急性氧化应激初期人晶状体上皮细胞株SRA01/04中衰老标记蛋白30抗氧化作用的研究
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摘要
目的初步探讨衰老标记蛋白30(senescence marker protein 30,SMP30)的增加对处于急性氧化应激初期的人晶状体上皮细胞株SRA01/04的保护作用。方法将处于对数生长期的SRA01/04接种于96孔板,分别使用含150μmol·L~(-1)、200μmol·L~(-1)、250μmol·L~(-1)、300μmol·L~(-1)、350μmol·L~(-1)、400μmol·L~(-1)、450μmol·L~(-1)H2O2的培养基作用2 h,CCK-8法选取最佳H2O2浓度;使用慢病毒对SRA01/04进行转染建立SMP30高表达模型,实验分3组:SMP30过表达(overexpression,OE)组、SMP30过表达相应空载(negative control over expression,NCOE)组及对照(control,CON)组。通过超氧化物歧化酶(superoxide dismutase,SOD)定量检测试剂盒、氧化型谷胱甘肽(oxidized glutathione,GSSG)/总谷胱甘肽(total glutathi-one,T-GSH)测定试剂盒测量细胞内SOD活力及GSSG/T-GSH比值的变化。结果经分析,建立模型的最佳H2O2浓度为300μmol·L~(-1)。在此浓度下,与CON组(5. 783±0. 192) U·mg~(-1)及NCOE组(5. 837±0. 182) U·mg~(-1)相比,OE组细胞内SOD活力升高,为(10. 251±0. 110) U·mg~(-1);与CON组(29. 943±0. 241)及NCOE组(30. 037±0. 433)相比,OE组细胞内GSSG/T-GSH比值(20. 990±0. 342)降低;差异均有统计学意义(均为P <0. 05)。结论当SRA01/04处于H2O2诱导的急性氧化应激初期时,SMP30表达的增加可通过增加SOD活力和降低GSSG/T-GSH比值,加强SRA01/04的抗氧化能力。
Objective To investigate the protective effect of increased senescence marker protein 30 (SMP3 0) on the human lens epithelial cell (HLEC)SRA01/04 in the early stage of acute oxidative stress preliminarily. Methods The SRA01/04 in the period of logarithmic phase were seeded into 96-well plates,treated with 150 μmol· L~(-1),200 μmol · L~(-1),25 0 μmol · L~(-1),300 μmol · L~(-1),350μmol · L~(-1), 400 μmol · L~(-1),45 0 μmol · L~(-1) hydrogen peroxide (H_2O_2) for 2 h,respectively, and CCK-8 assay was used to select the optimum concentration. Then the SRA01/04 were transfected with lentivirus to establish a high-expressed model of SMP30. There were three groups: over expression (OE) SMP30 group,negative control over expression (NCOE) group, control group (CON). The levels of intracellular superoxide dismutase(SOD) and total glutathione(T-GSH)/oxidized glutathione(GSSG) were determined by the SOD activity assay kit and T-GSH/GSSH assay kit.Results The optimum concentration of H_2O_2 in the acute oxidative stress cell model was 3 00 μmol · L~(-1). At this concentration, compared with the CON group(5. 783±0.192) U·mg~(-1) and the NCOE group(5.837 ±0. 182) U · mg~(-1),the intracellular SOD value of OE group(10.251 ±0. 110) U · mg~(-1) was increased. W hen compared with the CON group (29. 943 ± 0. 24 1) and the NCOE group (30. 037± 0. 433),the GSSG/T-GSH value in the OE group (20. 990 ± 0. 342) was decreased, and the difference was statistically significant (all P < 0. 05). Conclusion During the early stage of acute oxidative stress induced by H_2O_2, the up-regulation of SMP30 expression can enhance the antioxidant capacity of SR A01/04, by means of increasing the activity of SOD and decreasing the GSSG/T-GSH ratio.
