【作者】 李宪花；

【导师】 高海青；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 研究背景和目的:随着生活水平的提高,糖尿病发病率迅速增高,目前世界糖尿病患者大约1亿7千万人,其并发症在全世界范围内急剧增加。在糖尿病各种慢性并发症中,糖尿病肾病（Diabetic nephropathy,DN）是最主要的血管并发症,不仅发病率高,且最终发展为终末期肾衰而致死,死亡率亦高。DN已成为发达国家终末期肾衰的首位病因,在我国为终末期肾衰第二大病因。据美国肾脏病资料系统（USRDS）的统计显示,超过30%的终末期肾功能衰竭为DN所致。DN严重威胁人类的生命和健康,已成为世界关注的重要问题。DN基本病理特征包括系膜基质合成增多,肾小球基底膜增厚,从而引起肾小球硬化及肾小管间质纤维化。生长因子和细胞因子的过度表达如转化生长因子-β1（Transforming growth factorβ1,TGF-β1）,结缔组织生长因子（Connective tissuegrowth factor,CTGF）,生长激素,胰岛素样生长因子和血管内皮生长因子在DN发生和进展中起重要作用。目前认为,TGF-β1是已知的在DN中致硬化作用最强的细胞因子。肾脏多种实质细胞,尤其是系膜细胞合成分泌TGF-β1,并拥有其特异性受体。TGF-β1有自我诱生作用,即在损伤部位释放的TGF-β1可诱导细胞产生更多的TGF-β1,从而在局部放大了其生物学作用。TGF-β1可上调葡萄糖转运蛋白（glucose transporter,GLUT）-1,引起细胞内糖摄入增加和右旋（D）-葡萄糖增加;增加细胞外基质蛋白合成,如胶原Ⅰ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、纤连蛋白、层连蛋白等,增强细胞表面细胞外基质（Extracellular matrix,ECM）受体-整合素的表达,使细胞与基质黏附增强,促使ECM在肾小球的沉积,促进系膜扩张,基底膜增厚;足细胞凋亡、分离,诱发基底膜剥脱;刺激足细胞分泌血管内皮生长因子,并抑制ECM降解酶的合成与活性,使ECM降解减少,从而促使肾小球硬化。阻断TGF-β1活性能抑制ECM的增加和减轻肾脏纤维化。但由于TGF-β1同时具有抗免疫、抗增生效应,通过基因敲除的方法长期完全阻断TGF-β的作用,动物不能生存。因而人们不得不继续探寻其更加特异的作用途径。CTGF在DN发病过程中作为TGF-β1的下游调节因子,介导TGF-β1的上述作用。体外实验显示,CTGF能诱导ECM的合成增加,并且在高糖、机械张力、非酶糖基化终产物（Advanced glycation end products,AGEs）和TGF-β1下,CTGF在肾脏细胞表达明显增加。CTGF可以和胰岛素样生长因子、血管内皮生长因子、TGF-β1和骨形态蛋白（Bone morphogenetic proteins,BMPs）相互作用,并影响上述因子的信号传导。已有专家提出CTGF可作为DN治疗的新靶点。AGEs及其受体在DN的发病及进展中有很重要的作用。AGEs在半衰期长的蛋白质（胶原蛋白、晶体蛋白、弹性蛋白等）及细胞壁上蓄积,使血管壁增厚,弹性降低。当肾小球基底膜增厚时,即使有效纠正高血糖,已被糖化的蛋白质也不能恢复正常。此外,糖化的蛋白质与基膜中重要的阴离子蛋白聚糖成分硫酸乙酰肝素的亲和力降低,清除增加,一方面损伤基膜的电荷屏障,同时丧失抑制基膜及系膜增生的作用,从而导致基膜和系膜增生。AGEs通过两种方式发挥作用:受体依赖性信息交联通路和非受体依赖性通路对肾脏造成损害。AGEs与非酶糖基化终产物受体（Receptor for advancedglycation end product,RAGE）结合,可激活第二信号系统,产生大量细胞因子包括TGF-a、血小板源细胞因子、白介素-1等,对肾小球及肾小管间质造成不可逆转的损伤。除了这些促纤维化的生长因子外,已有报道抗纤维化的生长因子骨形态蛋白质-7（Bone morphogenetic proteins-7,BMP-7）在DN中是下调的,并能拮抗实验性DN的纤维化。BMP-7作为纤维化的负性调节因子,通过维持上皮细胞表型、抑制肾脏上皮细胞的凋亡、促进ECM的降解、减少多种促炎症因子的表达、影响TGF-β/Smads传导途径以及与TGF-β1的互逆作用,对肾间质纤维化起到预防及逆转作用。近年来,大量研究提示足细胞损伤与缺失可能是导致肾小球硬化的一个中心环节。蛋白尿和ECM积聚较多的DN病人足细胞少于有着相同病程但没有蛋白尿和肾小球硬化的糖尿病病人。提示足细胞损伤在DN的发病及进展过程中亦有很重要的作用。Nephrin,podocin,CD2AP是足细胞裂孔隔膜上的跨膜蛋白,肾小球滤过屏障的分子成分,具有多种生物活性功能。它们的正常表达及分布对维持滤过膜完整的形态结构和屏障功能有着重要意义。若发生异常将导致大量蛋白尿的产生。已有研究证实并发肾病综合征的糖尿病患者肾小球nephrin染色明显减少,糖尿病大鼠模型中亦证实有nephrin表达的下降。目前防治DN的措施包括有效控制血糖,控制系统性高血压,抑制肾素-血管紧张素系统（RAS）,控制血脂障碍等。虽然这些措施可以减缓蛋白尿的增加和肾功能减退,减少死亡率,DN仍然是一个巨大的临床课题,迫切需要更有效的治疗措施。葡萄子原花青素（Grape seed pmanthocyanidin extracts,GSPE）是一类在植物界广泛存在的具有抗氧化作用的多酚化合物,是一种新型高效抗氧化剂,是目前为止所发现的最强效的自由基清除剂,具有非常强的体内活性。其抗自由基氧化能力是维生素E的50倍、维生素C的20倍。近年来国外研究发现GSPE有心血管保护作用,它的自由基清除能力可抗心肌缺血再灌注损伤,通过抑制低密度脂蛋白的氧化作用而抗动脉粥样硬化。针对糖尿病,GSPE可抑制糖尿病大鼠体内非酶糖基化反应。国内外对DN的防治方法多种多样,但目前为止尚无GSPE预防及治疗DN的深入研究。本研究拟以糖尿病大鼠为动物模型,从肾脏病理水平,细胞,分子生物学水平（包括CTGF,TGF-β1,BMP-7,AGEs,RAGE,nephrin,podocin）研究GSPE对糖尿病大鼠肾脏的保护作用及作用机制,为临床应用GSPE预防及治疗DN提供更深入的理论依据,为减少DN的发生,减缓DN的发展开辟一种崭新的治疗方法。材料和方法:雄性Wistar大鼠,禁食12小时,尾静脉注射STZ 55mg/kg（即5.5ml/kg）,72h后测定血糖,以空腹血糖水平≥16.7mmol/l（300mg/kg）为糖尿病模型成功的标准。