促红细胞生成素抑制氧化损伤诱导的人眼Müller细胞凋亡

doi:10.6040/j.issn.1673-3770.0.2014.293

摘要/Abstract

摘要： 目的探讨促红细胞生成素(EPO)抑制氧化损伤导致的Müller凋亡的可能性及其分子机制。方法对培养的人眼Müller细胞系MIO-M1细胞采用BrdU标记法和MTT比色法观察正常及过氧化氢(H₂O₂)或葡萄糖氧化酶(GO)氧化损伤条件下0、0.01、0.1、1、10、30、100 U/mL EPO作用24、48、72 h后对Müller细胞增殖、迁移的影响;采用MTT比色法观察加PI3K/PDK1/PKB(Akt)信号传导通路阻断剂LY294002后Müller细胞增殖的变化;采用ELISA实验观察Müller细胞对EPO的表达和分泌;通过Western-blotting技术检测体外不同条件培养下EPO对ERK1/2及Akt信号传导通路的作用。结果正常培养条件下, EPO有轻度促进Müller细胞增殖迁移的作用, 但差异无统计学意义;正常培养条件下, Müller细胞自身不分泌EPO, 0.4 mmol/L H₂O₂致Müller细胞损伤80%时, 其培养液内EPO的表达量为正常培养液下的1.42倍;氧化损伤状态下, 0.08 mmol/L H₂O₂或8 U/L GO作用Müller细胞24 h后导致其半数死亡且Akt信号通路激活;提前2 h加入外源性EPO后, 发现30 U/mL EPO对抗氧化所致Müller细胞的损伤作用最明显;同时提前2 h加入Akt信号通路阻断剂LY294002后, Akt信号传导通路的保护作用被阻断减弱。结论 EPO对体外正常培养的Müller细胞不具有促进增殖迁移作用;正常培养条件下Müller细胞自身不表达并且不分泌EPO, 氧化损伤条件下Müller细胞自身可低分泌EPO;加入外源性EPO后, EPO可能通过Akt信号传导通路对H₂O₂损伤的Müller细胞发挥保护作用。

关键词: ller细胞, Mü, 增殖, 年龄相关性黄斑变性, 促红细胞生成素

Abstract: Objective To demonstrate whether EPO can inhibit Müller cell apoptosis induced by oxidative damage and to investigate the molecular mechanism. Methods The proliferation effect of EPO on cultured human Müller cell-MIO-M1 was determined by BrdU marking and MTT assay both under normal condition and exposed to H₂O₂ or GO with different concentration (0 U/mL, 0.01 U/mL, 0.1 U/mL, 1 U/mL, 10 U/mL, 30 U/mL and 100 U/mL) at 24 h, 48 h and 72 h after exposure. The Müller cell proliferation changes after the use of PI3K/PDK1/PKB(Akt) signal pathway blocker-LY294002 was surveyed by MTT assay. The Müller cell function on EPO expression and secretion was observed by ELISA. The EPO effect on ERK1/2 and Akt signal pathway was detected under different cultured condition in vitro by Western-blotting. Results EPO had no effect on Müller cell proliferation under normal culture condition. The 80% damaged Müller cell caused by 0.4 mmol/L H₂O₂ can secrete 1.42 times EPO as that under normal culture condition. Under oxidative damage condition, 0.08 mmol/L H₂O₂ or 8 U/L GO could cause half of Müller cells to be dead and activate Akt signal pathway. While adding EPO 2 h earlier, 30 U/mL of EPO can reduce the oxidative damage to the minimum. The protection effect of Akt signal pathway could be reduced by its blocker LY294002 spontaneously. Conclusion EPO has no proliferation and migration effects on Müller cells cultured in vitro. Under normal culture condition, Müller cells did not express and secrete EPO. While under oxidative damage, Müller cells can secret EPO which can protect Müller cells by Akt signal pathway.

Key words: ller cells, Age-related macular degeneration, Mü, Erythropoietin, Proliferation

中图分类号:

R774.1

陈春丽, 宋宗明, 贾新国, 周仲楼, 汪朝阳. 促红细胞生成素抑制氧化损伤诱导的人眼Müller细胞凋亡[J]. 山东大学耳鼻喉眼学报, 2015, 29(3): 65-71.

CHEN Chunli, SONG Zongming, JIA Xinguo, ZHOU Zhonglou, WANG Zhaoyang. Erythropoietin inhibits Müller cell apoptosis induced by oxidative damage[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2015, 29(3): 65-71.