Objective To investigate the protective effect of increased senescence marker protein 30 (SMP3 0) on the human lens epithelial cell (HLEC)SRA01/04 in the early stage of acute oxidative stress preliminarily. Methods The SRA01/04 in the period of logarithmic phase were seeded into 96-well plates,treated with 150 μmol· L~(-1),200 μmol · L~(-1),25 0 μmol · L~(-1),300 μmol · L~(-1),350μmol · L~(-1), 400 μmol · L~(-1),45 0 μmol · L~(-1) hydrogen peroxide (H_2O_2) for 2 h,respectively, and CCK-8 assay was used to select the optimum concentration. Then the SRA01/04 were transfected with lentivirus to establish a high-expressed model of SMP30. There were three groups: over expression (OE) SMP30 group,negative control over expression (NCOE) group, control group (CON). The levels of intracellular superoxide dismutase(SOD) and total glutathione(T-GSH)/oxidized glutathione(GSSG) were determined by the SOD activity assay kit and T-GSH/GSSH assay kit.Results The optimum concentration of H_2O_2 in the acute oxidative stress cell model was 3 00 μmol · L~(-1). At this concentration, compared with the CON group(5. 783±0.192) U·mg~(-1) and the NCOE group(5.837 ±0. 182) U · mg~(-1),the intracellular SOD value of OE group(10.251 ±0. 110) U · mg~(-1) was increased. W hen compared with the CON group (29. 943 ± 0. 24 1) and the NCOE group (30. 037± 0. 433),the GSSG/T-GSH value in the OE group (20. 990 ± 0. 342) was decreased, and the difference was statistically significant (all P < 0. 05). Conclusion During the early stage of acute oxidative stress induced by H_2O_2, the up-regulation of SMP30 expression can enhance the antioxidant capacity of SR A01/04, by means of increasing the activity of SOD and decreasing the GSSG/T-GSH ratio.
引文
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[8]YANG M,ZHANG Q Y,HUANG H W,MA X,WANG J,MA L.Protective effect of lutein on human RPE cells from oxidative stress[J].Food Nutr CHN,2017,23(8):57-61.杨敏,张倩玉,黄宏伟,马譞,王娇,马乐.叶黄素对人RPE细胞氧化应激损伤的保护作用[J].中国食物与营养,2017,23(8):57-61.
[9]HAN F,ZHOU M L.Optimal action concentration of H2O2for Hep G2 cells to produce oxidative stress[J].Sci Techn Cer Oils Foods,2011,19(1):62-64.韩飞,周孟良.过氧化氢诱导人肝癌细胞产生氧化应激最佳作用浓度的研究[J].粮油食品科技,2011,19(1):62-64.
[10]CHEN X,LI S M,LI Y W,HAN Z H,LIANG H.Effect of senescence marker protein 30 on the proliferation and apoptosis of human lens epithelial cells SRA01/04[J].Int J Ophthalmol,2018,11(4):553.
[11]INDARAM M,AGRON E,CLEMONS T E,SPERDUTO RD,WONG W T,FERRIS F L,et al.Changes in lens opacities on the age-related eye disease study grading scale predict progression to cataract surgery and vision loss:age-related eye disease study report no.34[J].Ophthalmology,2015,122(5):888-896.
[12]ORIC'L,MIRIC D,KISIC B.Basic review of the oxidative stress role in age-related cataractogenesis[J].Oxid Stress Appl Bas Res Clin Pract,2015,56(1):147-154.
[13]WANG W J,CHEN J,WANG Y Y,LIU X Y,ZHANG X L,QU YX,et al.Research progress on relationship between oxidative stress and ocular surface diseases[J].Rec Adv Ophthalmol,2014,34(6):585-588.王雯婕,陈剑,王园园,刘小勇,张晓玲,曲艺欣,等.氧化应激与眼表疾病关系的研究进展[J].眼科新进展,2014,34(6):585-588.
[14]DE-QIAN K,YUE L,LI L,GUANGYING Z.Downregulation of Smac attenuates H2O2-induced apoptosis via endoplasmic reticulum stress in human lens epithelial cells[J].Medicine,2017,96(27):e7419.
[15]MA T J,LAN D H,HE S Z,YE Z,LI P,ZHAI W,et al.Nrf2 protects human lens epithelial cells against H2O2-induced oxidative and ER stress:The ATF4 may be involved[J].Exp Eye Res,2018,169:28-37.
[16]PENG J,ZHENG T T,LIANG Y,DUAN L F,ZHANG Y D,WANG L J,et al.p-Coumaric acid protects human lens epithelial cells against oxidative stress-induced apoptosis by MAPK signaling[J].Oxid Med Cellul Long,2018,2018:8549052.
[17]FUJITA T,UCHIDA K,MARUYAMA N.Purification of senescence marker protein-30(SMP30)and its androgen-independent decrease with age in the rat liver[J].Biochimica Et Biophysica Acta,1992,1116(2):122-128.
[18]SCOTT S H,BAHNSON B J.Senescence marker protein 30:Functional and structural insights to its unknown physiological function[J].Biomol Concepts,2011,2(6):469-480.
[19]KONDO Y,ISHIGAMI A.Involvement of senescence marker protein-30 in glucose metabolism disorder and non-alcoholic fatty liver disease[J].Geriatr Gerontol Int,2016,16(Suppl 1):4-16.