实验分组和处理:（1）正常对照组（C）:取健康雄性Wistar大鼠12只,生理盐水灌胃。（2）GSPE正常治疗组（CT）:取健康雄性Wistar大鼠12只,GSPE 250mg·kg^-1·d^-1灌胃。（3）模型组（DM）:糖尿病模型大鼠15只,生理盐水灌胃。（4）GSPE小剂量治疗组（T1）:糖尿病模型大鼠15只,GSPE250mg·kg^-1·d^-1灌胃。（5）GSPE大剂量治疗组（T2）:糖尿病模型大鼠15只,GSPE 500mg·kg^-1·d^-1灌胃。动物实验结束,取血,留尿,取肾组织,检测血白蛋白（Albumin,Alb）,肌酐（Serum creatinine,Scr）,AGEs,HDL,LDL,血尿酸（Uric acid,UA）,血糖（fastingplasma glucose,FPG）,糖化血红蛋白(hemoglobin A1c,HbA_1c),总胆固醇（Cholesterol,CH）,甘油三酯（Triglyceride,TG）,尿查24h尿蛋白总量。肾组织病理检查包括光镜PAS染色、Masson染色、电镜观察系膜细胞、内皮细胞、足细胞超微结构的变化及进行TGF-β1,CTGF,BMP-7,RAGE,nephrin,podocin免疫组化染色。采用RT-PCR技术检测TGF-β1,CTGF,BMP-7,RAGE,nephrin,podocinmRNA,Western印迹法检测其蛋白表达。比较各组间差异。结果:DM组FPG、HbA_1（?）、血清AGEs、血压、24h尿蛋白定量、内生肌酐清除率（Ccr）、肾重/体重水平均显著高于C组（p＜0.01）;DM组ECM蓄积增多,固有细胞超微结构受损;DM组肾组织中TGF-β1,CTGF,RAGEmRNA及蛋白表达水平增加,与C组相比,差异有统计学意义（p＜0.05或p＜0.01）;DM组nephrin,BMP-7mRNA及蛋白水平较C组显著降低（p＜0.05）。治疗组（T1组和T2组）血清AGEs、血压、24h尿蛋白定量、Ccr、肾重/体重水平较DM组显著降低（p＜0.05或p＜0.01）,且ECM蓄积减少,固有细胞超微结构受损情况减轻;治疗组（T1组和T2组）与DM组相比,肾组织中nephrin,BMP-7mRNA及蛋白表达水平升高,差异有统计学意义（p＜0.05或p＜0.01）,TGF-β1、CTGF、RAGEmRNA及蛋白显著降低（p＜0.05或p＜0.01）。T1组与T2组之间相比,Ccr、肾重/体重和CTGF mRNA水平差异有统计学意义（p＜0.05或p＜0.01）。结论:应用GSPE干预DN大鼠,证实GSPE能够显著降低糖尿病大鼠蛋白尿、血压,改善肾小球高滤过状态,减轻肾脏ECM蓄积及固有细胞超微结构的损害,对DN具有明确的治疗作用。其作用机制与降低血清AGEs、下调RAGE、TGF-β1、CTGF表达及上调BMP-7表达有关;对足细胞有明确的保护作用,可上调足细胞跨膜蛋白nephrin的表达从而改善足突结构减轻蛋白尿。本研究为DN的治疗提供了新思路,为临床应用GSPE预防及治疗DN提供更深入的理论依据,为减少DN的发生,减缓DN的发展开辟一种崭新的治疗方法。更多还原

【Abstract】 Background and Objective With the increasing of living standard,morbidity and complications of diabetes mellitus rise sharply in the world.Now patients of diabetes are about 1,710,000,000 throughout the world.Diabetic nephropathy（DN） is emerging as the leading cause of end-stage renal failure in developed country and the second cause in our country.United State Renal Disease Statistic（USRDS）indicated that exceed 30%of the end-stage renal failure were induced by DN.DN threatens severely the life and health of mankind,it is an important problem that is followed with interest by all over the world.The structural changes of kidney in diabetes consist of glomerular and tubuloepithelial hypertrophy,followed by thickening of glomerular and tubular basement membranes and progressive accumulation of extracellular matrix proteins in the mesangium and the interstitium.This contributes to glomerulosclerosis and tubulointerstitial fibrosis.Overexpression of cytokines and growth factors play a central role in the initiation and progression of DN,for example:transforming growth factorβ1（TGF-β1）,connective tissue growth factor（CTGF）,growth hormone,insulin-like growth factor（IGF）and vascular endothelial growth factor（VEGF）.Recent studies have suggested that TGF-β1 could be the most important cytokine in the nephrosclerosis. There are many intrinsic cells in the