从SIRT1探讨白藜芦醇抑制肾小球足细胞表型改变的作用机制
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摘要
目的探讨SIRT1在白藜芦醇抑制肾小球足细胞表型改变中的作用,研究白藜芦醇维持足细胞表型的重要机制。方法体外分化条件下培养小鼠足细胞10天,用TGF-β_1诱导作为研究模型,分别用白藜芦醇、SRT1720和EX527以及白藜芦醇与EX527联合干预等。采用RT-PCR和Western blot法等检测足细胞α-平滑肌肌动蛋白(α-smooth muscle-actin,α-SMA)、snail等分子基因表达;NEPH1、结蛋白Desmin和α-SMA等分子蛋白表达。结果 TGF-β1诱导的足细胞α-SMA、snail基因表达较正常组增高,白藜芦醇、SRT1720干预组则较模型组降低。模型组NEPH1蛋白表达较正常组显著降低,而Desmin和α-SMA蛋白表达水平则显著增高。白藜芦醇组NEPH1蛋白表达较模型组显著上调,Desmin和α-SMA表达显著降低,白藜芦醇与EX527同时干预或EX527单独干预,对足细胞表型分子表达则未表现出显著干预作用。结论激活SIRT1是白藜芦醇显著抑制TGF-β1诱导的足细胞表型异常的重要机制。
Objective To investigate the role of SIRT1(silent mating type information regulation 2 homolog 1) in the inhibition of podocyte transdifferentiation by resveratrol,and explore the mechanism of resveratrol to maintain podocyte phenotype.Methods Immortalized mouse podocytes were cultured under differentiating conditions for 10 d in vitro.The podocytes incubated with TGF-β1 were served as research model,and they were treated with resveratrol or SRT1720 or EX527 or resveratrol combined with EX527,respectively.The genetic expressions of α-SMA and snail were verified by RT-PCR.The protein levels of NEPH1,α-SMA and Desmin were determined by western-blotting.Results Compared with normal group,the genetic expression of α-SMA and snail in the podocytes induced with TGF-β1(model group) increased.While the resveratrol and SRT1720 intervention group was lower than that of the model group.The protein expression of NEPH1 decreased significantly in model group compared with normal group,however,the expression of α-SMA and Desmin increased obviously.Besides,the expression of NEPH1 protein in Resveratrol group was significantly higher than that of the model group,while the expression of α-SMA and Desmin decreased significantly.In co-ordinated intervention of resveratrol and EX527 group and EX527 group,the expression of podocyte phenotype molecules including NEPH1,α-SMA and Desmin all showed no significant difference with model group.Conclusion Activating SIRT1 is an important mechanism of resveratrol to significantly inhibit transdifferentiation of podocytes induced by TGF-β1.
Objective To investigate the role of SIRT1(silent mating type information regulation 2 homolog 1) in the inhibition of podocyte transdifferentiation by resveratrol,and explore the mechanism of resveratrol to maintain podocyte phenotype.Methods Immortalized mouse podocytes were cultured under differentiating conditions for 10 d in vitro.The podocytes incubated with TGF-β1 were served as research model,and they were treated with resveratrol or SRT1720 or EX527 or resveratrol combined with EX527,respectively.The genetic expressions of α-SMA and snail were verified by RT-PCR.The protein levels of NEPH1,α-SMA and Desmin were determined by western-blotting.Results Compared with normal group,the genetic expression of α-SMA and snail in the podocytes induced with TGF-β1(model group) increased.While the resveratrol and SRT1720 intervention group was lower than that of the model group.The protein expression of NEPH1 decreased significantly in model group compared with normal group,however,the expression of α-SMA and Desmin increased obviously.Besides,the expression of NEPH1 protein in Resveratrol group was significantly higher than that of the model group,while the expression of α-SMA and Desmin decreased significantly.In co-ordinated intervention of resveratrol and EX527 group and EX527 group,the expression of podocyte phenotype molecules including NEPH1,α-SMA and Desmin all showed no significant difference with model group.Conclusion Activating SIRT1 is an important mechanism of resveratrol to significantly inhibit transdifferentiation of podocytes induced by TGF-β1.
