【作者】 张浩军；

【导师】 李平；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的糖尿病肾病(DN)是糖尿病常见的微血管并发症,约20%-40%的DN患者进展为终末期肾病,严重危害人类健康,DN发病机制非常复杂,目前尚未完全阐明,现代医学主要依靠控制血糖、降脂以及血管紧张素转化酶抑制剂和血管紧张素受体阻断剂类药物的应用控制DN的发展。中医药在防治DN方面显示出明显的特色与优势。寻找安全、高效的治疗DN中药及其机制探讨是目前研究热点之一。本研究以链脲佐菌素诱导的1型糖尿病肾病大鼠和自发性2型糖尿病肾病动物模型OLETF大鼠为研究对象,在验证糖肾方药物作用基础上,从致糖尿病肾病的重要生长因子TGF-β1及细胞外基质合成降解失衡为切入点,探讨糖肾方防治糖尿病肾病的作用机理,为临床应用提供理论依据。方法1.采用单侧肾切除加链脲佐菌素腹腔注射建立1型糖尿病肾病大鼠模型,随机分为模型组、糖肾方大剂量组、糖肾方小剂量和蒙诺组,另设假手术组为空白组。分别于第0、4、8、12、16、20周动态观察大鼠一般状态、体重、血糖、尿蛋白/肌酐,第20周时动物取材,血液流变仪检测全血粘度及血浆粘度,血生化仪检测血脂、血清总蛋白、白蛋白及肾功的变化,半定量评价肾组织肾小球及肾小管间质病理变化;采用免疫组化法检测肾组织及Ⅳ型胶原表达变化,RT-PCR方法测定肾组织TGF-β1、MMP-2及MMP-9表达。2.从国外引进自发性2型糖尿病肾病模型OLETF大鼠,随机分为模型组、糖肾方组和蒙诺组,以遗传背景相同、同周龄、性别的LETO大鼠为空白组。每4周观察大鼠一般状态、体重、血糖、24h尿蛋白定量,分别连续给药24周及44周,于这两个时间点分别取材,血液流变仪检测全血粘度及血浆粘度,血生化仪检测血脂、血清总蛋白、白蛋白及肾功的变化,半定量评价肾组织肾小球及肾小管间质病理变化。结果1.糖肾方对链脲佐菌素诱导的1型糖尿病肾病大鼠的作用:模型组从实验第0周起,状态与体重即明显差于空白组,且随实验的进行差异持续增加,到第20周差异达峰值,与模型组相比,各给药组大鼠状态均有明显改善,糖肾方小剂量组体重从第4周起显著升高并持续至实验结束;模型组大鼠尿蛋白/肌酐从12周起显著升高,糖肾方组与蒙诺组从16周开始能显著降低模型组大鼠尿蛋白/肌酐,模型组大鼠血糖、胆固醇、甘油三酯、血浆粘度,血尿素氮、肾小球硬化指数及肾小管间质纤维化指数均显著高于空白组,糖肾方大剂量组能显著降低模型动物血浆粘度(P＜0.05),胆固醇(P＜0.05)及甘油三酯含量(P＜0.01),降低肾小球硬化指数(P＜0.01)和肾小管间质纤维化指数(P＜0.01),糖肾方对模型组大鼠血糖及尿素氮无明显影响,各实验组全血粘度、血清总蛋白、白蛋白及肌酐无显著性差异。2.糖肾方对链脲佐菌素诱导的1型糖尿病肾病大鼠的TGF-β1、MMP-2及MMP-9、Ⅳ型胶原表达影响:免疫组化结果显示:模型组肾组织在肾小球及小管间质表达均显著升高,与空白组比较有显著性差异,糖肾方大、小剂量组与蒙诺组TGF-β1及Ⅳ型胶原表达与模型组相比显著降低。RT-PCR实验表明:与空白组相比,模型组肾组织TGFβ1 mRNA表达水平显著升高,MMP-9 mRNA显著降低,糖肾方大量组与蒙诺组能显著降低模型组大鼠肾组织TGFβ1 mRNA表达,升高MMP-9 mRNA水平,各实验组肾组织MMP-2 mRNA表达无显著性差异。2.糖肾方对自发性2型糖尿病肾病OLETF大鼠的作用:模型组大鼠体重从6周龄起显著高于空白组,在40周龄时差异达最大,随后差异逐渐减小,至52周龄时与空白组体重无显著性差异。糖肾方在40、44、48周龄能显著降低模型组大鼠体重;模型组血糖及24h尿蛋白水平从6周龄起与空白组显著升高,且随实验周期呈渐进性进展,至56周龄时差异最大,糖肾方从36周龄起与24h尿蛋白水平与模型组相比显著降低,血糖水平从52周龄起显著降低;在36周龄时模型组大鼠血浆粘度、甘油三酯、肾小球硬化指数及肾小管间质纤维化指数均显著高于空白组;糖肾方能显著降低模型组大鼠血浆粘度,效果优于蒙诺,蒙诺组能显著降低模型组大鼠血清甘油三酯水平,优于糖肾方。两给药组对模型组大鼠肾小球硬化指数与肾小管间质纤维化指数均有显著降低作用;56周龄时模型组大鼠胆固醇、甘油三酯、全血粘度高切与低切,血浆粘度,尿素氮、肾小球硬化指数及肾小管间质纤维化指数均显著高于空白组,血清总蛋白与白蛋白水平均显著低于空白组,糖肾方大剂量组能显著降低模型动物全血粘度高切与低切,血浆粘度,胆固醇,升高血清总蛋白与白蛋白水平,降低肾小球硬化指数和肾小管间质纤维化指数,各给药组对模型组大鼠尿素氮无明显影响。结论1.糖肾方能够降低1型糖尿病肾病尿蛋白排泄,改善肾组织病理损伤,其作用机制与纠正脂代谢紊乱及血液流变学异常有关,但与血糖相关性不强。2.细胞外基质积聚是糖尿病肾病发病的重要环节。糖肾方能抑制1型糖尿病肾病大鼠TGF-β1mRNA与蛋白表达,升高MMP-9 mRNA表达,减少细胞外基质主要组分Ⅳ型胶原的表达,这可能是其对1型糖尿病肾病大鼠保护作用的重要机制。3.糖肾方能降低2型自发性糖尿病肾病模型OLETF大鼠早期与中晚期24h尿蛋白水平,显著改善肾小球与肾小管间质病理改变,有着良好的肾保护作用。纠正糖、脂代谢紊乱与血液流变学异常是其可能的作用环节。4.纠正脂代谢紊乱和血液流变学异常可能时糖肾方防治1型与2型糖尿病肾病的共同作用机制。更多还原

【Abstract】 ObjectiveDiabetic nephropathy（DN） is a common microvascular complication of diabetes mellitus.About 20 to 40%patients develop end stage renal disease.This disease do harm to human health.The pathogenesy of DN is very complicated,ACEI and angiotensin receptor to control developing of DN.traditional chinese medicine have evident special feature and superiority in treating DN.One of the hot spot of studying DN is seeking for safe and high-performance chinese medicine.this study set up two DN model.Type 1 DN rat model was induced streptozotocin.Spontaneous type 2 DN is the other model.In the base of prove the drug action of Tangshen Formula,we study the mechanism of action of Tangshen formula on DN in TGF-β1 and unbalance of synthesis and degradation of extracellular matrix which is important factor in inducing DN,and provide theory evidence for clinical application.Methods:1.Type 1 DN rat model was induced by right-kidney nephrectomy plus