[1]冯绮婷,梁瑜钦,黄雄飞,等.P-糖蛋白对H2O2诱导的视网膜色素上皮细胞氧化损伤的抑制作用[J].眼科新进展,2020,40(10):915-919.[doi:10.13389/j.cnki.rao.2020.0206]
　FENG Qiting,LIANG Yuqin,HUANG Xiongfei,et al.Inhibition effect of P-glycoprotein functional expression on oxidative damage of retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2020,40(10):915-919.[doi:10.13389/j.cnki.rao.2020.0206]
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P-糖蛋白对H2O2诱导的视网膜色素上皮细胞氧化损伤的抑制作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年10期
页码:
915-919
栏目:
实验研究
出版日期:
2020-10-05

文章信息/Info

Title:
Inhibition effect of P-glycoprotein functional expression on oxidative damage of retinal pigment epithelial cells
作者:
冯绮婷; 梁瑜钦; 黄雄飞; 张跃红
510080　广东省广州市，广州市第一人民医院（华南理工大学附属医院）眼科
Author(s):
FENG Qiting; LIANG Yuqin; HUANG Xiongfei; ZHANG Yuehong
Department of Ophthalmology，Guangzhou First People’s Hospital，School of Medicine，South China University of Technology，Guangzhou 510080，Guangdong Province，China
关键词:
P-糖蛋白; 氧化应激; 视网膜色素上皮细胞; 老年性黄斑变性
Keywords:
P-glycoprotein;  oxidative stress;  retinal pigment epithelium cells;  age-related macular degeneration
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2020.0206
文献标志码:
A
摘要:
目的　探讨P-糖蛋白对H2O2诱导的视网膜色素上皮（retinal pigment epithelium，RPE）细胞氧化损伤的抑制作用。方法　取人RPE细胞株D407细胞，放入体积分数5% CO2培养箱中用含体积分数10%胎牛血清和双抗的DMEM培养基于37 ℃传代培养。先将细胞按H2O2浓度梯度处理，使用免疫印迹实验和P-糖蛋白荧光性底物罗丹明123蓄积实验确定H2O2对P-糖蛋白表达的最佳诱导浓度。预先使用1 μmol·L-1P-糖蛋白抑制剂Tariquidar处理细胞，通过测量细胞内罗丹明123蓄积量检测P-糖蛋白功能活性。将细胞分成3组：对照组、H2O2模型组（400 μmol·L-1）、H2O2 + P-糖蛋白抑制剂组（H2O2 + Tariquidar），通过检测细胞内ATP水平以及采用Annexin V-FITC凋亡检测试剂盒检测细胞凋亡率来验证氧化应激下P-糖蛋白功能性表达对细胞氧化损伤的抑制作用。结果　免疫印迹实验结果显示，随着H2O2浓度的增加，P-糖蛋白相对表达量呈剂量依赖性增加并在400 μmol·L-1时达到最高值。H2O2在浓度400 μmol·L-1时对P-糖蛋白表达和功能均有最明显的诱导作用，该浓度为最佳处理浓度。H2O2 + P-糖蛋白抑制剂组中细胞内罗丹明123水平较H2O2模型组增加，差异有统计学意义（P<0.01），提示Tariquidar能有效抑制P-糖蛋白功能。H2O2模型组ATP水平较对照组显著下降，差异有统计学意义（P<0.001）；而H2O2 + P-糖蛋白抑制剂组ATP水平则较H2O2模型组更低，差异亦有统计学意义（P<0.05），提示抑制P-糖蛋白功能可进一步加剧氧化应激导致的ATP水平下降。与对照组相比，H2O2模型组细胞凋亡率显著增加，而H2O2 + P-糖蛋白抑制剂组细胞凋亡率则较H2O2模型组增加，提示抑制P-糖蛋白功能可加重氧化应激下的细胞凋亡。结论　P-糖蛋白对H2O2诱导的RPE细胞氧化损伤具有一定的抑制作用。
Abstract:
