儿茶素没食子酸酯对人视网膜色素上皮细胞氧化应激损伤的机制研究
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摘要
目的探讨儿茶素没食子酸酯(EGCG)对过氧化氢(H_2O_2)诱导人视网膜色素上皮(ARPE-19)细胞氧化应激损伤及TRAF6/TAK1信号通路活化的影响。方法采用H_2O_2(100μmol/L)体外诱导ARPE-19细胞氧化应激损伤模型,将ARPE-19细胞分为正常对照组、H_2O_2诱导组、H_2O_2+低剂量(40μmol/L)EGCG组、H_2O_2+中剂量(80μmol/L)EGCG组、H_2O_2+高剂量(160μmol/L)EGCG组;CCK8法检测ARPE-19细胞相对存活率,相关试剂盒检测氧化应激产物MDA、NO水平及SOD1活性,Western Blot方法分析HO-1、SOD1、TRAF6、TAK1及p-TAK1蛋白表达。结果与H_2O_2诱导组比较,不同浓度EGCG干预的ARPE-19细胞相对存活率明显升高(P<0.05),细胞匀浆氧化应激产物MDA、NO水平明显降低(P<0.05),匀浆中SOD1活性显著升高(P<0.05),细胞中HO-1及SOD1表达水平显著升高(P<0.05),细胞中TRAF6及p-TAK1蛋白表达显著降低(P<0.05),并表现出剂量依赖性。结论 EGCG能明显抑制H_2O_2诱导的ARPE-19细胞氧化应激损伤,其作用机制可能与抑制TRAF6/TAK1信号通路活化有关。
OBJECTIVE To investigate the inhibitory effects of epigallocatechin gallate(EGCG) on oxidative stress injury induced by H_2O_2 in human retinal pigment epithelial(ARPE-19)cells and the activation of TRAF6/TAK1 signaling pathway. METHODS The H_2O_2(100 μmol/L)was used to induce the model of oxidative stress injury in ARPE-19 cells in vitro. All ARPE-19 cells were divided into five groups, including control group, H_2O_2-induced group, H_2O_2+low dose(40 μmol/L) EGCG group, H_2O_2+medium dose(80 μmol/L) EGCG group, and H_2O_2+high dose(160μmol/L) EGCG group. And CCK8 assay was used to measure the relative survival rates of ARPE-19 cells. Related kits were used to detect the levels of MDA, NO and the activity of SOD1. Western Blot was used to determine the expression levels of HO-1, SOD1, TRAF6, TAK1 and p-TAK1 proteins. RESULTS Compared with H_2O_2 induced group, the relative survival rates of ARPE-19 cells were greatly increased after treatment with different concentrations of EGCG(P<0.05). The levels of oxidative stress productions(MDA and NO) were significantly decreased, while the activity of SOD1 was greatly increased(P<0.05). The expression levels of HO-1 and SOD1 were also greatly increased, while the expression levels of TRAF6 and p-TAK1 were greatly decreased(P <0.05) with a dose-dependent manner.CONCLUSIONS The EGCG significantly inhibited the oxidative stress injury of ARPE-19 cells induced by H_2O_2 via suppressing the activation of TRAF6/TAK1 signaling pathway.
OBJECTIVE To investigate the inhibitory effects of epigallocatechin gallate(EGCG) on oxidative stress injury induced by H_2O_2 in human retinal pigment epithelial(ARPE-19)cells and the activation of TRAF6/TAK1 signaling pathway. METHODS The H_2O_2(100 μmol/L)was used to induce the model of oxidative stress injury in ARPE-19 cells in vitro. All ARPE-19 cells were divided into five groups, including control group, H_2O_2-induced group, H_2O_2+low dose(40 μmol/L) EGCG group, H_2O_2+medium dose(80 μmol/L) EGCG group, and H_2O_2+high dose(160μmol/L) EGCG group. And CCK8 assay was used to measure the relative survival rates of ARPE-19 cells. Related kits were used to detect the levels of MDA, NO and the activity of SOD1. Western Blot was used to determine the expression levels of HO-1, SOD1, TRAF6, TAK1 and p-TAK1 proteins. RESULTS Compared with H_2O_2 induced group, the relative survival rates of ARPE-19 cells were greatly increased after treatment with different concentrations of EGCG(P<0.05). The levels of oxidative stress productions(MDA and NO) were significantly decreased, while the activity of SOD1 was greatly increased(P<0.05). The expression levels of HO-1 and SOD1 were also greatly increased, while the expression levels of TRAF6 and p-TAK1 were greatly decreased(P <0.05) with a dose-dependent manner.CONCLUSIONS The EGCG significantly inhibited the oxidative stress injury of ARPE-19 cells induced by H_2O_2 via suppressing the activation of TRAF6/TAK1 signaling pathway.
