤多甙可减轻DM大鼠及患者尿蛋白排泄,但对其机制未作进一步研究。本研究进一步探讨雷公藤多甙的有效组分—雷公藤甲素（Triptolide）是否通过足细胞的保护作用减轻DN大鼠尿白蛋白排泄,并探讨其可能机制。观察雷公藤甲素联合厄贝沙坦对T2DM大鼠足细胞的影响,探讨雷公藤甲素对DN肾脏保护作用的机制。方法:高脂高糖喂养8周联合小剂量STZ(30mg.kg^-1)建立T2DM大鼠模型,将DM模型大鼠随机分为4组:雷公藤甲素治疗组（DT）、厄贝沙坦治疗组（DI）、雷公藤甲素联合厄贝沙坦治疗组（DTI）及模型对照组（DM）;另设正常对照组（NC）。治疗各组分别药物干预8周后,观察大鼠体重（BW）、肾重（KW）、肾肥大指数（KW/BW）、尿白蛋白排泄量（UAL）、血糖、血清尿素氮（BUN）、肌酐（Scr）、内生肌酐清除率（Ccr）、总胆固醇（CH）、甘油三酯（TG）变化;利用光镜、电镜观察肾脏病理改变;应用免疫组化技术观察足细胞相关蛋白Nephrin、Podocin及其相关细胞因子骨形态发生蛋白（BMP-7）、结缔组织生长因子（CTGF）、转化生长因子-β₁（TGF-β₁）在肾组织分布与表达;采用实时定量PCR及Westernblot技术测定肾皮质Nephrin、Podocin、BMP-7、CTGF及TGF-β₁ mRNA及蛋白的表达。结果:（1）高脂高糖喂养联合小剂量诱导成功建立T2DM模型,模型具有中等高血糖、高血脂、高血压、胰岛素抵抗及肾脏损害等特点。（2）较NC组,DM组UAL明显升高（P＜0.01）,肾肥大指数及Ccr增加（P＜0.01）,肾脏光镜及电镜病理明显病变,肾小球硬化指数（GSI）及肾间质纤维化指数（RIFI）明显升高（P＜0.01）,Nephrin、Podocin、BMP-TmRNA及蛋白的表达下调（P＜0.01）,CTGF及TGF-β₁ mRNA及蛋白的表达显著上调（P＜0.01）。相关分析表明,UAL与Nephrin、Podocin、BMP-7 mRNA存在负相关,相关系数分别为-0.825、-0.769、-0.784（P＜0.01）;与CTGF、TGF-β₁ mRNA存在正相关,相关系数分别为0.537、0.628（P＜0.01）;UAL与GSI及RIFI存在正相关（r₁=0.518,P＜0.01;r₂=0.646,P＜0.01）。Nephrin与BMP-7 mRNA表达存在正相关,与CTGF、TGF-β₁ mRNA呈负相关,相关系数分别为0.737、-0.675、-0.802（P＜0.01）;Podocin与BMP-7表达也存在正相关,与CTGF、TGF-β₁ mRNA呈负相关,相关系数分别为0.641、-0.508、-0.752（P＜0.01）。（3）应用雷公藤甲素、厄贝沙坦及雷公藤甲素联合厄贝沙坦干预8周后,DN大鼠较DM组UAL均明显减少、肾功能改善、肾脏病理改变显著减轻、足细胞数量增多,足细胞Nephrin、Podocin、BMP-7mRNA及蛋白表达量明显上调（P＜0.01）,CTGF及TGF-β₁ mRNA及蛋白的表达明显抑制（P＜0.01）,以雷公藤甲素联合厄贝沙坦组变化更显著（P＜0.05）。结论:在DN早期则出现的足细胞损伤,足细胞损伤与炎症反应及细胞因子表达失衡有关。雷公藤甲素联合厄贝沙坦能够更有效减轻足细胞损伤,降低蛋白尿,延缓肾功能损害的进展,可能与其抗炎及细胞因子的表达有关。提示雷公藤甲素的治疗可通过不同途径改善足细胞损伤,为临床DN新的治疗方法提供实验性理论基础。第三部分雷公藤甲素对2型DM大鼠肾小管-间质损伤的保护作用研究背景和目的:新近研究表明,糖尿病肾病（DN）是一种代谢紊乱诱导的炎症性疾病,其中巨噬细胞浸润是DN炎症的特征性表现之一,也是DN发生、发展的中心环节。以往研究表明免疫抑制剂—霉酚酸酯可通过其抗炎作用减轻STZ诱导的1型DM大鼠肾组织的炎症反应和减轻肾脏损害,本文旨在观察具有免疫抑制作用的中药—雷公藤多甙的有效组分雷公藤甲素（Triptolide）对T2DM大鼠肾小管-三间质损伤的保护作用,并探讨其机制。方法:高脂高糖喂养联合小剂量链脲佐菌素STZ(30mg.kg^-1)建立T2DM大鼠模型,将DM模型大鼠随机分为2组:雷公藤甲素治疗组（DT）及模型对照组（DM）;另设正常对照组（NC）。雷公藤甲素干预8周后,观察大鼠体重（BW）、肾重（KW）、肾肥大指数（KW/BW）、尿白蛋白排泄量（UAL）、血糖（FBG）、血清尿素氮（BUN）、肌酐（Scr）等变化,利用光镜观察肾脏病理改变,应用免疫组化技术观察骨形态发生蛋白（BMP-7）、结缔组织生长因子（CTGF）、骨桥蛋白（OPN）、转化生长因子-β₁（TGF-β₁）、单核/巨噬细胞表面特异性标识抗原（ED-1）及纤连蛋白（FN）在肾组织表达及定位情况。采用实时定量PCR及Western blot技术测定肾皮质BMP-7、CTGF、OPN及TGF-β₁ mRNA及蛋白的表达。结果:（1）较NC组,DM组UAL及肾肥大指数明显升高（P＜0.01）,肾脏病理明显病变。肾组织BMP-7mRNA及蛋白的表达下调（P＜0.01）,FN、CTGF、OPN及TGF-β₁ mRNA及蛋白的表达显著上调（P＜0.01）。肾组织ED-1阳性细胞数明显增多,以肾小管-间质为主（P＜0.01）。相关分析表明,尿蛋白与BMP-7 mRNA存在负相关,（r=-0.784,P＜0.01）;与CTGF、TGF-β₁、OPN mRNA存在正相关,相关系数分别为0.537、0.628、0.502（P＜0.01）;肾小管-间质内ED-1阳性细胞数与OPN和TGF-β₁的表达明显相关（r₁=0.492,P＜0.05,r₂=0.406,P＜0.05）;TGF=β₁与CTGF的表达呈正相关（r=0.662,P＜0.01）,而与BMP-7存在负相关（r=-0.604,P＜0.01）。萎缩肾小管比例及间质纤维化均与TGF-β₁、CTGF及OPN表达存在明显正相关（P＜0.05）,与BMP-7存在负相关（P＜0.05）。（2）应用雷公藤甲素干预8周后,DN大鼠UAL明显减少、肾脏小管-间质脖湎灾跚帷⑸鲎橹疎D-1阳性细胞数明显减少,BMP-7表达上调,而FN、CTGF、OPN及TGF-β₁表达被抑制（P＜0.05）。结论:雷公藤甲素治疗后DM大鼠肾组织TGF-β₁、CTGF、OPN mRNA和蛋白及ED-1、FN表达明显降低,BMP-7的mRNA和蛋白表达上调,萎缩肾小管及间质纤维化明显减少,并可减轻蛋白尿,不论从基因水平还是从蛋白水平,均可说明雷公藤甲素抑制促纤维化细胞因子的表达及巨噬细胞的浸润,并上调抑制纤维化因子,减少细胞外基质的合成,促进其降解,提示雷公藤甲素对肾小管-间质损害具有明显的保护作用,其机制为多方位,多靶点。更多还原

