甘草黄酮拮抗高糖诱导的视网膜色素上皮细胞损伤及分子机制
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摘要
目的探讨甘草黄酮对高糖刺激下人视网膜色素上皮细胞(RPE)氧化应激的影响。方法采用40 mmol·L~(-1)的葡萄糖培养RPE细胞建立细胞氧化损伤模型,用10~(-9)mol/L、10~(-8)mol/L浓度的甘草黄酮干预,观察细胞活性、细胞内活性氧(ROS)水平、凋亡细胞比例及凋亡相关蛋白表达的变化。结果 MTT试验结果显示,用10-9mol/L、10-8mol/L浓度的甘草黄酮处理后,RPE细胞存活率分别提高到67.86%±3.23%和78.67%±2.57%,与高糖组(40.45%±3.27%)比较,差异具有统计学意义(P<0.01);2',7'-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针染色结果显示,甘草黄酮可不同程度抑制40 mmol/L葡萄糖处理后RPE细胞内ROS水平;此外,甘草黄酮可以抑制高糖诱导的RPE细胞凋亡及细胞内Caspase-3及Caspase-9的表达,其抑制效应与浓度呈正相关。结论甘草黄酮可以抑制高糖诱导的人眼RPE细胞氧化损伤及凋亡的发生,对糖尿病性视网膜病变具有预防作用。
OBJECTIVE To investigate the effect of licorice flavonoids on high glucose-induced human retina pigment epithelium(RPE) cells under oxidative stress. METHODS The oxidative damage model of RPE cells was induced by 40 mmol·L~(-1)glucose, and intervened with different concentrations of licorice flavonoids, cell survival rate was detected by MTT colorimetry, intracellular reactive oxygen species(ROS) were detected by H_2 DCFDA staining, nucleus apoptosis was observed by Hoechst staining, the expression of caspase-3 and caspase-9 were tested by Western blot. RESULTS The MTT results showed that RPE cell survival rate increased to67.86% ±3.23% and 78.67% ±2.57% after treated with 10~(-9)mol·L~(-1), 10~(-8)mol·L~(-1)licorice flavonoids, the difference was statistically significant when compared with result(40.45%±3.27%)of high glucose group(P <0.01); The H2 DCFDA fluorescent probe dying showed that licorice flavonoids reduced ROS generation in RPE cells and decreased oxidative-damaged cells; Besides,licorice flavonoids inhibited RPE cells apoptosis and the expression of high glucose-induced caspase-3 and caspase-9 expression in RPE cells, the inhibitory effect was positively correlated with the dose. CONCLUSIONS Licorice flavonoids could inhibit high glucose-induced oxidative damage and apoptosis in human RPE cells,which had preventive effect on diabetic retinopathy.
OBJECTIVE To investigate the effect of licorice flavonoids on high glucose-induced human retina pigment epithelium(RPE) cells under oxidative stress. METHODS The oxidative damage model of RPE cells was induced by 40 mmol·L~(-1)glucose, and intervened with different concentrations of licorice flavonoids, cell survival rate was detected by MTT colorimetry, intracellular reactive oxygen species(ROS) were detected by H_2 DCFDA staining, nucleus apoptosis was observed by Hoechst staining, the expression of caspase-3 and caspase-9 were tested by Western blot. RESULTS The MTT results showed that RPE cell survival rate increased to67.86% ±3.23% and 78.67% ±2.57% after treated with 10~(-9)mol·L~(-1), 10~(-8)mol·L~(-1)licorice flavonoids, the difference was statistically significant when compared with result(40.45%±3.27%)of high glucose group(P <0.01); The H2 DCFDA fluorescent probe dying showed that licorice flavonoids reduced ROS generation in RPE cells and decreased oxidative-damaged cells; Besides,licorice flavonoids inhibited RPE cells apoptosis and the expression of high glucose-induced caspase-3 and caspase-9 expression in RPE cells, the inhibitory effect was positively correlated with the dose. CONCLUSIONS Licorice flavonoids could inhibit high glucose-induced oxidative damage and apoptosis in human RPE cells,which had preventive effect on diabetic retinopathy.
引文
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[12]GONZALEZ DE VEGA R,GARCIA M,FERNANDEZ-SANCHEZML,et al.Protective effect of selenium supplementation following oxidative stress mediated by glucose on retinal pigment epithelium[J].Metallomics,2018,10(1):83-92.
[13]JIANG Y,SANG Y,QIU Q.microRNA-383 mediates high glucoseinduced oxidative stress and apoptosis in retinal pigment epithelial cells by repressing peroxiredoxin 3[J].Am J Transl Res,2017,9(5):2374-2383.
[2]赵海燕,杨少娟,马永平,等.甘草黄酮对2型糖尿病大鼠抗氧化能力的影响[J].中国现代医学杂,2011,21(35):4359-4363.
[3]SHAO J,YAO Y.Transthyretin represses neovascularization in diabetic retinopathy[J].Mol Vis,2016,22:1188-1197.
[4]肖扬,李裕舒,董平栓.乳鼠成纤维细胞在人血清中的存活与增殖的研究[J].中国医学创新,2014,11(31):15-17.
[5]胡流芳,王迎,任汝静,等.Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制[J].国际药学研究杂志,2016,43(1):146-152,166.
[6]ZHANG ZZ,MA R,ZHANG J,et al.Photodynamic therapy of a 2methoxyestradiol tumor-targeting drug delivery system mediated by asn-gly-arg in breast cancer[J].Int J Nanomedicine,2013,2013(NULL):1551-1562.
[7]TU G,ZHANG YF,WEI W,et al.Allicin attenuates H_2O_2-induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species[J].Mol Med Rep,2016,13(3):2320-2326.
[8]FARNOODIAN M,HALBACH C,SLINGER C,et al.High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression[J].Am J Physiol Cell Physiol,2016,311(3):C418-C436.
[9]司俊康,郭俊国,郭大东,等.黄芪多糖对过氧化氢诱导人视网膜色素上皮细胞氧化损伤的保护作用[J].眼科新进展,2015,35(1):18-21.
[10]CHEN YM,LIU ZQ,FENG ZH,et al.Adhesive protein-free synthetic hydrogels for retinal pigment epithelium cell culture with low ROSlevel[J].J Biomed Mater Res A,2014,102(7):2258-2267.
[11]MINASYAN L,SREEKUMAR PG,HINTON DR,et al.Protective mechanisms of the mitochondrial-derived peptide humanin in oxidative and endoplasmic reticulum stress in rpe cells[J].Oxid Med Cell Longev,2017,2017:1675230.
[12]GONZALEZ DE VEGA R,GARCIA M,FERNANDEZ-SANCHEZML,et al.Protective effect of selenium supplementation following oxidative stress mediated by glucose on retinal pigment epithelium[J].Metallomics,2018,10(1):83-92.
[13]JIANG Y,SANG Y,QIU Q.microRNA-383 mediates high glucoseinduced oxidative stress and apoptosis in retinal pigment epithelial cells by repressing peroxiredoxin 3[J].Am J Transl Res,2017,9(5):2374-2383.
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