Urocortin对抗糖尿病肾病的发展及其机制研究

【作者】 李璇；

【导师】 李胜男；

【作者基本信息】 南京医科大学 ， 药理学， 2008， 博士

【摘要】 高糖诱导内皮细胞线粒体活性氧族(reactive oxygen species,ROS)的过度生成是包括糖尿病肾病(diabetic nephropathy,DN)在内的糖尿病并发症的最主要的分子学机制。在2型糖尿病状态下内皮依赖的血管舒张作用受到损害。肾小球胞外基质(extracelluar matrix,ECM)过度生成和降解障碍导致ECM沉积。Urocortin(UCN)是一个40个氨基酸的多肽,属于促肾皮质激素释放激素(corticotrophin releasing factor,CRF)家族的一员。UCN抑制内皮细胞活性氧族的释放,诱导内皮依赖的大鼠冠状动脉的舒张;抑制内皮细胞增殖,下调VEGF表达;诱导绒毛膜滋养层细胞、羊膜上皮细胞、合包体滋养层细胞分泌基质金属蛋白酶-9(Matrix metalloproteinase,MMP-9),后者与基质降解密切相关。这些报道提示UCN可能与DN的发生发展有一定关联。本文采用肾小球系膜细胞(mesangial cell,MC)作为离体研究模型,db/db鼠和链佐星-弗氏完全佐剂(streptozotocin-complete Freund’sadjuvant,STZ-CFA)联合诱导的DN模型作为整体动物模型,研究了UCN对抗DN发展的作用及可能的作用机制。本文的第一部分采用了体外糖基化终产物(advanced glycationend products,AGEs)生成实验和MC作为研究对象,研究了UCN对AGEs生成及高糖/AGEs作用下MC增殖、组织转化生长因子-β1(transforming growth factor-β1,TGF-β1)、结缔组织生长因子(connective tissue growth factor,CTGF)、Ⅳ型胶原(collagen typeⅣ,collaⅣ)表达的影响,及对TGF-β1作用下血管肉皮生长因子(vascularendothelium growth factor,VEGF)分泌的影响。以荧光法测定AGEs的生成,~3H-TDR掺入法测定细胞增殖状态,免疫细胞化学法测定TGF-β1、CTGF、collaⅣ的表达,ELISA测定VEGF的分泌。结果表明,UCN对AGEs生成没有明显影响,但可以抑制高糖/AGEs诱导的MC的异常增殖和TGF-β1、CTGF、collaⅣ的过度表达和TGF-β1诱导的VEGF过度分泌。而CRF受体阻断剂astressin(AS)能减弱UCN的上述作用。本文第二部分研究了UCN对db/db鼠肾病动物模型DN发展的影响。采用公认的2型DN模型db/db鼠为研究对象,给药六周,连续监测了UCN对血糖、体重、进食量的影响,并检测了UCN对db/db鼠血浆胰岛素、尿素氮(blood urine nitrogen,BUN)、肌酐(creatinine,Crea)、AGEs、红细胞内山梨醇(sorbitol,SB)含量、肾组织匀浆中超氧化物岐化酶(superoxide dismutase,SOD)活性、丙二醛(malonaldehyde,MDA)水平的影响及对肾组织病理学、肾小球ECM沉积的影响。