蔗糖铁对肾小管上皮细胞氧化应激及纤维化指标的影响
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摘要
目的探讨蔗糖铁刺激对肾小管上皮细胞纤维化的影响及其机制。方法使用0.1 mmol/L的蔗糖铁分别刺激HK-2细胞12 h、24 h、36 h,亚铁离子探针FeRhoNox及羟自由基探针(hydroxyphenyl fluorescein,HPF)检测HK-2细胞内亚铁及羟自由基含量,Western blot法检测细胞TGF-β1、CollagenⅠ、CollagenⅣ、Fibronectin蛋白表达。结果荧光半定量分析显示,0.1 mmol/L蔗糖铁刺激HK-2细胞12 h后,细胞内亚铁离子是对照组的(8.7±1.2)倍(P <0.01),细胞内羟基是对照组(9.0±1.0)倍(P <0.01);24 h后细胞内亚铁离子是对照组的(8.6±1.2)倍(P <0.01),细胞内羟基是对照组的(9.2±0.5)倍(P <0.01);36 h后细胞内亚铁离子是对照组的(8.8±0.8)倍(P <0.01),细胞内羟基是对照组的(8.6±1.0)倍(P <0.01)。0.1mmol/L蔗糖铁刺激HK-2细胞24 h和36 h后,TGF-β1、CollagenⅠ、CollagenⅣ、Fibronectin的表达明显增加。结论 0.1mmol/L的蔗糖铁刺激HK-2细胞增加细胞内氧化应激,与细胞纤维化相关。
Objective To investigate the effect of iron sucrose stimulation on renal tubular epithelial cell fibrosis and its mechanism.Methods HK-2 cells were stimulated with 0.1 mmol/L sucrose iron for 12 h, 24 h, 36 h. Ferrous ion probe FeRhoNox and hydroxyl radical probe hydroxyphenyl fluorescein(HPF) were used to detect ferrous and hydroxyl radicals in HK-2 cells. The expression of TGF-β1, collagenⅠ, collagenⅣand fibronectin were detected by western blotting. Results After stimulation of HK-2 cells with0.1 mmol/L sucrose iron for 12 h, 24 h and 36 h, the intracellular ferrous ions and the intracellular hydroxyl increased(all P<0.01).And the expression of TGF-β1, collagenⅠ, collagenⅣ, fibronectin also increased significantly. Conclusion After the stimulation of sucrose iron(0.1 mmol/L), intracellular ferrous and hydroxyl radicals increase, which is associated with cell fibrosis.
Objective To investigate the effect of iron sucrose stimulation on renal tubular epithelial cell fibrosis and its mechanism.Methods HK-2 cells were stimulated with 0.1 mmol/L sucrose iron for 12 h, 24 h, 36 h. Ferrous ion probe FeRhoNox and hydroxyl radical probe hydroxyphenyl fluorescein(HPF) were used to detect ferrous and hydroxyl radicals in HK-2 cells. The expression of TGF-β1, collagenⅠ, collagenⅣand fibronectin were detected by western blotting. Results After stimulation of HK-2 cells with0.1 mmol/L sucrose iron for 12 h, 24 h and 36 h, the intracellular ferrous ions and the intracellular hydroxyl increased(all P<0.01).And the expression of TGF-β1, collagenⅠ, collagenⅣ, fibronectin also increased significantly. Conclusion After the stimulation of sucrose iron(0.1 mmol/L), intracellular ferrous and hydroxyl radicals increase, which is associated with cell fibrosis.
引文
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2 Koskenkorva-Frank TS,Weiss G,Koppenol WH,et al.The complex interplay of iron metabolism,reactive oxygen species,and reactive nitrogen species:insights into the potential of various iron therapies to induce oxidative and nitrosative stress[J].Free Radic Biol Med,2013,65:1174-1194.
3 Huang ML,Lane DJ,Richardson DR.Mitochondrial mayhem:the mitochondrion as a modulator of iron metabolism and its role in disease[J].Antioxid Redox Signal,2011,15(12):3003-3019.
4 Naito Y,Fujii A,Sawada H,et al.Effect of iron restriction on renal damage and mineralocorticoid receptor signaling in a rat model of chronic kidney disease[J].J Hypertens,2012,30(11):2192-2201.
5 Shah SV,Baliga R,Rajapurkar M,et al.Oxidants in chronic kidney disease[J].J Am Soc Nephrol,2007,18(1):16-28.
6 Duffield JS.Cellular and molecular mechanisms in kidney fibrosis[J].J Clin Invest,2014,124(6):2299-2306.
7 Naito Y,Fujii A,Sawada H,et al.Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease[J].Hypertens Res,2015,38(7):463-470.
8 Ikeda Y,Ozono I,Tajima S,et al.Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction[J].PLoS One,2014,9(2):e89355.
9 Ikeda Y,Horinouchi Y,Hamano H,et al.Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice[J].Sci Rep,2017,7(1):10621.
10 Huynh P,Chai Z.Transforming growth factor beta(TGFbeta)and related molecules in chronic kidney disease(CKD)[J].Clin Sci(Lond),2019,133(2):287-313.
11 Nogueira A,Pires MJ,Oliveira PA.Pathophysiological Mechanisms of Renal Fibrosis:A Review of Animal Models and Therapeutic Strategies[J].In Vivo,2017,31(1):1-22.
12 Veuthey T,D'Anna MC,Roque ME.Role of the kidney in iron homeostasis:renal expression of Prohepcidin,Ferroportin,and DMT1 in anemic mice[J].Am J Physiol Renal Physiol,2008,295(4):F1213-1221.
13 Alfrey AC.Toxicity of tubule fluid iron in the nephrotic syndrome[J].Am J Physiol,1992,263(4 Pt 2):F637-641.
14 Van Raaij SEG,Rennings AJ,Biemond BJ,et al.Iron handling by the human kidney:glomerular filtration and tubular reabsorption both contribute to urinary iron excretion[J].Am J Physiol Renal Physiol,2019,316(3):F606-f614.
15 Smith CP,Thevenod F.Iron transport and the kidney[J].Biochim Biophys Acta,2009,1790(7):724-730.
16 Sheetz M,Barrington P,Callies S,et al.Targeting the hepcidinferroportin pathway in anaemia of chronic kidney disease[J/OL].https://doi.org/10.1111/bcp.13877.
17 Nankivell BJ,Chen J,Boadle RA,et al.The role of tubular iron accumulation in the remnant kidney[J].J Am Soc Nephrol,1994,4(8):1598-1607.
18 Ramm GA,Ruddell RG.Iron homeostasis,hepatocellular injury,and fibrogenesis in hemochromatosis:the role of inflammation in a noninflammatory liver disease[J].Semin Liver Dis,2010,30(3):271-287.