kidney,especially mesangial cells that can synthetize and secrete TGF-β1,and with special receptors of TGF-β1.TGF-β1 is one effector molecule that has been studied extensively as a major mediator of the hypertrophic and prosclerotic changes in diabetic kidney disease.TGF-β1 stimulates the synthesis of key extracellular matrix molecules including typeⅠcollagen,typeⅢcollagen,typeⅣcollagen,typeⅤcollagen,typeⅥcollagen,fibronectin,and laminin.TGF-β1 also decreases matrix degradation by inhibiting proteases as well as activating protease inhibitors（e.g.,plasminogen activator inhibitor-l）.TGF-β1 promotes cell-matrix interactions by upregulating integrins,the cell surface receptors for matrix.In addition,high ambient glucose increases TGF-β1 mRNA and protein level in cultured proximal tubular cells and glomerular epithelial and mesangial cells.In experimental and human diabetes mellitus,several reports describe overexpression of TGF-β1 in the glomeruli and tubulointerstitium.The data presented here strongly support the consensus that the TGF-βsystem mediates the renal hypertrophy, glomerulosclerosis,and tubulointerstitial fibrosis of diabetic kidney disease.Inhibiting renal TGF-β1 activity can partially reverse the glomerular basement membrane（GBM） thickening and mesangial matrix expansion,attenuate the nephrosclerosis.But TGF-β1 can regulate immune and inhibit proliferation,inhibiting TGF-β1 completely by gene knock-out,the animal can’t live.People have to search for more special pathway. CTGF is a cysteine-rich member of a new family of growth regulators.It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis in DN.In development of progressive extracellular matrix accumulation,CTGF may act as a downstream mediator of TGF-β1.Some mediators upregulates the expression and bioactivity of CTGF in renal intrinsic cell.These mediators encompass the following:high glucose concentration,early and advanced products of nonenzymatic glycation of proteins,mechanical tension and TGF-β.CTGF acts on IGF,VEGE,TGF-β,Bone morphogenetic proteins（BMPs）,and affects their signaling pathways.Some experts suggesting that CTGF blockade could be a new therapeutic target against DN.Several lines of evidence suggest that advanced glycation end products（AGEs）may be involved in the development of diabetic glomerular lesions.AGEs mediate their effects through two different pathways via a receptor-independent AGEs cross-link formation pathway and a receptor-dependent pathway where AGEs bind to specific cell surface-associated receptors,such as Receptor for advanced glycation end product（RAGE）.AGEs modification indeed alters the structure and function of matrix tissue proteins and,more interestingly, AGE-modified proteins stimulate a variety of cellular responses via