【Abstract】 PART ONE: The study of type 2 diabetic rat model and nephropathyBackground and Objective:Diabetic nephropathy is the major cause of end-stage renal disease（ESRD） in the worldwide.The majority of these patients are affected by type 2 diabetes.Animal models of diabetes lay particular emphasis on large dose streptozocin induced type 1 diabetes.For this reason,it is important to establish animal models which simulate the common manifesitation of type 2 diabetes in human population,but the methods of animal models of type 2 diabetes differ,and their characteristic differ too.The study aims at study the feasibility of the high-sucrose-high-fat diet and injected with the low dose STZ induced the model of type 2 diabetic rat,and observed the characteristic of nephropathy.Methods:Wistar rats were fed with regular chow or high-sucrose-high-fat diet. After 8 weeks,rats fed with high-sucrose-high-fat diet were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce hyperglycemia.Pats fed with regular chow receive vehicle.Determine systolic blood pressure,serum glucose, fasting seruminsulin,serum fat,serum creatinine,blood urine nitrogen,urinary albumin excretion and kidney construction by optical microscope and electron microscope.Results:The rats fed with high-sucrose-high-fat diet and injected with a low dose STZ showed increase in body weight,systolic blood pressure,blood glucose,fat and serum insulin levels in comparison with normal control group,while insulin seneitivity index（ISI） was reduced.At the 8 week after DM,kidney weight/body weight,urinary albumin excretion,blood urea nitrogen,serum creatinine,mean glomerular area,mean mesangial matrix area,mean mesangial matrix area/mean glomerular area ratio were significantly increased as compared with normal control group,but the capillary plexus area/mean glomerular area ratio was reduced. Ultrastructure of kidney damaged.Conclusion:Rats fed a high fat diet and given a low dose of STZ develop type 2 diabete with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.We present a nongenetic rat model of type 2 diabetes mellitus and nephropathy.The Type 2 diabetic model rats were successed.PART TWO: The protective effect of the combination of triptolide and irbesartan on the podocytes in type 2 diabetic rat model and its mechanismBackground and Objective:Diabetic nephropathy is one of the most common complications,and also is the major cause of end-stage renal disease（ESRD） in the worldwide.Recently,the researches show that diabetic nephropathy is inflammatory disease induced by metabolic disorder and macrophages have been thought to play a central role in the progression of diabetic nephropathy.Inflammation of renal tissue relates to glomerular filtration barrier injury and proteinuria in diabetic nephropathy. Podocytes locate in the exothecium of filtration barrier.Podocyte injury participates in the early stage of diabetic nephropathy.The studies indicated immune depressant-Tripterygium wilfordii can decrease proteinuria,but its mechanism is not still clear. The study aims to investigate the protective effect of the combination of triptolide