[1]许雲,陈泽君,权卓娅,等.线粒体靶向肽SS31及RIP抑制剂Necrostatin-1 (Nec-1)对H2O2诱导的氧化应激损伤ARPE-19细胞的保护作用[J].眼科新进展,2020,40(7):607-611.[doi:10.13389/j.cnki.rao.2020.0139]
　XU Yun,CHEN Zejun,QUAN Zhuoya,et al.Protective effect of mitochondrial targeted peptide SS31 and the RIP1 inhibitor necrostatin-1 (Nec-1) on ARPE-19 cells after oxidative stress injury induced byH2O2[J].Recent Advances in Ophthalmology,2020,40(7):607-611.[doi:10.13389/j.cnki.rao.2020.0139]
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线粒体靶向肽SS31及RIP抑制剂Necrostatin-1 (Nec-1)对H2O2诱导的氧化应激损伤ARPE-19细胞的保护作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年7期
页码:
607-611
栏目:
实验研究
出版日期:
2020-07-05

文章信息/Info

Title:
Protective effect of mitochondrial targeted peptide SS31 and the RIP1 inhibitor necrostatin-1 (Nec-1) on ARPE-19 cells after oxidative stress injury induced byH2O2
作者:
许雲; 陈泽君; 权卓娅; 张瑞雪; 何蓓蕾; 何媛
710038　陕西省西安市，西安医学院第二附属医院眼科（许雲，陈泽君，权卓娅,张瑞雪，何蓓蕾，何媛）；710021　陕西省西安市，西安医学院（许雲，陈泽君，权卓娅,张瑞雪，何蓓蕾）
Author(s):
XU Yun1; 2;  CHEN Zejun1; 2;  QUAN Zhuoya1; 2;  ZHANG Ruixue1; 2;  HE Beilei1; 2;  HE Yuan1
1.Department of Ophthalmology, the Second Affiliated Hospital of Xi’an Medical University, Xi’an 710038, Shaanxi Province, China
2.Xi’an Medical University, Xi’an 710068, Shaanxi Province, China
关键词:
线粒体靶向肽SS31; 氧化应激; ARPE-19细胞; 程序性坏死
Keywords:
mitochondrial targeted peptide SS31;  oxidative stress; ARPE-19 cells;  necroptosis
分类号:
R774
DOI:
10.13389/j.cnki.rao.2020.0139
文献标志码:
A
摘要:
目的　探讨线粒体靶向肽SS31及RIP抑制剂Necrostatin-1 (Nec-1)对H2O2诱导的氧化应激损伤ARPE-19细胞的保护作用。方法　选择400 μmol·L-1 H2O2构建氧化应激损伤模型；根据MTT结果筛选出1 μmol·L-1 SS31、40 μmol·L-1 Nec-1作为实验最佳浓度。将培养的细胞分为对照组、SS31+Nec-1组、H2O2组、SS31+H2O2 组、SS31+Nec-1+H2O2组、Nec-1+H2O2组。利用MTT测定细胞存活率，双氯荧光素染色测定细胞活性氧自由基（ROS）水平，JC-1染色测定线粒体膜电位，AnnexinV/PI双染检测细胞PI阳性率，Western blot 法检测RIP3蛋白表达水平。结果　与对照组相比，H2O2 组细胞存活率明显降低（P<0.05）。SS31 + H2O2组、Nec-1+ H2O2组细胞存活率高于H2O2 组，差异均有统计学意义（均为P<0.05）。荧光显微镜下双氯荧光素染色结果显示，与H2O2组相比，SS31+H2O2 组、Nec-1+H2O2组绿色荧光明显减弱，细胞内ROS含量明显减少。流式细胞仪结果显示：与对照组相比，H2O2 组线粒体膜电位降低的细胞比例升高，差异有统计学意义（P<0.05）；与H2O2 组相比，SS31+H2O2组、Nec-1+H2O2 组线粒体膜电位降低的细胞比例降低，差异均有统计学意义（均为P<0.05）。AnnexinV/PI双染色结果显示：与对照组相比，H2O2 组PI阳性率增加；与H2O2 组相比，SS31+ H2O2组、Nec-1+H2O2组PI阳性率均明显降低，差异均有统计学意义（均为P<0.05）。Western blot检测结果显示，与对照组相比，H2O2组RIP3 蛋白表达水平明显升高，差异有统计学意义（P<0.05）；与H2O2 组相比，SS31+ H2O2 组、Nec-1+H2O2 组RIP3 蛋白表达上调作用显著减弱，差异均有统计学意义（均为P<0.05）。结论　SS31及Nec-1对H2O2诱导的ARPE-19细胞氧化应激损伤具有明显的保护作用。
Abstract:
