【作者】 陈晶波；

【导师】 徐向进；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 硕士

【摘要】 目的泛素蛋白酶复合体系统是细胞内氧化蛋白降解的主要途径,糖尿病氧化应激可引起细胞内蛋白空间结构异常,从而进入泛素蛋白酶复合体系统降解。因此,本研究旨在揭示高糖高脂喂养结合小剂量STZ诱导的2型糖尿病大鼠肾脏组织泛素蛋白酶复合体系统介导NF-κB的活化与24小时尿蛋白、肾脏病理改变之间的关系;并通过观察吡格列酮、复方丹参滴丸浸膏、槲皮素药物干预对2型糖尿病大鼠肾脏组织泛素、NF-κB表达的影响,以揭示上述三种药物对2型糖尿病肾脏保护作用的机制。方法清洁级SD雄性大鼠52只,适应性喂养1周,随机分为2组,正常对照组(NC组,n=8只)给予普通饲料喂养;造模组44只,给予高糖高脂饲料。喂养4周后,腹腔注射链脲佐菌素(STZ,30mg·kg-1)制备2型糖尿病大鼠模型。72小时后,尾静脉采血随机两次血糖≥16.7mmol/L,视为造模成功,成模大鼠继续给予高糖高脂饲料喂养,并再次随机分为4组:糖尿病对照组(DM组,n=9只)、吡格列酮治疗组(PIO组,n=9只,4 mg·kg-1·d-1)、复方丹参滴丸浸膏治疗组(DSP组,n=9只,500 mg·kg-1·d-1)、槲皮素治疗组(QUE组,n=9只,100 mg·kg-1·d-1);上述三种药物均溶于蒸馏水等量灌胃,NC组和DM组给予等量蒸馏水灌胃。治疗8周后各组大鼠分别脱臼处死,处死前一日收集24小时尿,检测24小时尿蛋白定量;下腔静脉取血检测空腹血糖(GLU)、空腹胰岛素(INS)、胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、尿素氮(BUN)、肌酐(Scr)。取双肾称重,计算肾脏肥大指数(肾重/体重),10%福尔马林固定,石蜡切片, PASM染色观察肾小球细胞外基质的堆积,并分别采用免疫组化、Western-blot方法检测肾脏组织中泛素、NF-κB p65蛋白表达。数据运用SPSS13.0统计软件分析。结果实验结束时高糖高脂喂养结合小剂量STZ诱导的2型糖尿病SD大鼠,均表现为持续蛋白尿及肾小球体积增大、基底膜、系膜基质增生、局灶节段性硬化,肾小管空泡变性等糖尿病肾病典型变化。与NC组相比较,高糖高脂喂养结合小剂量STZ诱导的2型糖尿病大鼠空腹血糖、空腹胰岛素、肌酐、尿素氮、血清胆固醇、甘油三酯、低密度脂蛋白胆固醇水平均明显增高(P<0.01);高密度脂蛋白胆固醇水均明显降低(P<0.05);24小时尿蛋白总量、肾脏肥大指数、肾小球基质堆积指数亦均明显增高(P<0.01);肾脏组织泛素、NF-κB p65蛋白的表达水平均明显增高(P<0.01)。与DM组比较,三个治疗组空腹血糖、空腹胰岛素、肌酐、尿素氮、血清胆固醇、甘油三酯、低密度脂蛋白胆固醇水平均较低(P﹤0.01);高密度脂蛋白胆固醇水平无明显差异(P﹥0.05);24小时尿蛋白总量、肾脏肥大指数、肾小球基质堆积指数均较低(P﹤0.01);肾脏组织泛素、NF-κB p65蛋白的表达水平均较低(P﹤0.01)。三种药物干预治疗后,上述指标均有不同程度改善,但仍未降至正常,三个治疗组组间上述指标无明显差异(P﹥0.05)。各组大鼠24小时尿蛋白定量与Western-blot检测泛素、NF-κB p65蛋白表达水平呈正相关(P﹤0.01)。各组大鼠肾小球基质堆积指数与Western-blot检测泛素、NF-κB p65蛋白表达水平呈正相关(P﹤0.01)。结论1、2型糖尿病大鼠出现持续蛋白尿、糖尿病肾病早期病理改变,与泛素、NF-κB p65表达呈正相关;2、吡格列酮、复方丹参浸膏及槲皮素三种药物干预后均可改善糖脂代谢,减轻氧化应激,下调肾组织泛素、NF-κB p65表达,从而减轻肾脏炎症反应,降低24小时尿蛋白,减轻糖尿病肾病病理改变,延缓糖尿病肾病进展。更多还原