and irbesartan on the podocytes in type 2 diabetic rat model,and evaluates its machanism.Methods:Wistar rats were fed with high-sucrose-high-fat diet for 8 weeks,and rats were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce type 2 diabetic rat model.DM rats were randomly divided into 4 groups:2 diabetic model group（DM）,triptolide treatment group（DT）,irbesartan treatment group（DI） and triptolide combined with irbesartan treatment group（DTI）.In addition,the normal rats served as a normal control group（NC）.All the rats were received daily gavage respectively for 8 weeks.The urinary albumin excretion（UAL）,body weight（BW）, kidney weight（KW）,KW/BW,glucemia,urea nitrogen,creatinine,Ccr,total cholesterol,triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expressions of Nephrin,Podocin and BMP-7, CTGF,TGF-β₁ mRNA and proteins were detected by immunohistochemistry, real-time PCR and Western blot.Results:（1） Rats fed a high fat diet and given a low dose of STZ develop type 2 diabete with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.（2） Compared to NC,UAL in 24 hours,KW and Ccr were increased significantly （P＜0.01）.Kidney lesion was severe,and GSI,RIFI were increased than NC （P＜0.01）.The expressions of Nephrin,Podocin and BMP-7 mRNA and proteins were down-regulated in glumorular,and CTGF,TGF-β₁ mRNA and proteins were up-regulated significantly（P＜0.01）.Correlation analysis showed that there were negative correlation between UAL and Nephrin,Podocin,BMP-7 mRNA（r₁=-0.825, r₂=-0.769,r₃=-0.784,P＜0.01） and there were positive correlation between UAL and CTGF,TGF-β₁ mRNA（r₁=0.537,r₂=0.628,P＜0.01）.Nephrin correlated positively with BMP-7 mRNA（r=0.737,P＜0.01）,and correlated negatively with CTGF,TGF-β₁ （r₁=-0.675,r₂=-0.802,P＜0.01）.Podocin correlated positively with BMP-7 mRNA（r=0.641,P＜0.01）,and correlated negatively with CTGF,TGF-β₁（r₁=-0.508, r₂=-0.752,P＜0.01）.（3） Decrease UAL occurred in all treatment groups.A better improvement on the ultrastructure of the podcytes and alleviated the damage of kidney（P＜0.01）. Meanwhile,the expressions of Nephrin,Podocin and BMP-7 were up-regulated remarkably in renal tissue,and CTGF,TGF-β₁ were down-regulated remarkably in renal tissue.These changes were the best in DTI（P＜0.05）.Conclusion:There was podocyte damage in the early stage of DN,which related to inflammatory reaction and cytokine disbalance.Renoprotection of triptolide on type 2 diabetic rats may be mediated,at least partly,suppressing inflammatory reaction and balancing cytokine and attenuating podocyte injury.PART THREE: The protective effect of triptolide on the renal tubule-interstitium in type 2 diabetic rat model and its machanismsBackground and Objective:Recently,the researches indicated that diabetic nephropathy was inflammatory disease induced by metabolic disorder and macrophages have been thought to play a central role in the progression of diabetic nephropathy.Mycophenolate mofetil（MMF）,an anti-inflammatory agent,has been shown to suppress