peritoneal injection of low-dosage streptozotocin（40 mg/kg） a week later,rat were randomly divided into five groups:normal,model,high-dose TSF,low-dose TSF and monopril,10 rats in each group.general states,weigh,blood glucose,Urine protein/creatinine were observed in the 0^th, 4^th,8^th,12^th,16^th,20th week.The rats were sacrificed at the end of the 20th week.Whole Blood viscosity and blood plasma viscosity were detected by blood rheometer.Blood fat total serum protein,albumin and function of kidneys were detected by blood biochemistrymeter. Pathological change of glomerular and renal tubularinterstitium were observed by semiquantitative assessment.CollagenⅣand TGF-β1 of kidneys were detected by immunohistochemistry.the mRNA expression of TGF-β1、MMP-2 and MMP-9 were mesured by RT-PCR.2.Spontaneous animal model of type 2 DN--OLETF rats were introduced abroad. The rats were randomly divided into model group,TSF group and Monopril group,LETO rats with same genetic background but not developing diabetic symdromes,same week age and sex were used as control group.General states,weight,blood glucose,Urine protein in 24 hours of the rats are observed in every 4 weeks.And successive administration was made for 24 weeks and 44 weeks.The rats were sacrificed at 36 and 56 weeks of age respectively. Blood viscosity and plasma viscosity were detected by blood rheometer.Serum total serum protein,albumin,lipid and function of kidneys were detected by blood biochemistrymeter. Pathological change of glomerular and renal tubularinterstitium were observed by semiquantitative assessment.Result:1 Effects of Tangshen fomula on type 1 DN rats induced by streptozotocin:1) General state and weight of model group was significantly worse than that of control group,and the difference was more significant followed by the progress of the experiment, and the differece was peaking in 20^th week.The general state of rats of every medication administration group was better than that of model group.Body weight of low-dose TSF group was increased from 4^th week and continued until the end of the experiment.Urine protein/creatinine of the rats of model group were signicantly increased from 12^th week.Urine protein / creatinine of rats of TSF group and Monopril group was signicantly lower than that of model group from 16^th week.Blood glucose,cholesterol,triglyceride,plasma viscosity, Blood urea nitrogen,Glomerulosclerosis index（GSI） and renal tubularinterstitial fibrosis index（RIFI） of model group were higher significantly than that of control group.Plasma viscosity,cholesterol,triglyceride,GSI and RIFI of high dose TSF group was lower than that of model group.Blood glucose,urea nitrogen,whole blood viscosity,total serum protein, albumin,creatinine of rats of TSF group were the same as that of model group.2) The result of immunohistochemistry shows that the expression of renal tissue of model group was higher significantly than that of control group.TGF-βlandⅣcollagen of low dose TSF and high dose TSF group were lower than that of mdel group.Experiment of RT-PCR shows that expression of mRNA of TGFβ1 of renal tissue of model group was higher significantly than that of control group.Expression of mRNA of MMP-9 of renal tissue of model group was lower