引文
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14 赵敬,王颖超,赵宗江,等.糖肾平通过TGF-β1-Smad2/3-ILK信号通路干预高糖+LPS诱导足细胞上皮间质转分化的分子机制研究[J].中国中西医结合肾病杂志,2014,5:392-396
15 Mahmood MQ,Reid D,Ward C,et al.Transforming growth factor(TGF)β1 and Smad signalling pathways:a likely key to EMT-associated COPD pathogenesis[J].Respirology,2017,22(1):133
16 Huang XZ,Wen D,Zhang M,et al.Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-β/Smad3 pathway[J].J Cell Biochem,2014,115(5):996
17 曹端方.白藜芦醇促进SIRT1去乙酰化酶活性的结构生物学研究[D].中国科学院大学,2015
18 Barletta GM,Kovari IA,Verma RK,et al.Nephrin and Neph1 colocalize at the podocyte foot process intercellular junction and form cis hetero-oligomers[J].J Biol Chem,2003,278(21):19266
19 Sellin L,Huber TB,Gerke P,et al.NEPH1 defines a novel family of podocin interacting proteins[J].FASEB J,2003,17(1):115
2 Tagawa A,Yasuda M,Kume S,et al.Impaired podocyte autophagy exacerbates proteinuria in diabetic nephropathy[J].Diabetes,2016,65(3):755-767
3 Yingjian L,Young Sun K,Chunsun D,et al.Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria[J].Am J Pathol,2008,172:299-308
4 Ying Q,Wu G.Molecular mechanisms involved in podocyte EMT and concomitant diabetic kidney diseases:an update[J].Renal Fail,2017,39(1):474.5
5 李明红.白藜芦醇对低氧内皮细胞的保护作用及机制研究[D].广州:广东药科大学,2016
6 Kur2vietiene L,Stanevicien 6)e I,Mongirdien 6)e A,et al.Multiplicity of effects and health benefits of resveratrol[J].Medicina,2016,52(3):148-155
7 张志全,高辉,刘莹男,等.白藜芦醇对糖尿病肾病大鼠氧化应激反应的影响研究[J].黑龙江医药科学,2015,38(1):88-90
8 梁瑾.白藜芦醇对肾脏纤维化的保护作用及相关机制研究[D].苏州:苏州大学,2015
9 杨汝春,朱晓玲,张华琴,等.白藜芦醇对TGF-β1诱导的肾小球足细胞转分化抑制作用研究[J].中国中西医结合杂志,2013,33(12):1677-1682
10 Grahammer F.New structural insights into podocyte biology[J].Cell Tissue Res,2017,369(1):1-6
11 Lin X,Zhen X,Huang H,et al.Role of MiR-155 signal pathway in regulating podocyte injury induced by TGF-β1[J].Cell Physiol Biochem,2017,42(4):1469-1480
12 叶迅,侯鹏超,朱伶俐,等.雷公藤内酯醇干预高糖诱导足细胞转分化作用机制的研究[J].浙江医学,2015,37(17):1428-1431
13 杨莉莉,刘章锁.GSK-3β在高糖环境下足细胞转分化效应中的作用[J].重庆医学,2015,44(12):1609-1612
14 赵敬,王颖超,赵宗江,等.糖肾平通过TGF-β1-Smad2/3-ILK信号通路干预高糖+LPS诱导足细胞上皮间质转分化的分子机制研究[J].中国中西医结合肾病杂志,2014,5:392-396
15 Mahmood MQ,Reid D,Ward C,et al.Transforming growth factor(TGF)β1 and Smad signalling pathways:a likely key to EMT-associated COPD pathogenesis[J].Respirology,2017,22(1):133
16 Huang XZ,Wen D,Zhang M,et al.Sirt1 activation ameliorates renal fibrosis by inhibiting the TGF-β/Smad3 pathway[J].J Cell Biochem,2014,115(5):996
17 曹端方.白藜芦醇促进SIRT1去乙酰化酶活性的结构生物学研究[D].中国科学院大学,2015
18 Barletta GM,Kovari IA,Verma RK,et al.Nephrin and Neph1 colocalize at the podocyte foot process intercellular junction and form cis hetero-oligomers[J].J Biol Chem,2003,278(21):19266
19 Sellin L,Huber TB,Gerke P,et al.NEPH1 defines a novel family of podocin interacting proteins[J].FASEB J,2003,17(1):115