macrophage infiltration and to improve renal injury in streptozotocin-induced diabetic kidneys.We examined whether triptolide,which acted through immunosupres- sive mechanisms,inhibits progression of diabetic nephropathy in 2 type diabetic model rats.The study aimed to investigate the protective effect of triptolide on renal tubule-interstitium in type 2 diabetic rat model, and evaluate its machanism.Methods:Wistar rats were fed with high-sucrose-high-fat diet for 8 weeks,and rats were injected with a low dose of STZ(30mg.kg^-1) into abdominal cavity to induce type 2 diabetic rat model.DM rats were randomly divided into 2 groups:type 2 diabetic model group（DM）,triptolide treatment group（DT）.In addition,the normal rats served as a normal control group（NC）.All the rats were received daily gavage respectively for 8 weeks.The urinary albumin excretion（UAL）,bodyweight（BW）, kidneyweight（KW）,KW/BW,glucemia,urea nitrogen,creatinine,total cholesterol, triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expression of BMP-7,CTGF,OPN and TGF-β₁ mRNA and proteins were detected by immunohistochemistry,real-time PCR and Western blot.Results:（1） Rats fed a high fat diet and given a low dose of STZ developed type 2 diabetes with insulin resistance,hyperinsulinemia,moderate hyperglycemia, hyperlipidemia,hypertension and proteinuria,kidney pathology lesions.（2） Compared to NC,UAL,KW and KW/BW were increased significantly （P＜0.01）.The expression of BMP-7 mRNA and proteins were down-regulated in renal tubule-interstitium,and CTGF,OPN and TGF-β₁ mRNA and proteins were up-regulated significantly（P＜0.01）.Correlation analysis showed that there were negative correlation between UAL and BMP-7 mRNA（r=-0.784,P＜0.01） and there were positive correlation between UAL and CTGF,OPN and TGF-β₁ mRNA （r₁=0.537,r₁=0.502,r₃=0.628,P＜0.01）.The number of ET-lpositive cells were directly correlated with RIFI（r=0.585,P＜0.01）,also correlated with mRNA expression of OPN,TGF-β₁ in renal interstitium（r₁=0.492,P＜0.05,r₂=0.406,P＜0.05）. mRNA expression of TGF-β₁ correlated positively with CTGF（r₁=0.662,P＜0.01）,and correlated negatively with BMP-7（r=-0.604,P＜0.01）.The ratio of atrophic renal tubule and interstitium fibrosis all correlated positively with CTGF,OPN and TGF-β₁ （P＜0.05）,and correlated negatively with BMP-7（P＜0.05）.（3） Decrease in UAL occurred in triptolide treatment group.Triptolide treatment alleviated the damage of renal tubule-interstitium（P＜0.01）.Meanwhile,the expression of BMP-7 was up-regulated remarkably in renal tubule-interstitium,and CTGF,OPN and TGF-β₁ were down-regulated remarkably in renal tubule-interstitium.Conclusion:Renoprotection of triptolide on renal tubule-interstitium in type 2 diabetic rats may be mediated,at least partly,suppressing macrophage accumulation, down-regulated the expression of CTGF,OPN and TGF-β₁ in renal tissue,meanwhile up-regulated the expression of BMP-7.Triptolide had protective effect on diabetic nephropathy by multitarget,polyols pathway.更多还原