引文
[1] SCHWARTZ SD, REGILLO CD, LAM BL, et al.Human embryonic stem cell-derived retinal pigment epithelium in patients with agerelated macular degeneration and Stargardt's macular dystrophy:follow-up of two open-label phase 1/2 studies[J] Lancet,2015,385(9967):509-516.
[2] KAARNIRANTA K, SINHA D, BLASIAK J, et al.Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration[J].Autophagy,2013,9(7):973-984.
[3] ARNAULT E, BARRAU C, NANTEAU C, et al.Phototoxic action spectrum on a retinal pigment epithelium model of age-related macular degeneration exposed to sunlight normalized conditions[J].PLoS One,2013,8(8):e71398.
[4] XIAO J, HO CT, LIONG EC, et al.Epigallocatechin gallate attenuates fibrosis, oxidative stress, and inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3 K/Akt/FoxO1,and NF-kappa B pathways[J].Eur J Nutr, 2014, 53(1):187-99.
[5] CASANOVA E, BASELGA-ESCUDERO L, RIBAS-LATRE A, et al.Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes[J].Biochim Biophys Acta,2014,1837(6):783-791.
[6] CIA D, VERGNAUD-GAUDUCHON J,JACQUEMOT N, et al.Epigallocatechin gallate(EGCG)prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells[J].Curr Eye Res,2014,39(9):944-952.
[7]叶盼盼,姚克,张丽.表没食子儿茶素没食子酸酯对氧化应激诱导人晶状体上皮细胞凋亡的保护研究[J].中国医院药学杂志,2007,12(5):56-58.
[8] WEI ZF, TONG B, XIA YF, et al. Norisoboldine suppresses osteoclast differentiation through preventing the accumulation of TRAF6-TAK1 complexes and activation of MAPKs/NF-κB/c-Fos/NFATc1pathways[J].PLoS One,2013,8(3):e59171.
[9] SONG Z, ZHU X, JIN R, et al. Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury[J].PLoS One,2014,9(7):e101671.
[10] LETTNER A. Age-related macular degeneration-biology and treatment[J].Med Monatsschr Pharm,2015,38(7):258-264.
[11]阮熵,汪枫桦.干细胞定向分化视网膜色素上皮治疗年龄相关性黄斑变性的研究进展[J].上海交通大学学报:医学版,2018,38(9):1123-1128.
[12] WANG X, LIU X, REN Y, et al. PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression[J].Mol Med Rep, 2019,19(1):59-74.
[13] KAMAT CD, GADAL S, MHATRE M, et al. Antioxidants in central nervous system diseases:preclinical promise and translational challenges[J].J Alzheimers Dis, 2008,15(3):473-493.
[14] SEN T, DUTTA A, CHATTERJEE A. Epigallocatechin-3-gallate(EGCG)downregulates gelatinase-B(MMP-9)by involvement of FAK/ERK/NFjB and AP-1 in the human breast cancer cell line MDA-MB-231[J].Anti-cancer Drugs,2010,21(6):632-644.
[15] CAO Y, BAO S, YANG W, et al. Epigallocatechin gallate prevents inflammation by reducing macrophage infiltration and inhibiting tumor necrosis factor-αsignaling in the pancreas of rats on a high-fat diet[J].Nutr Res,2014,34(12):1066-1074.
[16] OMORI E, INAGAKI M, MISHINA Y, et al. Epithelial transforming growth factorβ-activated kinase 1(TAK1)is activated through two independent mechanisms and regulates reactive oxygen species[J].Proc Natl Acad Sci USA.2012,109(9):3365-3370.