【Abstract】 ObjectiveThe Ubiquitin-proteasome system is principal degration route of intracellular and oxidized proteins, thus diabetes-induced oxidative stress can lead to protein misfolding and degradation by the ubiquitin-proteasome system. The aim of this study was to evaluate the activity of ubiquitin-proteasome system in renal tissues of type 2 diabetic rats in relation to inflammation and its renal structural lesions, and to observe the effecs of Pioglitazone, Danshen dripping pill and Quercetin on the renal expression of ubiquitin and NF-κB p65 and to investigate its renoprotective effects.MethodAfter breed adaptively for 1 week, a total of 52 specific pathogen-free male SD rats were randomly divided into 2 groups:normal group for comparison purpose ( group NC, n=8 ) was fed for regular diet and experimental model group ( n=44 ) was fed with fat enriched diets.The rats of experimental model groups were intraperitoneally given low-dose streptozotocin ( STZ, 30 mg/kg ) after having the sucrose and fat enriched diets for 4 weeks. After 72 hours, measured randomly blood glucose twice and the one whose blood glucose was equal or above 16.7mmo1/L was enlisted. The modeling rats were further randomly divided into four groups: type 2 diabetic group ( Group DM, n=9 ) , pioglitazone group ( Group PIO, n=9, 4 mg·kg-1·d-1 ) , danshen dripping pill group ( Group DSP, n=9, 500 mg·kg-1·d-1 ) and quercetin group ( Group QUE, n=9, 100 mg·kg-1·d-1). The rats of treatmental group were performed by intragastric administration for 8 weeks. The rats of two control groups were performed by intragastric administration with equivalent distilled water for 8 weeks. Before the rats were sacrificed, we collected 24 hours urine for measurement of 24h urinary protein, then killed the rats, gathered blood from vein immediately, and measured their fasting plasma glucose, fasting serum insulin, serum creatinine, blood urea nitrogen, triglyceride, total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol.After that, the kidneys were removed and weighed to calculate the ratio of kidney weight vs body weight ( KW/BW ) . Put the kidney into 10% formalin to be fixed and made paraffinslices. Observed the pathological changes of the kidney and studied the expression of ubiquitin and NF-κB p65 in the kidney with immunohistochemical and Western-blot . The data were analyzed by SPSS13.0 system.ResultsThe features of renal involvement in the model included overt persistent proteinuria and typical morphological changes, Histologically the earliest abnormality was mainly glomerular hypertrophy, Vascular changes, mild mesangial expansion and vacuolation of tubularelinal cell were came forth at the end of exprement.Compared with group NC, the level of fasting plasma glucose, fasting serum insulin, triglyceride, total cholesterol, low density lipoprotein cholesterol, serum creatinine, blood urea nitrogen, 24h urinary protein, the ratio of kidney weight to body weight ( KW/BW ), extracellular matrix accumulation index and the expressions of ubiquitin and NF-κB p65 in type 2 diabetic rats were significantly increased ( P﹤0.01 ), the level of high density lipoprotein cholesterol in group DM was decrease( P﹤0.05 ).Compared with group DM, the level of fasting plasma glucose, fasting serum insulin, triglyceride, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, serum creatinine, blood urea nitrogen, 24h urinary protein, the ratio of kidney weight to body weight ( KW/BW ), extracellular matrix accumulation index and the expressions of ubiquitin and NF-κB p65 in group PIO, DSP and QUE were significantly decrease( P﹤0.01 ). the level of high density lipoprotein cholesterol in group PIO, DSP and QUE was no significant difference with group DM( P﹥0.05 ). Afer treamwnt, each index was improved, but not reversed to normal level. It has no significant difference between group PIO, DSPand QUE (P﹥0.05).The expression of ubiquitin and NF-κB p65 were positively related with the level of 24h urinary protein and extracellular matrix accumulation index.Conclusion①The features of renal involvement in the type 2 diabetic rats included overt persistent proteinuria and typical morphological changes. Increasing expression of ubiquitin and NF-κB p65 in type 2 diabetic rats were positively related with functional and pathological damage in rats renal.②Pioglitazone, danshen dripping pill and quercetin can improve glucolipid metabolism, reduce oxidative stress and decrease the expression of ubiquitin and NF-κB p65 in renal tissues of type 2 diabetic rats. Thus they exerts the actions against inflammatory response and to decrease 24h urinary protein, which may have benefit in diabetic nephropathy.更多还原