白芍总苷对糖尿病肾脏足细胞相关蛋白的影响及可能机制
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摘要
背景与目的糖尿病肾病是糖尿病的严重并发症及引起终末期肾病的主要病因,其发病率逐年增加。蛋白尿既是DN肾小球损伤的标志,同时也是加速肾脏损害的重要因素。足细胞损伤是糖尿病肾病发生、发展的重要因素,足细胞裂孔隔膜(slit diaphragm,SD)相关蛋白(主要为Nephrin)在维持肾小球滤过屏障结构与功能完整性中发挥重要作用。体外研究发现,巨噬细胞及其分泌的炎症因子可以下调Nephrin基因启动子的活性与蛋白的表达,提示炎症在足细胞损伤过程中起重要作用。白芍总苷(total glucosides of paeony,TGP)为我国传统中草药,目前已应用于多种自身免疫性疾病的治疗。本研究以Nephrin为切入点,探讨白芍总苷对糖尿病大鼠肾脏足细胞相关蛋白Nephrin蛋白表达的影响及机制。
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50,100,200 mg.kg~(-1)不同剂量每日灌胃给药,正常组和模型组每日给予等量溶媒,共8 wk。血糖、肝功能、肾功能与血脂由全自动生化分析仪检测,尿蛋白检测采用酶联免疫法,应用间接免疫荧光法检测肾组织Nephrin分布、Western杂交检测肾组织Nephrin、肿瘤坏死因子-α(TNF-α)与NF-κB-p65蛋白表达。
结果1、各组大鼠一般指标变化与对照组相比,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,TGP(50,100,200 mg.kg~(-1))给药8 wk没有防止模型组大鼠血糖升高与体重下降。TGP(50,100,200 mg.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。模型组大鼠尿白蛋白排泄率(Urinary albumin excretion rate,UAER)明显高于对照组,TGP 50,100,200mg.kg~(-1)给药8 wk大鼠UAER水平明显低于模型组。模型组大鼠Ccr明显低于对照组,TGP(50,100,200 mg.kg~(-1))给药组Ccr水平与模型组相比无明显差异。模型组大鼠血TC、TG水平明显高于对照组,TGP(50,100,200 mg.kg~(-1))给药组血TC、TG水平与模型组相比无明显差异。另外,模型组、各给药组与对照组相比,血谷丙转氨酶、谷草转氨酶无明显变化,提示TGP对肝功能指标无明显影响。模型组大鼠24hAER明显高于给药组,TGP治疗组大鼠24hAER明显减低。2、各组大鼠肾组织Nephrin分布及表达变化免疫荧光显示Nephrin在对照组大鼠肾小球呈线状均匀分布,糖尿病组大鼠肾小球表达明显减少、且呈颗粒状不均匀分布;TGP不同剂量干预组Nephrin表达不同程度的增加,呈线状及颗粒状分布。Western印迹条带光密度分析显示糖尿病模型组Nephrin较对照组表达下降81.1%;TGP干预组Nephrin量较糖尿病组有明显增加,与剂量不成正相关,TGP 50,100,200mg.kg~(-1)给药8 wk使肾组织Nephrin蛋白表达分别上调25.2%,89.2%与73.9%。3、各组大鼠肾组织TNF-α蛋白表达变化Western印迹条带光密度分析显示模型组肾组织TNF-α蛋白表达较对照组增加9.2倍,TGP 50,100,200 mg.kg~(-1)给药8 wk使肾组织TNF-α蛋白表达分别下降46.5%,86%与93.8%。4、各组大鼠肾组织NF-κB-p65蛋白表达变化Western印迹条带光密度分析显示模型组肾组织NF-κB-p65蛋白表达明显高于对照组,TGP给药(50,100与200 mg.kg~(-1))组肾组织NF-κB-p65蛋白表达较模型组分别降低38.4%,53%与59%。
结论TGP降低糖尿病大鼠尿蛋白机制可能部分与增加肾小球Nephrin表达有关。
BACKGROUND AND OBJECTIVE Diabetic nephropathy is a serious complication of diabetes mellitus and the major cause of end-stage renal disease,its incidence has increased year by year.Proteinuria is not only a cardinal manifestation of glomerular injury in diabetes,but also an important pathogenetic factor in the progression of renal dysfunction.Podocyte injury is an important factor in the occurance and development of diabetic nephropathy,podocyte slit diagrame associated protein play an important role in the maintentance of glomerular filtration barrier integrity of the structure and function.In vitro study found that macrophages and their secretion of inflammatory factors may reduce Nephrin gene promoter activity and protein expression,suggesting that inflammation plays an important role in podocyte injury.Total glucosides of paeony(TGP) was the traditional Chinese herbal medicine in our country,it has been used in a variety of autoimmune diseases. In the present study,we investigate the effect of total glucosides of paeony(TGP) on the expression of Nephrin and its mechanism in the kidney from diabetic rats.