[20]ZHUANG Y,LI X L,ZHENG S X,WANG F,MO Z J,BIN X Y,et al.The effect of SMP30 on the behavior of hepatocellular carcinoma cells and its relationship with glucose metabolism[J].Gen Appl Biol,2017,36(2):450-455.庄园,李晓龙,郑顺心,王峰,莫之婧,宾晓芸,等.SMP30对肝癌细胞行为学影响及其与糖代谢关系研究[J].基因组学与应用生物学,2017,36(2):450-455.
[21]LI S,CHEN X,LAI W,HU M,ZHONG X,TAN S,et al.Downregulation of SMP30 in senescent human lens epithelial cells[J].Mol Med Rep,2017,16(4):4022-4028.
[22]TENG H,HUANG L Y,TIAN F,DONG L J,ZHANG H.Effects of SMP-30 overexpression on apoptosis of human lens epithelial cells induced by ultraviolet B irradiation[J].Chin J Ophthalmol,2017,53(11):835-841.
[23]ISHIKAWA Y,HASHIZUME K,KISHIMOTO S,TEZUKA Y,NISHIGORI H,YAMAMOTO N,et al.Effect of vitamin C depletion on UVR-B induced cataract in SMP30/GNL knockout mice[J].Exp Eye Res,2012,94(1):85-89.
[24]XUE D X,ZHANG Y,DU L L,YUE C Y,LIU P.Relationship of superoxide dismutases and age-related cataract[J].Rec Adv Ophthalmol,2013,33(8):740-742.薛大喜,张毅,杜玲玲,岳超英,刘平.超氧化物歧化酶与年龄相关性白内障关系的研究[J].眼科新进展,2013,33(8):740-742.
[25]YU G,YAN H.Protective effects of thioltransferase in rat lens incubated with high glucose[J].Int Eye Sci,2014,14(11):1927-1930.于罡,严宏.硫醇转移酶对高糖诱导大鼠晶状体氧化应激的保护作用[J].国际眼科杂志,2014,14(11):1927-1930.
[2]LI W C,KUSZAK J R,DUNN K,WANG R R,MA W,WANG GM,et al.Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals[J].J Cell Biol,1995,130(1):169-181.
[3]SU D,HU S,GUAN L,WU X,SHI C,YANG X,et al.Down-regulation of GJA3 is associated with lens epithelial cell apoptosis and age-related cataract[J].Biochem Biophys Res Commun,2017,484(1):159-164.
[4]BEEBE D C,HOLEKAMP N M,SHUI Y B.Oxidative damage and the prevention of age-related cataracts[J].Ophthalmic Res,2010,44(3):155-165.
[5]LAI W X,TAN S J,LI X,ZOU W J,JIANG L Z,LIANG H.Comparison of expression levels of senescence marker protein 30 in lens epithelial cells among different ages of cataract patients[J].Chin Exp Ophthalmol,2014,32(6):521-524.赖伟霞,谭少健,李霞,邹文进,蒋林志,梁皓.衰老标记蛋白30在不同年龄白内障患者晶状体上皮细胞中的表达变化[J].中华实验眼科杂志,2014,32(6):521-524.
[6]HU M X,LUO G R,ZHOU S F,MO F R,XIE X X,LIANGH.Expression and significance of senescence marker protein-30 in human lens epithelial cells[J].Rec Adv Ophthalmol,2008,28(1):13-15.胡美霞,罗国容,周素芳,莫发荣,谢小薰,梁皓.衰老标记蛋白-30在人晶状体上皮细胞中的表达及意义[J].眼科新进展,2008,28(1):13-15.
[7]LI S N,CHEN X,ZHANG H K,JIANG L Z,LIANG H,TANS J.Effects of acute oxidative stress induced by H2O2on expression of SMP30 and morphology,survival rate of human lens epithelial cells[J].Rec Adv Ophthalmol,2017,37(4):310-313.李舒凝,陈曦,张鸿侃,蒋林志,梁皓,谭少健.过氧化氢诱导的急性氧化应激对人晶状体上皮细胞形态、生存率及衰老标记蛋白30(SMP30)表达的影响[J].眼科新进展,2017,37(4):310-313.
[8]YANG M,ZHANG Q Y,HUANG H W,MA X,WANG J,MA L.Protective effect of lutein on human RPE cells from oxidative stress[J].Food Nutr CHN,2017,23(8):57-61.杨敏,张倩玉,黄宏伟,马譞,王娇,马乐.叶黄素对人RPE细胞氧化应激损伤的保护作用[J].中国食物与营养,2017,23(8):57-61.
[9]HAN F,ZHOU M L.Optimal action concentration of H2O2for Hep G2 cells to produce oxidative stress[J].Sci Techn Cer Oils Foods,2011,19(1):62-64.韩飞,周孟良.过氧化氢诱导人肝癌细胞产生氧化应激最佳作用浓度的研究[J].粮油食品科技,2011,19(1):62-64.