结果表明,UCN对模型动物血糖、进食量、血浆胰岛素水平没有明显影响,但从给药第三周起能够降低模型动物体重,并且能降低血浆BUN、Crea、AGEs水平、红细胞内SB含量、肾匀浆中MDA含量,提高肾匀浆中SOD活性,减轻肾小球病理变化及肾小球系膜基质聚积和系膜区扩张。AS除不能对抗UCN降低红细胞内SB含量的效应外,可逆转UCN对抗db/db鼠肾病发展的保护作用。本文第三部分研究了UCN对STZ-CFA联合诱导的糖尿病模型大鼠DN发展的影响。采用STZ-CFA联合诱导辅以高脂饮食,连续喂养14周制作DN模型。连续给药8周(自实验开始第7周起给药),监测UCN对体重、血糖的影响;生化检测UCN对血清血糖、胰岛素(放免)、BUN、Crea、总胆固醇、HDL-胆固醇、LDL-胆固醇、SOD、MDA水平的影响,收集24 h尿液,测定尿液体积、尿液中Crea、尿素氮、尿微量白蛋白(放免)水平,计算肌酐清除率;观察了UCN对肾脏病理组织学的影响及对系膜基质聚积的影响;免疫组织化学方法研究了肾小球TGF-β1、CTGF、VEGF的表达,RT-PCR方法检测肾组织VEGF mRNA的表达。结果表明,UCN对DN大鼠血糖、胰岛素、体重没有明显影响,但减轻肾脏重量;可以改善DN状态下的脂代谢紊乱:降低血清总胆固醇、LDL水平,提高HDL水平;改善氧化还原失衡状态:提高血清SOD水平、降低血清MDA水平,降低血清AGEs水平和红细胞内SB含量;改善肾脏功能:减少24 h尿液体积和微白蛋白排出量,提高尿液尿素氮、Crea水平,降低血清BUN、Crea水平,提高肌酐清除率(creatinine clearance rate,Ccr);减轻肾小球胞外基质聚积和系膜区增宽程度,抑制肾小球TGF-β1、CTGF、VEGF、VEGF mRNA、collaⅣ的过度表达。除对SB的影响外,AS能够减弱UCN对STZ-CFA诱导的DN模型的作用。此外,本文还研究了STZ-CFA辅以高脂饮食诱导的DN模型肾脏UCN表达的变化。采用RT-PCR和免疫组织化学方法分别检测UCN mRNA和UCN在肾脏组织的表达。结果表明,UCN mRNA在DN模型大鼠肾脏表达有增加的趋势,但无统计学意义,而UCN在肾脏组织中的蛋白表达水平没有明显差异,由于时间所限,此结果仅为初步结果,尚需进一步实验研究。综上所述,UCN能够改善肾脏功能,减少尿微白蛋白排出,这与UCN通过多途径对抗DN发展的机制有关:抑制MC异常增殖,抑制促纤维化因子(TGF-β1、CTGF、VEGF、VEGF mRNA)的过度表达从而抑制肾小球ECM聚积和系膜基质增生(collaⅣ),减少AGEs堆积、抑制PP活性、纠正机体氧化还原失衡,降低血清总胆固醇、LDL、提高HDL水平。除对SB的影响外,UCN改善DN的作用与CRF受体相关。此外,UCN在DN肾脏中表达变化,尚需更多的数据支持。更多还原