Objective　To investigate the inhibition role of P-glycoprotein on oxidative damage of retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H2O2).Methods　The human RPE cell line D407 was cultured in 5% CO2 incubator with DMEM containing 10% fetal bovine serum and double antibody, and subcultured at 37 ℃. Cells were treated with different concentration of H2O2, and the optimal concentration of H2O2 on p-glycoprotein expression was determined by western blot and rhodamine 123 accumulation tests. P-glycoprotein inhibitor tariquidar (1 μmol·L-1) was pretreated prior to H2O2 exposure and the inhibition efficiency was detected by measuring the intracellular accumulation of rhodamine 123, a fluorescent substrate of P-glycoprotein. Cells were divided into three groups: control group, H2O2 model group (400 μmol·L-1) and H2O2+ P-glycoprotein inhibitor group (H2O2+ tariquidar group). The inhibition effect of P-glycoprotein on oxidative damage was verified by detecting intracellular ATP level, and the apoptosis rate was detected by Annexin V-FITC apoptosis detection kit.Results　The relative expression level of P-glycoprotein increased in a dose-dependent manner with the increase of H2O2 concentration, and it reached the highest value at 400 μmol·L-1. H2O2 could induce the expression and function of P-glycoprotein at the concentration of 400 μmol·L-1. The level of rhodamine 123 in the H2O2+ tariquidar group was higher than that in H2O2 model group (P< 0.01), which suggested that tariqudar can effectively inhibit the function of P-glycoprotein. The ATP level of the H2O2 model group was significantly lower than that of the control group, and the difference was statistically significant (P<0.001); while the ATP level of the H2O2 model group was lower than that of the H2O2 model group, and the difference was also statistically significant (P<0.05), suggesting that inhibiting the function of P-glycoprotein can further aggravate the decline in ATP levels caused by oxidative stress. Compared with the control group, the apoptosis rate of H2O2 model group was significantly increased, while that of H2O2+ tariquidar group was higher than that of H2O2 model group, suggesting that inhibition of H2O2 model group aggravate apoptosis under oxidative stress.Conclusion　P-glycoprotein can inhibit RPE cells from oxidative damage induced by H2O2.