[17] OMORI E, MORIOKA S, MATSUMOTO K, et al. TAKI regulates reactive oxygen species and cell death in keratinocytes, which is essential for skin integrity[J].J Biol Chem,2008,283(38):26161-26168.
[18] ZHANG J, LEI Z, HUANG Z,et al. Epigallocatechin-3-gallate(EGCG)suppresses melanoma cell growth and metastasis by Targeting TRAF6 activity[J]. Oncotarget,2016, 7(48):79557-79571.
[2] KAARNIRANTA K, SINHA D, BLASIAK J, et al.Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration[J].Autophagy,2013,9(7):973-984.
[3] ARNAULT E, BARRAU C, NANTEAU C, et al.Phototoxic action spectrum on a retinal pigment epithelium model of age-related macular degeneration exposed to sunlight normalized conditions[J].PLoS One,2013,8(8):e71398.
[4] XIAO J, HO CT, LIONG EC, et al.Epigallocatechin gallate attenuates fibrosis, oxidative stress, and inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3 K/Akt/FoxO1,and NF-kappa B pathways[J].Eur J Nutr, 2014, 53(1):187-99.
[5] CASANOVA E, BASELGA-ESCUDERO L, RIBAS-LATRE A, et al.Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes[J].Biochim Biophys Acta,2014,1837(6):783-791.
[6] CIA D, VERGNAUD-GAUDUCHON J,JACQUEMOT N, et al.Epigallocatechin gallate(EGCG)prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells[J].Curr Eye Res,2014,39(9):944-952.
[7]叶盼盼,姚克,张丽.表没食子儿茶素没食子酸酯对氧化应激诱导人晶状体上皮细胞凋亡的保护研究[J].中国医院药学杂志,2007,12(5):56-58.
[8] WEI ZF, TONG B, XIA YF, et al. Norisoboldine suppresses osteoclast differentiation through preventing the accumulation of TRAF6-TAK1 complexes and activation of MAPKs/NF-κB/c-Fos/NFATc1pathways[J].PLoS One,2013,8(3):e59171.
[9] SONG Z, ZHU X, JIN R, et al. Roles of the kinase TAK1 in CD40-mediated effects on vascular oxidative stress and neointima formation after vascular injury[J].PLoS One,2014,9(7):e101671.
[10] LETTNER A. Age-related macular degeneration-biology and treatment[J].Med Monatsschr Pharm,2015,38(7):258-264.
[11]阮熵,汪枫桦.干细胞定向分化视网膜色素上皮治疗年龄相关性黄斑变性的研究进展[J].上海交通大学学报:医学版,2018,38(9):1123-1128.
[12] WANG X, LIU X, REN Y, et al. PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression[J].Mol Med Rep, 2019,19(1):59-74.
[13] KAMAT CD, GADAL S, MHATRE M, et al. Antioxidants in central nervous system diseases:preclinical promise and translational challenges[J].J Alzheimers Dis, 2008,15(3):473-493.
[14] SEN T, DUTTA A, CHATTERJEE A. Epigallocatechin-3-gallate(EGCG)downregulates gelatinase-B(MMP-9)by involvement of FAK/ERK/NFjB and AP-1 in the human breast cancer cell line MDA-MB-231[J].Anti-cancer Drugs,2010,21(6):632-644.
[15] CAO Y, BAO S, YANG W, et al. Epigallocatechin gallate prevents inflammation by reducing macrophage infiltration and inhibiting tumor necrosis factor-αsignaling in the pancreas of rats on a high-fat diet[J].Nutr Res,2014,34(12):1066-1074.
[16] OMORI E, INAGAKI M, MISHINA Y, et al. Epithelial transforming growth factorβ-activated kinase 1(TAK1)is activated through two independent mechanisms and regulates reactive oxygen species[J].Proc Natl Acad Sci USA.2012,109(9):3365-3370.
[17] OMORI E, MORIOKA S, MATSUMOTO K, et al. TAKI regulates reactive oxygen species and cell death in keratinocytes, which is essential for skin integrity[J].J Biol Chem,2008,283(38):26161-26168.
[18] ZHANG J, LEI Z, HUANG Z,et al. Epigallocatechin-3-gallate(EGCG)suppresses melanoma cell growth and metastasis by Targeting TRAF6 activity[J]. Oncotarget,2016, 7(48):79557-79571.
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