【作者】 方芳；

【导师】 吴永贵；

【作者基本信息】 安徽医科大学 ， 内科学， 2008， 硕士

【摘要】 背景与目的DN已成为ESRD的主要病因之一,其发生与发展的内在机制尚未完全阐明。近年研究表明,氧化应激在糖尿病并发症包括DN的发生和发展中起重要作用,应用抗氧化剂可有效延缓DN的进展。TGP是中药白芍根中提取的有效成分,它主要含有芍药苷、羟基芍药苷、芍药花苷、芍药内酯苷及苯甲酰芍药苷等,具有抗炎、抗氧化和免疫调节等活性。我们既往的研究表明,TGP对DN具有明显保护作用,但不降低血糖和血脂的水平。本研究进一步观察TGP对实验性糖尿病大鼠肾脏氧化应激的影响,以探讨其抗DN的作用机制。方法应用链脲佐菌素诱导大鼠糖尿病模型,随机分:正常组、模型组、TGP给药组。TGP按50,100,200mg·kg^-1不同剂量每日灌胃给药,对照组和糖尿病组给予等量溶媒。所有大鼠在整个实验期间喂标准饮食,自由饮水,不应用胰岛素,观察8wk。血糖由全自动生化分析仪检测,PAS染色对肾组织作病理形态学检查同时采用彩色病理图像分析系统测定肾小球平均容积V_G,TⅡ,肾组织T-AOC,SOD,CAT和GSH-PX活性由分光光度法检测,肾组织NT蛋白的表达采用Western印迹方法检测,肾小管-间质OPN,α-SMA表达采用免疫组化方法检测。结果1.各组大鼠一般指标变化与对照组相比,糖尿病模型组大鼠表现为血糖升高、体重下降、相对肾重（肾重/体重）增加（P＜0.05,P＜0.01）,TGP(50,100,200mg·kg^-1)给药8wk没有防止模型组大鼠血糖升高与体重下降。TGP(50,100,200mg·kg^-1)给药组大鼠相对肾重与模型组相比有所下降,但差异无统计学意义。2.各组大鼠肾脏病理形态学变化模型组大鼠V_G与TⅡ明显高于对照组,TGP 50mg·kg^-1给药组大鼠V_G明显低于模型组,TⅡ较模型组有所下降,但差异未达统计学意义,TGP 100,200mg·kg^-1给药组V_G与TⅡ均明显低于模型组（P＜0.05,P＜0.01）。3.肾组织T-AOC,SOD,CAT和GSH-PX活性变化与正常对照组比较,糖尿病模型组大鼠肾组织T-AOC,SOD与CAT活性明显降低（P＜0.01）,GSH-PX活性无明显变化;TGP 200mg·kg^-1给药8wk肾组织T-AOC、SOD与CAT活性均明显高于模型组（P＜0.05,P＜0.01）,对GSH-PX活性无明显影响。4.各组大鼠肾组织NT蛋白表达变化Western印迹结果表明,糖尿病模型组肾组织NT蛋白表达较正常对照组增加3.4倍,TGP 50,100和200 mg·kg^-1给药8wk后肾组织NT蛋白表达与模型组相比分别下降41.2%、43.8%和57.5%。5.各组大鼠肾小管-间质OPN蛋白、α-SMA蛋白表达变化免疫组化显示照组大鼠肾小管-间质有微弱OPN蛋白表达,模型组肾小管-间质OPN与α-SMA蛋白表达明显高于对照组（p＜0.01）,TGP 100,200mg·kg^-1给药组肾小管-间质OPN表达明显低于模型组（p＜0.01）。免疫组化显示对照组大鼠肾小管-间质有微弱α-SMA蛋白表达,模型组。肾小管-间质α-SMA蛋白表达明显高于对照组（p＜0.01）,TGP 50,100,200mg·kg^-1给药组肾小管-间质α-SMA蛋白表达明显低于模型组（p＜0.01）。结论糖尿病大鼠肾脏存在氧化应激反应,TGP抗糖尿病肾病的作用可能与其抗氧化活性有关。更多还原