METHODS Fifty adult male Sprague-Dawley rats were devideded into five groups at random.Control group(n=10),model group(n=10),model group treated with TGP (50 mg kg~(-1),by gavage,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 the experimental model of diabetes,rats received a single intraperitoneal injection of STZ(60mg kg ~(-1.) d~(-1)).TGP was orally administered.Control group and model group were treated with the same vehicle alone.Eight weeks after STZ injection,the following determinations were done in samples:(1) plasma glucose(BG),liver function,kidney function and plasma lipid were determined according to standard methods;(2) 24 hours urinary albumin excretion rate(UAER) were measured by enzyme immunoassay(EIA); The expression of Nephrin,tumor necrosis factor-α(TNF-α) and NF-κB-p65 protein were determined by immunofluorescence or western blot.
RESULTS Elevated AER was markedly attenuated by TGP treated.(p＜0.05). There was a finely dotted linear epithelial staining of Nephrin in control group glomeruli.In contrast,the staining of glomeruli from untreated diabetic rats was attenuated,more dispersed and clustered,this diabetic-induced loss of glomerular Nephrin expression was largly prevented in TGP-treated diabetic rats.Western blot analysis showed that the expression of Nephrin protein was reduced in the kidney from diabetic rats,but significantly increased with TGP(p＜0.01,p＜0.05).The expression of TNF-αand NF-κB-p65 protein in the kidney were significantly increased in diabetic rats,which were all significantly inhibited by TGP treated(p＜0.01).
CONCLUSION TGP could decrease AER in diabetic rat,which mechanism may be at least partly correlated with upregulated the expression of Nephrin in the kidney.
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50,100,200 mg.kg~(-1)不同剂量每日灌胃给药,正常组和模型组每日给予等量溶媒,共8 wk。血糖、肝功能、肾功能与血脂由全自动生化分析仪检测,尿蛋白检测采用酶联免疫法,应用间接免疫荧光法检测肾组织Nephrin分布、Western杂交检测肾组织Nephrin、肿瘤坏死因子-α(TNF-α)与NF-κB-p65蛋白表达。
结果1、各组大鼠一般指标变化与对照组相比,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,TGP(50,100,200 mg.kg~(-1))给药8 wk没有防止模型组大鼠血糖升高与体重下降。TGP(50,100,200 mg.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。模型组大鼠尿白蛋白排泄率(Urinary albumin excretion rate,UAER)明显高于对照组,TGP 50,100,200mg.kg~(-1)给药8 wk大鼠UAER水平明显低于模型组。模型组大鼠Ccr明显低于对照组,TGP(50,100,200 mg.kg~(-1))给药组Ccr水平与模型组相比无明显差异。模型组大鼠血TC、TG水平明显高于对照组,TGP(50,100,200 mg.kg~(-1))给药组血TC、TG水平与模型组相比无明显差异。另外,模型组、各给药组与对照组相比,血谷丙转氨酶、谷草转氨酶无明显变化,提示TGP对肝功能指标无明显影响。模型组大鼠24hAER明显高于给药组,TGP治疗组大鼠24hAER明显减低。2、各组大鼠肾组织Nephrin分布及表达变化免疫荧光显示Nephrin在对照组大鼠肾小球呈线状均匀分布,糖尿病组大鼠肾小球表达明显减少、且呈颗粒状不均匀分布;TGP不同剂量干预组Nephrin表达不同程度的增加,呈线状及颗粒状分布。Western印迹条带光密度分析显示糖尿病模型组Nephrin较对照组表达下降81.1%;TGP干预组Nephrin量较糖尿病组有明显增加,与剂量不成正相关,TGP 50,100,200mg.kg~(-1)给药8 wk使肾组织Nephrin蛋白表达分别上调25.2%,89.2%与73.9%。3、各组大鼠肾组织TNF-α蛋白表达变化Western印迹条带光密度分析显示模型组肾组织TNF-α蛋白表达较对照组增加9.2倍,TGP 50,100,200 mg.kg~(-1)给药8 wk使肾组织TNF-α蛋白表达分别下降46.5%,86%与93.8%。4、各组大鼠肾组织NF-κB-p65蛋白表达变化Western印迹条带光密度分析显示模型组肾组织NF-κB-p65蛋白表达明显高于对照组,TGP给药(50,100与200 mg.kg~(-1))组肾组织NF-κB-p65蛋白表达较模型组分别降低38.4%,53%与59%。
结论TGP降低糖尿病大鼠尿蛋白机制可能部分与增加肾小球Nephrin表达有关。
BACKGROUND AND OBJECTIVE Diabetic nephropathy is a serious complication of diabetes mellitus and the major cause of end-stage renal disease,its incidence has increased year by year.Proteinuria is not only a cardinal manifestation of glomerular injury in diabetes,but also an important pathogenetic factor in the progression of renal dysfunction.Podocyte injury is an important factor in the occurance and development of diabetic nephropathy,podocyte slit diagrame associated protein play an important role in the maintentance of glomerular filtration barrier integrity of the structure and function.In vitro study found that macrophages and their secretion of inflammatory factors may reduce Nephrin gene promoter activity and protein expression,suggesting that inflammation plays an important role in podocyte injury.Total glucosides of paeony(TGP) was the traditional Chinese herbal medicine in our country,it has been used in a variety of autoimmune diseases. In the present study,we investigate the effect of total glucosides of paeony(TGP) on the expression of Nephrin and its mechanism in the kidney from diabetic rats.