[10]CHEN X,LI S M,LI Y W,HAN Z H,LIANG H.Effect of senescence marker protein 30 on the proliferation and apoptosis of human lens epithelial cells SRA01/04[J].Int J Ophthalmol,2018,11(4):553.
[11]INDARAM M,AGRON E,CLEMONS T E,SPERDUTO RD,WONG W T,FERRIS F L,et al.Changes in lens opacities on the age-related eye disease study grading scale predict progression to cataract surgery and vision loss:age-related eye disease study report no.34[J].Ophthalmology,2015,122(5):888-896.
[12]ORIC'L,MIRIC D,KISIC B.Basic review of the oxidative stress role in age-related cataractogenesis[J].Oxid Stress Appl Bas Res Clin Pract,2015,56(1):147-154.
[13]WANG W J,CHEN J,WANG Y Y,LIU X Y,ZHANG X L,QU YX,et al.Research progress on relationship between oxidative stress and ocular surface diseases[J].Rec Adv Ophthalmol,2014,34(6):585-588.王雯婕,陈剑,王园园,刘小勇,张晓玲,曲艺欣,等.氧化应激与眼表疾病关系的研究进展[J].眼科新进展,2014,34(6):585-588.
[14]DE-QIAN K,YUE L,LI L,GUANGYING Z.Downregulation of Smac attenuates H2O2-induced apoptosis via endoplasmic reticulum stress in human lens epithelial cells[J].Medicine,2017,96(27):e7419.
[15]MA T J,LAN D H,HE S Z,YE Z,LI P,ZHAI W,et al.Nrf2 protects human lens epithelial cells against H2O2-induced oxidative and ER stress:The ATF4 may be involved[J].Exp Eye Res,2018,169:28-37.
[16]PENG J,ZHENG T T,LIANG Y,DUAN L F,ZHANG Y D,WANG L J,et al.p-Coumaric acid protects human lens epithelial cells against oxidative stress-induced apoptosis by MAPK signaling[J].Oxid Med Cellul Long,2018,2018:8549052.
[17]FUJITA T,UCHIDA K,MARUYAMA N.Purification of senescence marker protein-30(SMP30)and its androgen-independent decrease with age in the rat liver[J].Biochimica Et Biophysica Acta,1992,1116(2):122-128.
[18]SCOTT S H,BAHNSON B J.Senescence marker protein 30:Functional and structural insights to its unknown physiological function[J].Biomol Concepts,2011,2(6):469-480.
[19]KONDO Y,ISHIGAMI A.Involvement of senescence marker protein-30 in glucose metabolism disorder and non-alcoholic fatty liver disease[J].Geriatr Gerontol Int,2016,16(Suppl 1):4-16.
[20]ZHUANG Y,LI X L,ZHENG S X,WANG F,MO Z J,BIN X Y,et al.The effect of SMP30 on the behavior of hepatocellular carcinoma cells and its relationship with glucose metabolism[J].Gen Appl Biol,2017,36(2):450-455.庄园,李晓龙,郑顺心,王峰,莫之婧,宾晓芸,等.SMP30对肝癌细胞行为学影响及其与糖代谢关系研究[J].基因组学与应用生物学,2017,36(2):450-455.
[21]LI S,CHEN X,LAI W,HU M,ZHONG X,TAN S,et al.Downregulation of SMP30 in senescent human lens epithelial cells[J].Mol Med Rep,2017,16(4):4022-4028.
[22]TENG H,HUANG L Y,TIAN F,DONG L J,ZHANG H.Effects of SMP-30 overexpression on apoptosis of human lens epithelial cells induced by ultraviolet B irradiation[J].Chin J Ophthalmol,2017,53(11):835-841.
[23]ISHIKAWA Y,HASHIZUME K,KISHIMOTO S,TEZUKA Y,NISHIGORI H,YAMAMOTO N,et al.Effect of vitamin C depletion on UVR-B induced cataract in SMP30/GNL knockout mice[J].Exp Eye Res,2012,94(1):85-89.
[24]XUE D X,ZHANG Y,DU L L,YUE C Y,LIU P.Relationship of superoxide dismutases and age-related cataract[J].Rec Adv Ophthalmol,2013,33(8):740-742.薛大喜,张毅,杜玲玲,岳超英,刘平.超氧化物歧化酶与年龄相关性白内障关系的研究[J].眼科新进展,2013,33(8):740-742.
[25]YU G,YAN H.Protective effects of thioltransferase in rat lens incubated with high glucose[J].Int Eye Sci,2014,14(11):1927-1930.于罡,严宏.硫醇转移酶对高糖诱导大鼠晶状体氧化应激的保护作用[J].国际眼科杂志,2014,14(11):1927-1930.