a specific cell-surface receptor on several cell types,including glomerular cells.RAGE is one of AGE-specific cellular receptors.RAGE has been proposed to play an important role in the development of DN.AGEs can activate RAGE gene through nuclear transcription factor-k B（NF-kB）,AGEs accumulation may involve upregulation and activation of RAGE.The AGE-RAGE interaction is associated with the pathogenesis of DN. Except these growth factors that cause fibrosis,there is anti-fibrotic growth factor: Bone morphogenetic proteins-7（BMP-7）that expresses lower in DN and can inhibit fibrosis of experimental DN.BMP-7 has a protective effect on the kidney,by decreasing apoptosis,maintaining and restoring the epithelial phenotype,increasing matrix degradation,decreasing lots of inflammation factors,affecting TGF-β/Smads pathway and displaying anti-fibrotic activity.Recent studies have demonstrated that injury and loss of podocyte is one central mediator of glomerulosclerosis in DN.Podocyte molecules（nephrin,podocin, CD2-associated protein）make a pivotal contribution to the maintenance of the selective filtration barrier of the normal glomerulus.A marked reduction was observed at the protein levels of nephrin and podocalyxin in DN.This suggests that the alterations in the expression of podocyte-associated molecules represent a compensatory reaction of the podocyte that results from damage associated with proteinuria.Current therapeutics include intensified glycemic control,blood pressure lowering agents,inhibiting renin-angiotensin system（RAS）and intensified lipid control,et al. Though these can attenuate proteinuria and improve renal function in diabetes and decrease mortality of DN,renal disease continues to progress relentlessly in diabetic patients,DN is an important clinical problem that need be solved urgently.Grape seed proanthocyanidin extracts（GSPE）,which belong to a class of polyphenols,are widely distributed throughout the plant kingdom.GSPE have been reported to possess a wide range of biologic properties against oxidative stress.The antioxidative activities of GSPE were found to be much stronger than vitamin C or vitamin E in aqueous systems.Recent studies demonstrate that GSPE have potent antioxidant properties and should be considered a potential agent in the prevention of some diseases.GSPE has been reported to exert protective effects on various forms of cardiac disorders.The cardiovascular protective effects of GSPE are believed to be ascribed to its antioxidative properties.GSPE were showed to have preventive actions on diseases such as atherosclerosis,gastric ulcer,large bowel cancer,cataracts and diabetes.However,the effects of GSPE on DN have been little understood.The aim of this study was to determine the effect of GSPE