METHODS Fifty adult male Sprague-Dawley rats were devideded into five groups at random.Control group(n=10),model group(n=10),model group treated with TGP (50 mg kg~(-1),by gavage,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 the experimental model of diabetes,rats received a single intraperitoneal injection of STZ(60mg kg ~(-1.) d~(-1)).TGP was orally administered.Control group and model group were treated with the same vehicle alone.Eight weeks after STZ injection,the following determinations were done in samples:(1) plasma glucose(BG),liver function,kidney function and plasma lipid were determined according to standard methods;(2) 24 hours urinary albumin excretion rate(UAER) were measured by enzyme immunoassay(EIA); The expression of Nephrin,tumor necrosis factor-α(TNF-α) and NF-κB-p65 protein were determined by immunofluorescence or western blot.
RESULTS Elevated AER was markedly attenuated by TGP treated.(p＜0.05). There was a finely dotted linear epithelial staining of Nephrin in control group glomeruli.In contrast,the staining of glomeruli from untreated diabetic rats was attenuated,more dispersed and clustered,this diabetic-induced loss of glomerular Nephrin expression was largly prevented in TGP-treated diabetic rats.Western blot analysis showed that the expression of Nephrin protein was reduced in the kidney from diabetic rats,but significantly increased with TGP(p＜0.01,p＜0.05).The expression of TNF-αand NF-κB-p65 protein in the kidney were significantly increased in diabetic rats,which were all significantly inhibited by TGP treated(p＜0.01).
CONCLUSION TGP could decrease AER in diabetic rat,which mechanism may be at least partly correlated with upregulated the expression of Nephrin in the kidney.
引文
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[2]Koop K,Eikmans M,Baelde HJ,et al.Expression of podocyte - associated molecules in acquired human kidney diseases[J].J Am Soc Nephrol,2003,14(8):2063 -2071.
[3]管娜,邓江红,丁洁 足细胞分子分布和表达与足突形态变化和蛋白尿发生的关系[J].北京大学学报(医学版)2004,36(2):139-144
[4]Langham RG,Kelly DJ,Cox AJ,et al.Proteinuria and the expression of the podocyte slit diaphragm protein,nephrin,in diabetic nephropathy:effects of angiotensin converting enzyme inhibition.Diabetologia.2002,45(11):1572-6.
[5]Lee HB,Ha H,Kim SI,et al.Diabetic kidney disease research:where do we stand at the term of the century? Kidney Int,2000,58(supp177):S1-2
[6]Boydl -White J,Williams JC.Effects of cross linking on matripermeability:a model for AGE-modified basement membranes[J].Diabetes,1996,45:348
[7]Haslinger B,Mand-weber S,Sellmager A,et al.Effect of high glucose concentration on the synthesis of monocyte chemoattractant protein-1 in human perineal mesotherial cells;involvement of protein kinase C[J].Nephron,2001,87(4):346-351
[8]Kumar D,Robertson S,Bum K D.Evidence of apoptosis in human diabetic Kidney[J].Mol Cell Biochem,2004,259(1-2):67-70.
[9]Toyoda M,Najafian B,Kim Y,et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy[J].Diabetes,2007,56(8):2155-2160.
[10]Benzing T.Signaling at the slit diaphragm[J].J Am Soc Nephrol,2004,15(6):1382-1391.
[11]Roselli S,Heidet L,Sich M,et al.Early glomerular filtration defect and severe Immunopharmacol.2005;5(10):1560-73.
[23]王斌,陈敏珠,徐叔云.白芍总甙对大鼠腹腔巨噬细胞产生肿瘤坏死因子的调节机制[J].中国药理学通报,1997,13(3):255-257.
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