参考文献/References:

［1］　WONG W L，SU X，LI X，CHEUNG C M G，KLEIN R，CHENG C Y，et al.Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis ［J］.Lancet Glob Health,2014,2(2):e106-e116.
［2］　CHOPDAR A，CHAKRAVARTHY U，VERMA D.Age related macular degeneration ［J］.BMJ,2003，326(7387):485-488.
［3］　AO J，WOOD J P，CHIDLOW G，GILLIES M C，CASSON R J.Retinal pigment epithelium in the pathogenesis of age-related macular degeneration and photobiomodulation as a potential therapy［J］.Clin Exp Ophthalmol，2018，46(6):670-686.
［4］　俞永珍,邹秀兰,邹玉平.氧化应激及抗氧化物在年龄相关性黄斑变性中的作用 ［J］.眼科新进展,2015,35(5):489-492.
YU Y Z，ZOU X L，ZOU Y P.Roles of oxidative stress and antioxidants in age related macular degeneration ［J］.Rec Adv Ophthalmol，2015,35(5):489-492.
［5］　KEELING E，LOTERY A J，TUMBARELLO D A，RATNAYAKA J A.Impaired cargo clearance in the retinal pigment epithelium(RPE) underlies irreversible blinding diseases ［J］.Cells,2018,7(2):1-19.
［6］　VU K T，HULLEMAN J D.An inducible form of Nrf2 confers enhanced protection against acute oxidative stresses in RPE cells ［J］.Exp Eye Res,2017,164:31-36.
［7］　BAROT M，GOKULANDHI M R，MITRA A K.Mitochondrial dysfunction in retinal diseases ［J］.Curr Eye Res,2011,36(12):1069-1077.
［8］　JARRETT S G，BOULTON M E.Consequences of oxidative stress in age-related macular degeneration ［J］.Mol Aspects Med,2012,33(4):399-417.
［9］　SHADRACH K G，RAYBORN M E，HOLLYFIELD J G，BONILHA V L.DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium(RPE) ［J］.PLoS One,2013,8(7):1-14.
［10］　LEE W K，TORCHALSKI B，KOHISTANI N，THVENOD F.ABCB1 protects kidney proximal tubule cells against cadmium-induced apoptosis: roles of cadmium and ceramide transport ［J］.Toxicol Sci,2011,121(2):343-356
［11］　HULS M，KRAMERS C，LEVTCHENKO E N，WILMER M J，DIJKMAN H B，KLUIJTMANS L A，et al.P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury ［J］.Kidney Int,2007,72(2):1233-1241.
［12］　MILLERS D S.Regulation of ABC transporters blood-brain barrier: the good，the bad，and the ugly ［J］.Adv Cancer Res,2015,125(10):43-70.
［13］　WANG X，CAMPOS C R，PEART J C，SMITH L K，BONI J L，CANNON R E，et al.Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers ［J］.J Neurosci,2014,34(25):8585-8593.
［14］　KHANDHADIA S，CHERRY J，LOTERY A J.Age-related macular degeneration ［J］.Neurodegener Dis,2012,724(15):15-36.
［15］　DUNAIEF J L，DENTCHEY T，YING G S，MILAM A H.The role of apoptosis in age-related macular degeneration ［J］.Arch Ophthalmol，2002，120(11):1435-1442.
［16］　KARUNADHARMA P P，NORDGAARD C L，OLSEN T W，FERRINGTON D A.Mitochondrial DNA damage as a potential mechanism for age-related macular degeneration ［J］.Invest Ophthalmol Vis Sci,2010,51(11):5470-5479.
［17］　THVENOD F，FRIEDMANN J M，KATSEN A D，HAUSERI I A.Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis ［J］.J Biol Chem,2000,275(3):1887-1896.
［18］　CONSTABLE P A，LAWRENSEN J G，DOLMAN D E M，ARDEN G B，ABBOTT N J.P-glycoprotein expression in human retinal pigment epithelium cell lines ［J］.Exp Eye Res,2006,83(1):24-30.
［19］　王玉璘，王少峡，郭虹，胡利民.血脑屏障中P-糖蛋白的调节机制 ［J］.中国药理学通报,2011,27(9):1196-1200.
WANG Y L，WANG S X，GUO H，HU L M.Regulatory mechanisms of P-glycoprotein at the blood-brain barrier ［J］.Chin Pharmacol Bull，2011,27(9):1196-1200.
［20］　ZHANG Y H，LI J，YANG W Z，XIAN Z H，FENG Q T，RUAN X C.Mitochondrial expression and activity of P-glycoprotein under oxidative stress in outer blood-retinal barrier ［J］.Int J Ophthalmol,2017,10(7):1055-1063.
［21］　HARTZ A M，BAUER B，FRICKER G，MILLER D S.Rapid modulation of P-glycoprotein-mediated transport at the blood-brain barrier by tumor necrosis factor-α and lipopolysaccharide ［J］.Mol Pharmacol,2006,69(2):462-470.
［22］　MANTOVANI I，CAPPELLINI A，TAZZARI P L，PAPA V，COCCO L，MARTELLI A M.Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-lymphoblastoid CEM cells ［J］.J Cell Physiol,2006,207(9):836-844.
［23］　RUEFLI A，TAINTON K，DARCY P，SMYTH M，JOHNSHONE R.P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex(disc) following Fas ligation ［J］.Cell Death Differ,2002,9(8):1266-1272.

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备注/Memo

备注/Memo:
国家自然科学基金资助项目（编号: 81200709、81271196）；广州市科技创新发展专项资金资助项目（编号: 201607010119）；广州市卫生健康科技项目（编号：20181A010006）
更新日期/Last Update: 2020-10-05