on kidney in diabetic rats.Material and Methods 45 rats injected with streptozotocin（STZ）were randomly divided into diabetes group（DM）,small dosage GSPE treated group（T1） and big dosage GSPE treated group（T2）.Other 12 normal rats were used as control group（C）,12 normal rats were administrated GSPE at a dosage 250mg·kg^-1·d^-1as treated control group（CT）.The rats in T1 group were administrated GSPE at a dosage 250mg·kg^-1·d^-1,the rats in T2 group were administrated GSPE at a dosage 500mg·kg^-1·d^-1,and animals in other groups were treated with normal saline.The fasting plasma glucose（FPG）,hemoglobin Alc(HbA_1C),systolic blood pressure,24h urinary protein,serum creatinine（Scr）,urinary creatinine,serum AGEs and ratio of kidney weight to body weight were measured in each rat after 24 weeks.The renal pathological changes were examined with PAS staining,Masson staining and electron Microscope.The mRNA expression of TGF-β1,CTGF,RAGE,BMP-7,nephrin,and podocin in kidney were detected by reverse transcription-polymerase chain reaction（RT-PCR）,the protein expression were detected by immunohistochemical staining respectively and Western blot,and were quantified by computer image analysis system.Then these parameters of each group are compared.Results FPG,HbA_1C,AGEs,blood pressure,24h urinary protein,Ccr and ratio of kidney weight to body weight were significantly higher in DM group compared to C group（p＜0.01）.Extracellular matrix（ECM）accumulation were higher in DM group than that of C group（p＜0.01）,and intrisinc cells in the kidney were damaged seriously in DM group.Expression of mRNA and protein of TGF-β1,CTGF and RAGE increased in DM group compared to C group（p＜0.05 or p＜0.01）,whereas the expression of mRNA and protein of BMP-7 and nephrin was progressively down-regulated（p＜0.05）in DM group compared to C group.Diabetic rats treated with GSPE（T1 or T2 group）had less urinary protein,lower blood pressure,serum AGEs and Ccr compared to DM group（p＜0.05 or p＜0.01）.The expression of mRNA and protein of BMP-7 and nephrin were significantly higher in T1 and T2 group compared to DM group（p＜0.05 or p＜0.01）,whereas the expression of mRNA and protein of TGF-β1,CTGF and RAGE was progressively down-regulated in T1 and T2 group compared to DM group（p＜0.01）.Between T1 and T2 group the expression of mRNA of CTGF,Ccr and ratio of kidney weight to body weight were significantly different（p＜0.05 or p＜0.01）.Conclusions GSPE can decrease proteinuria,blood pressure and glomerular high filtration in diabetic rats,improve the diabetic nephropathy.GSPE has clear renal protective effects.Renoprotective mechanism of GSPE are correlated with suppression on AGEs,down-regulated overexpression of RAGE,TGF-β1,CTGF and up-regulated expression of BMP-7 and nephrin.更多还原