Objective　To investigate the protective effect of mitochondrial targeted peptide SS31 and the RIP1 inhibitor necrostatin-1 (Nec-1) on ARPE-19cells against oxidative stress injury induced by H2O2. Methods　The cell model of oxidative damage was established by adding 400 μmol·L-1 hydrogen peroxide (H2O2), According to the MTT results, 1 μmol·L-1 SS31 and 40 μmol·L-1 Nec-1 were the optimal concentration for sequential study. The cells were divided into control group, H2O2 group, SS31+Nec-1 group, SS31+H2O2 group, SS31+Nec-1+H2O2 group, and Nec-1+H2O2 group. MTT assay was used to detect the survival rate of the cells, while the level of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescin diacetate (DCFH-DA) staining. Mitochondrial membrane potential was measured using the JC-1 assay, and the PI positive rate was determined by flow cytometry after Annexin V/Propidium-dide (PI) staining. In addition，the protein levels of RIP3 (an indicator of necroptosis) were determined by Western blot. Results　The rate of cell viability was lower in the H2O2 group than in the control group（P<0.05）. The viability of the cells improved following treatment with SS31 and Nec-1 compared with H2O2 group, and the differences were statistically significant (all P<0.05). DCFH-DA staining showed that green fluorescence cells were much more in the H2O2 group compared with SS31+H2O2 group and Nec-1+H2O2 group, suggesting that treatment with SS31 and Nec-1 significantly decreased the quantity of ROS. Flow cytometry analysis confirmed that the proportion of cells with decreased mitochondrial membrane potential in the H2O2 group was higher than those in the control group, SS31+H2O2 group and Nec-1+H2O2 group, and the difference was statistically significant (all P<0.05). AnnexinV/PI double staining results showed that the PI-positive rate of ARPE-19 cells in H2O2 group was significantly higher than that in the control group, SS31+H2O2 group and Nec-1+H2O2 group (all P<0.05). Western blot analysis showed that compared with the control group, the relative expression levels of RIP3 in the H2O2 group was significantly increased（P<0.05）, but the relative expression levels of RIP3 in SS31 and Nec-1 treated H2O2 group were down-regulated when compared with H2O2 group (all P<0.05). Conclusion　SS31 and Nec-1 can obviously protect ARPE-19 cells from the sustained oxidative stress induced by H2O2.