significantly than that of control group.Expression of mRNA of TGFβ1 of renal tissue of high dose TSF group was lower significantly than that of model group.Expression of mRNA of MMP-9 of renal tissue of high dose TSF group was higher significantly than that of model group.Expressions of renal tissue of MMP-2 among the groups were no different.2.The effect of TSF on OLETF rats--a spontaneous animal model of type 2 DNBody weight of model group of rats was significantly lower than control group from 6 weeks old.The difference was most significant at 40 weeks of age,and then the difference becomes less.And at 52 weeks of age,there was no difference between two group.Body weight of TSF group was significantly lower than model group in 40,44 and 48 week of age, Blood glucose and urine protein of 24 hour of model group was significantly higher than those of control group from 6 week of age,and followed by the progression of the experiment, and the difference was biggest at 56 week of age.Urine protein of 24 hour of TSF group was significantly lower than that of model group from 36 week of age;blood glucose of TSF group was significantly lower than that of model group from 52 weeks of age.Plasma viscosity,triglyeride,GSI,RIFI of model group was significantly higher than those of control group in 36 week of age.Plasma viscosity of TSF group was significantly lower than that of model group,the effect is better than that of monopril.Triglyceride of monopril group was significantly lower than that of model group.The effect is better than that of TSF group.GSI and RIFI in TSF group and monopri group was significantly lower than that of model group. Cholesterol,triglyceride,whole blood viscosity,plasma viscosity,urea nitrogen,GSI,RIFI of model group were higher than those of control group in 56 week of age.Level of total serum protein and albumin of model group were lower than those of control group.Blood viscosity, plasma viscosity,cholesterol,GSI and RIFI of high dose TSF gorup were significantly lower than those of model group.The level of total serum protein and albumin of TSF group was significantly higher than those of model group.There was no difference between every group in the level of urea nitrogen.Conclusion:1 TSF can reduce urinary protein excretion,improve the impairment of kidney,the mechanism is related to improve unbalance of lipid metabolism and abnormal haemorheology,but it is not related with blood glucose.2 Accumulation of extracellular matrix is an key cause in the pathogenesis progression of DN.TSF can inhibit the expression of TGF-β1 mRNA and protein of type 1 DN,and increase the expression of MMP-9 mRNA,reduce the expression of collageⅣwhich is the main component of excellular matrix,this is the important mechanism of TSF of protect effect on rat with type 1 DN.3 TSF can reduce 24 hour urine protein of OLETF rats in early stage and advanced stage,and could improve pathological changes of glomerular and renal tubularinterstitium. Correcting disorder of lipid and glucose metabolism,improving blood rheology are renoprective mechanism of TSF on type 2 DN.4 Correcting disorder of lipid metabolism and abnormal haemorheology share common mechanism of treating effect of TSF on animal model of type 1 and type 2 DN.更多还原