参考文献

[1] Bird A C. The Bowman lecture. Towards an understanding of age-related macular disease[J]. Eye, 2003, 17(4):457-466.
[2] Harvey P T. Common eye diseases of elderly people: identifying and treating causes of vision loss[J]. Gerontology, 2003, 49(1):1-11.
[3] Mansoor S, Gupta N, Luczy-Bachman G, et al. Protective effects of memantine and epicatechin on catechol-induced toxicity on Müller cells in vitro[J]. Toxicology, 2010, 271(1):107-114.
[4] Sullivan R, Penfold P, Pow D V. Neuronal migration and glial remodeling in degenerating retinas of aged rats and in nonneovascular AMD[J]. Invest Ophthalmol Vis Sci, 2003, 44(2):856-865.
[5] Ambati J, Ambati B K, Yoo S H, et al. Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies[J]. Surv Ophthalmol, 2003, 48(3):257-293.
[6] Nowak J Z. Age-related macular degeneration (AMD): pathogenesis and therapy[J]. Pharmacol Re, 2006, 58(3):353-363.
[7] Kaur C, Foulds W S, Ling E A. Blood-retinal barrier in hypoxicischaemic ditions: basic concepts, clinical features and management[J]. Prog Retin Eye Res, 2008, 27(6): 622-647.
[8] Wang Z Y, Shen L J, Tu L, et al. Erythropoietin protects retinal pigment epithelial cells from oxidative damage[J]. Free Radic Biol Med, 2009, 46(8):1032-1041.
[9] Ye M, Hu D, Tu L, et al. Involvement of PI3K/Akt signaling pathway in hepatocyte growth factor—induced migration of uveal melanoma cells[J]. Invest Ophthalmol Vis Sci, 2008, 49(2):497-504.
[10] Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the pathogenesis of age-related macular degeneration[J]. Surv Ophthalmol, 2000, 45(2):115-134.
[11] Godley B F, Jin G F, Guo Y S, et al. Bcl-2 overexpression increases survival in human retinal pigment epithelial cells exposed to H₂O₂[J]. Exp Eye Res, 2002, 74(6):663-669.
[12] Kaur C, Foulds W S, Ling E A. Blood-retinal barrier in hypoxicischaemic conditions: basic concepts, clinical features and management[J]. Prog Retin Eye Res, 2008, 27(6): 622-647.
[13] Barbouti A, Doulias P T, Nousis L, et al. DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H₂O₂[J]. Free Radic Biol Med, 2002, 33(5): 691-702.
[14] Rollet-Labelle E, Grange M J, Elbim C, et al. Hydroxyl radical as a potential intracellular mediator of polymorphonuclear neutrophil apoptosis[J]. Free Radic Biol Med, 1998, 24(4):563-572.
[15] Halliwell B, Clement M V, Long L H. Hydrogen peroxide in the human body[J].FEBS Lett, 2000, 486(1):10-13.
[16] Cai J, Nelson K C, Wu M, et al. Oxidative damage and protection of the RPE[J]. Prog Ret Eye Res, 2000, 19(2):205-221.
[17] Fu Q L, Wu W, Wang H, et al. Up-regulated endogenous erythropoietin/erythropoietin receptor system and exogenous erythropoietin rescue retinal ganglion cells after chronic ocular hypertension[J]. Cell Mol Neurobiol, 2008, 28(2):317-329.
[18] Chong Z Z, Kang J Q, Maiese K. Erythropoietin is a novel vascular protectant through activation of AKT1 and mitochondrial modulation of cysteine proteases[J]. Circulation, 2002, 106(23):2973-2979.
[19] Katavetin P, Tungsanga K, Eiam-Ong S, et al. Antioxidative effects oferythropoietin[J]. Kidney Int Suppl, 2007, 107(1):10-15.
[20] Digicaylioglu M, Lipton S A. Erythropoietin-mediated neuroprotection involvescross-talk between Jak2 and NF-kappaB signalling cascades[J]. Nature, 2001, 412(6847):641-647.
[21] Parsa C J, Matsumoto A, Kim J, et al. Novel protective effect of erythropoietin in the infarcted heart[J]. J Clin Invest, 2003, 112(7):999-1007.
[22] Kilic U, Kilic E, Soliz J, et al. Erythropoietin protects from axotomy-induced degeneration of retinal ganglion cells by activating ERK-1/-2[J]. FASEB J, 2005, 19(3):249-251.
[23] Zhong L, Bradley J, Schubert W, et al. Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice[J]. Invest Ophthalmol Vis Sci, 2007, 48(3):1212-1218.
[24] Tsai J C, Wu L, Worgul B, et al. Intravitreal administration of erythropoietin and preservation of retinal ganglion cells in an experimental rat model of glaucoma[J]. Curr Eye Res, 2005, 30(11):1025-1031.
[25] Yamasaki M, Mishima H K, Yamashita H, et al. Neuroprotective effects of erythropoietin on glutamate and nitric oxide toxicity in primary cultured retinal ganglion cells[J]. Brain Res, 2005, 1050(1):15-26.
[26] Chong Z Z, Lin S H, Kang J Q, et al. Erythropoietin prevents early and late neuronal demise through modulation of AKT1 and induction of caspase 1, 3, and 8[J]. J Neurosci Res, 2003, 71(5):659-669.
[27] García-Ramírez M, Hernández C, Simó R. Expression of erythropoietin and its receptor in the human retina. A comparative study of diabetic and non-diabetic subjects[J]. Diabetes Care, 2008, 31(6):1189-1194.
[28] Barbouti A, Doulias P T, Nousis L, et al. DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H₂O₂[J]. Free Radic Biol Med, 2002, 33(5): 691-702.
[29] Zhang J, Wu Y, Jin Y, et al. Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes[J]. Invest Ophthalmol Vis Sci, 2008, 49(2):732-742.
[30] Bartesaghi S, Marinovich M, Corsini E, et al. Erythropoietin: a novel neuroprotective cytokine[J]. Neurotoxicology, 2005, 26(5):923-928.
[31] Hanada M, Feng J, Hemmings B A. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004, 1697(1):3-16.
[32] Cantley L C. The phosphoinositide 3-kinase pathway[J].Science, 2002, 296(5573):1655-1657.
[33] Yang P, Peairs J J, Tano R, et al. Oxidant-mediated Akt activation in human RPE cells[J]. Invest Ophthalmol Vis Sci, 2006, 47(10):4598-4606.
[34] Wang Z Y, Zhao K K, Song Z M, et al. Erythropoietin as a novel therapeutic agent for atrophic age-related macular degeneration[J]. Med Hypotheses, 2009, 72(4):448-450.

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