参考文献/References:

［1］　MEHTA S.Age-related macular degeneration［J］.Prim Care,2015,42(3): 377-391.
［2］　YILDIRIM Z,UCGUN N I,YILDIRIM F.The role of oxidative stress and antioxidants in the pathogenesis of age-related macular degeneration［J］.Clinics (Sao Paulo),2011,66(5): 743-746.
［3］　KHANDHADIA S,LOTERY A.Oxidation and age-related macular degeneration: insights from molecular biology［J］.Expert Rev Mol Med,2010,12: e34.
［4］　DUNAIEF J L,DENTCHEV T,YING G S,MILAM A H.The role of apoptosis in age-related macular degeneration［J］.Arch Ophthalmol,2002,120(11): 1435-1442.
［5］　HANUS J,ANDERSON C,WANG S.RPE necroptosis in response to oxidative stress and in AMD［J］.Ageing Res Rev,2015,24(Pt B): 286-298.
［6］　HANUS J,ZHANG H,WANG Z,LIU Q,ZHOU Q,WANG S.Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells［J］.Cell Death Dis,2013,4: e965.
［7］　ESCRIBANO-LOPEZ I,DIAZ-MORALES N,IANNANTUONI F,LOPEZ-DOMENECH S,DE MARANON A M,ABAD-JIMENEZ Z,et al.The mitochondrial antioxidant SS-31 increases SIRT1 levels and ameliorates inflammation,oxidative stress and leukocyte-endothelium interactions in type 2 diabetes［J］.Sci Rep,2018,8(1): 15862.
［8］　LIU Y,YANG W,SUN X,XIE L,YANG Y,SANG M,et al.SS31 ameliorates sepsis-induced heart injury by inhibiting oxidative stress and inflammation［J］.Inflammation,2019,42(6): 2170-2180.
［9］　ZHU L L,LI M Q,HE F,ZHOU S B,JIANG W.Mitochondria targeted peptide attenuates mitochondrial dysfunction,controls inflammation and protects against spinal cord injury-induced lung injury［J］.Cell Physiol Biochem,2017,44(1): 388-400.
［10］　ZHU Y,WANG H,FANG J,DAI W,ZHOU J,WANG X,et al.SS-31 provides neuroprotection by reversing mitochondrial dysfunction after traumatic brain injury［J］.Oxid Med Cell Longev,2018,2018: 4783602.
［11］　PANG Y,WANG C,YU L.Mitochondria-targeted antioxidant ss-31 is a potential novel ophthalmic medication for neuroprotection in glaucoma［J］.Med Hypothesis Discov Innov Ophthalmol,2015,4(3): 120-126.
［12］　CHEN M,LIU B,GAO Q,ZHUO Y,GE J.Mitochondria-targeted peptide MTP-131 alleviates mitochondrial dysfunction and oxidative damage in human trabecular meshwork cells［J］.Invest Ophthalmol Vis Sci,2011,52(10): 7027-7037.
［13］　LI J,CHEN X,XIAO W,MA W,LI T,HUANG J,et al.Mitochondria-targeted antioxidant peptide SS31 attenuates high glucose-induced injury on human retinal endothelial cells［J］.Biochem Biophys Res Commun,2011,404(1): 349-356.
［14］　GARG T K,CHANG J Y.Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12,14-PGJ2［J］.BMC Ophthalmol,2003,3: 5.
［15］　KIM J H,KIM J H,JUN H O,YU Y S,MIN B H,PARK K H,et al.Protective effect of clusterin from oxidative stress-induced apoptosis in human retinal pigment epithelial cells［J］.Invest Ophthalmol Vis Sci,2010,51(1): 561-566.
［16］　HANUS J,ANDERSON C,SARRAF D,MA J,WANG S.Retinal pigment epithelial cell necroptosis in response to sodium iodate［J］.Cell Death Discov,2016,2: 16054.
［17］　VANLANGENAKKER N,VANDEN BERGHE T,VANDENABEELE P.Many stimuli pull the necrotic trigger,an overview［J］.Cell Death Differ,2012,19(1): 75-86.
［18］　SZETO H H.Cell-permeable,mitochondrial-targeted,peptide antioxidants［J］.AAPS J,2006,8(2): E277-283.
［19］　ZHAO K,ZHAO G M,WU D,SOONG Y,BIRK A V,SCHILLER P W,et al.Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling,oxidative cell death,and reperfusion injury［J］.J Biol Chem,2004,279(33): 34682-34690.
［20］　ZHAO K,LUO G,GIANNELLI S,SZETO H H.Mitochondria-targeted peptide prevents mitochondrial depolarization and apoptosis induced by tert-butyl hydroperoxide in neuronal cell lines［J］.Biochem Pharmacol,2005,70(12): 1796-806.
［21］　MA W,ZHU X,DING X,LI T,HU Y,HU X,et al.Protective effects of SS31 on tBHP induced oxidative damage in 661W cells［J］.Mol Med Rep,2015,12(4): 5026-5034.
［22］　CAI M,LI J,LIN S,CHEN X,HUANG J,JIANG X,et al.Mitochondria-targeted antioxidant peptide ss31 protects cultured human lens epithelial cells against oxidative stress［J］.Curr Eye Res,2015,40(8): 822-829.

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备注/Memo

备注/Memo:
国家自然科学基金资助（编号：81100665、81770929）；陕西省教育厅2018年服务地方科学研究计划资助（编号：18JC026）；陕西省科技厅项目资助（编号：2019SF-162）
更新日期/Last Update: 2020-07-05