【Abstract】 Hyperglycemia induces process of overproduction of mitochondrial reactive oxygen stress (ROS) in endothelial cells, which is believed to be the main molecular mechanism of diabetic complications including diabetic nephropathy (DN). Impairment of endothelium-dependent vasodilatation is found in type 2 diabetes. Increased thickness of glomerular basement membrane and augmentation of glomerular extracellular matrix (ECM) are recognized as pathological hallmarks of diabetic nephropathy. ECM accumulation is due to the increased synthesis and decreased degradation of ECM. UCN is a 40-amino-acid peptide related to the corticotrophin-releasing factor (CRF) family, which suppresses production of ROS & expression of VEGF in endothelial cells and sustains endothelium-dependent rat coronary artery relaxation. Furthermore, UCN induces matrix metalloproteinase (MMP-9) expression of cultured cells from human placenta and fetal membranes, which is related to extracelluar matrix (ECM) degradation. These reports highly indicate that UCN may play an important role in the development of DN.We investigated effects of UCN on DN and the possible mechanisms by using cultured rat mesangial cells (MC), db/db mice and DN model rats induced streptozotocin-complete Freund’s adjuvant (STZ-CFA). In the first part, advanced glycation end products (AGEs) formation in vitro and cultured MC were used to investigate the effects of UCN on AGEs formation, high glucose/AGEs induced abnormal cell proliferation, overexpression of transforming growth factor (TGF-β1), connective tissue growth factor (CTGF), collagen type IV and TGF-β1 induced overproduction of vascular endothelial growth factor (VEGF). The results showed that UCN did not affect the AGEs formation process, however, it inhibited the over expression of TGF-β1, CTGF, collagen type IV induced by high glucose/AGEs and overproduction of VEGF induced by TGF-β1. Treatment with unselective CRF receptor blocker-astressin reversed such effects.In the second part, db/db mice were used to investigate the amelioration effect of UCN on type 2 DN. UCN was injected intraperitoneally to the mice for 6 weeks. The effects of UCN on blood glucose, body weight & food intake were recorded weekly. At the end of the experiment, plasma insulin, blood glucose, blood urine nitrogen (BUN), creatinine (Crea), AGEs level, sorbitol concentration in RBC and superoxide dismutase (SOD) activity & malonaldehyde (MDA) level in kidney homogenate were examined. The pathology change of kidney was observed by using HE- and PAS- stained paraffin section. The results showed that UCN significantly decreased body weight, plasma AGEs level, BUN level and Crea level. However, food intake, plasma insulin and plasma glucose level remained unaffected. Superoxide dismutase (SOD) activity was markedly increased, whereas malonaldehyde (MDA) level in kidney homogenate and concentration of sorbitol in red blood cells (RBC) were significantly decreased in UCN-treated group. UCN significantly decreased ECM expansion and accumulation in kidney. The beneficial effects of UCN on db/db mice could be abolished by astressin except its inhibition of sorbitol accumulation.In the third part, UCN was injected intraperitoneally to DN rat model induced by STZ-CFA (from the 7th week after the beginning of the experiment) daily for 8 weeks. Body weight and blood glucose were recorded weekly. At the end of the experiment, blood sample was collected to analyze effects of UCN on blood glucose, insulin, BUN, Crea, cholesterol, HDL, LDL, SOD, MDA level and sorbitol concentration in RBC. Urine of 24 h was collected and the effects of UCN on urine volume, Crea, urine nitrogen, microalbuminuria were detected and creatinine clearance rate (Ccr) was calculated. ECM accumulation & mesangial area expansion were observed by using HE- and PAS- stained paraffin section and percent mesangial area was calculated. Overexpression of TGF-β1、CTGF、VEGF and VEGF mRNA in the renal was presented by using immunohistochemistry & RT-PCR. Our results showed that UCN did not influence the serum blood glucose, insulin level and body weight; however, it decreased serum cholesterol, LDL, MDA, BUN, Crea, AGEs level, RBC sorbitol concentration and kidney weight significantly. Likewise, serum HDL level and SOD activity were obviously enhanced by UCN treatment. Total urine volume and microalbumin excretion of 24 h were diminished and Ccr increased significantly by UCN. Furthermore, UCN ameliorated ECM accumulation, mesangial area expansion and collagen type IV expression, which was related to its decreasing effects on overexpression of TGF-β1、CTGF、VEGF、VEGF mRNA in the kidney or glomeruli. Astressin treatment reversed the effects of UCN except its inhibitory effect on polyol pathway activity. RT-PCR and immunohistochemistry were used to compare the expression of UCN & UCN mRNA in kidney between normal rats and STZ-CFA induced DN rats. Although UCN mRNA expression appeared to increase in DN rats, UCN expression was not obviously enhanced. It needs further investigation to do other experiments to quantify the expression of UCN and UCN mRNA due to the time limitation.Taken together, UCN could significantly prevent DN development and this effect was related to CRF receptors. Inhibition of AGE accumulation, polyol pathway activation, ECM expansion & accumulation, TGF-β1, VEGF & CTGF over-expression, ROS over-production and regulation of lipid profiles may all contribute to the amelioration of DN by UCN.更多还原