【Abstract】 Background and objective Diabetic nephropathy（DN） has become one of the main cause of end-stage renal disease（ESRD）,but unfortunately the intimate mechanisms leading to the development and progression of renal injury are not yet fully known.The recent study in vivo and in vitro evidence demonstrated that oxidative stress play a major role in the development and progression of diabetic nephropathy,and that antioxidant treatment had significant protective effects on DN.Total glucosides of paeony（TGP） are active compounds extracted from the roots of the tradition herb Paeonia lactiflora Pall,its mainly composed of peoniflorin,hydroxide radical peoniflorin, peonin,albiflorin and benzoylpaeoniflorin.It possesses a variety of pharmacological functions,including anti-inflammation,anti-oxidative stress and immunoregulatory activity. The purpose of the present study was to investigate the effects of TGP on Oxidative stress in renal tissue of diabetic rats induced by STZ and explore the possible mechanism.Methods Fifty adult male Sprague-Dawley rats were separated into five groups at random.Control group（n=10）,model group（n=10）,model group treated with TGP 50 mg·kg^-1（n=10）,model group treated with TGP 100 mg·kg^-1（n=10） and model group treated with TGP 200 mg·kg^-1（n=10）.To induce an experimental model of diabetes, rats received a single intraperitoneal injection of STZ(60mg·kg^-1 d^-1).TGP was given by gavage.8 weeks after STZ injection,the following determinations were done in samples:（1） BG were determined according to standard methods;（2） Renal lesions were evaluated using PAS staining,the mean glomerular volume（V_G） and indices for tubulointerstitial injury（TII） were measured by using pathology image analysis system software;（3） Renal tissue T-AOC,GSH-PX,CAT,SOD were determined by spectrophotometric method;（4） Expression of nitrotyrosine（NT） was measured by Western blot analysis;（5） Expression of osteopontin（OPN） andα-smooth muscle actin （α-SMA） in renal tubulointerstitium were determined by immunohistochemistry method.Results 1.BG,Body weight（BW） ratio of KW to BW（KW/BW）:8 weeks after STZ injection,there was a significant increase in BG（P＜0.01） and significantly decreased in BW（p＜0.01） in diabetic rats compared with control group.The BG level of TGP group(50mg,100mg and200mg·kg^-1) have no statistically significant difference compared with model group.KW/BW in TGP group(50mg,100mg and 200mg·kg^-1) was decreased compared with model group,but there have no statistically significant.2. Renal pathologic morphology:Compared with control group,V_G and TII in model group was significantly increased.V_G in TGP group(50mg·kg^-1) was significantly lower than model group（P＜0.05）,TII in TGP group(50mg·kg^-1) was lower than model group,but there have no statistically significant.V_G and TII in TGP group(100,200mg·kg^-1) was significantly lower than model group（P＜0.05,P＜0.01）.3.T-AOC,GSH-PX,CAT,SOD activities in renal tissue:The T-AOC,SOD and CAT activities in renal tissue of model group significantly decreased compared with that of control group（P＜0.01）.TGP treatment with 200mg·kg^-1 increased the T-AOC,SOD and CAT activities（P＜0.01,P＜0.05）.In addition,GSH-PX have no difference between control group,model group,and TGP group(50,100,200mg·kg^-1).4.NT expression in renal tissue:Western blot analysis noted that the expression of NT protein in ranal tissue of model group increased by 3.4 folds to that of control group,TGP treatment with 50,100 and 200mg.kg^-1 reduced the increased expression of NT protein by 41.2%,43.8%and 57.5%,respectively.5.OPN andα-SMA protein expression in renal tubulointerstitium:There was minimal immunohistohemistrical staining for OPN in renal tubulointerstitium of control group rats.Immunostaining for OPN was markedly increased in model group in tubulointerstitium（P＜0.01）,elevated expression of OPN protein in renal tubulointerstitium in diabetic rats were only significantly inhibited by TGP treatment with 100 and 200mg.kg^-1（p＜0.01）.There was minimal immunohistohemistrical staining forα-SMA in renal tubulointerstitium of control group rats.Immunostaining forα-SMA was markedly increased in model group in tubulointerstitium（P＜0.01）,and increased expression ofα-SMA protein were significantly inhibited by TGP treatment with 50,100 and 200mg.kg^-1（p＜0.01）.Conclusion The oxidative stress is increased in the diabetic rat kidney,and TGP can prevent renal damage associated with diabetes by attenuating the oxidative stress.更多还原