[1]黄晓旭,苏荣健,刘华.青蒿素对过氧化氢诱导的人视网膜色素上皮（RPE）细胞氧化应激损伤的抑制作用[J].眼科新进展,2019,39(4):306-311.[doi:10.13389/j.cnki.rao.2019.0069]
　HUANG Xiao-Xu,SU Rong-Jian,LIU Hua.Inhibition of Artemisinin on oxidative stress injury induced by hydrogen peroxide in human retinal pigment epithelium cells[J].Recent Advances in Ophthalmology,2019,39(4):306-311.[doi:10.13389/j.cnki.rao.2019.0069]
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青蒿素对过氧化氢诱导的人视网膜色素上皮（RPE）细胞氧化应激损伤的抑制作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
39卷
期数:
2019年4期
页码:
306-311
栏目:
实验研究
出版日期:
2019-04-05

文章信息/Info

Title:
Inhibition of Artemisinin on oxidative stress injury induced by hydrogen peroxide in human retinal pigment epithelium cells
作者:
黄晓旭; 苏荣健; 刘华
121000　辽宁省锦州市，锦州医科大学附属第三医院，锦州医科大学基础医学院（黄晓旭）；121000　辽宁省锦州市，锦州医科大学基础医学院（苏荣健）；121000　辽宁省锦州市，锦州医科大学附属第三医院（刘华）
Author(s):
HUANG Xiao-Xu; SU Rong-Jian; LIU Hua
Third Affiliated Hospital of Jinzhou Medical University，School of Basic Medicine of Jinzhou Medical University （HUANG Xiao-Xu），Jinzhou 121000，Liaoning Province，China；the School of Basic Medicine of Jinzhou Medical University （SU Rong-Jian），Jinzhou 121000，Liaoning Province，China；the Third Affiliated Hospital of Jinzhou Medical University（LIU Hua），Jinzhou 121000，Liaoning Province，China
关键词:
青蒿素; 视网膜色素上皮细胞; 老年性黄斑变性; 氧化应激
Keywords:
Artemisinin; retinal pigment epithelium cells; age-related macular degeneration; oxidative stress
分类号:
R774
DOI:
10.13389/j.cnki.rao.2019.0069
文献标志码:
A
摘要:
目的　探讨青蒿素对过氧化氢诱导的人视网膜色素上皮（retinal pigment epithelium,RPE)细胞氧化应激损伤的抑制作用。方法　将ARPE-19细胞接种于96孔板，每孔7×103个细胞，细胞贴壁后分别加入不同浓度青蒿素，筛选对细胞增殖有效的最佳药物浓度。将APRE-19细胞分成4组。对照组：不做特殊处理；过氧化氢组：加入100 μmol·L-1过氧化氢作用细胞24 h；青蒿素组：加入青蒿素30 μmol·L-1，作用细胞24 h；青蒿素预保护组：加入30 μmol·L-1青蒿素预保护细胞24 h后加入100 μmol·L-1 过氧化氢作用24 h。利用MTT法筛选青蒿素对ARPE-19细胞作用的最佳药物浓度；通过Hoechst33342染色及线粒体膜电位观测细胞凋亡情况；检测细胞内超氧化物歧化酶（superoxide dismutase，SOD）含量和活性氧自由基（reactive oxygen species，ROS）阳性细胞数；Western blot法检测相关蛋白表达水平。结果　MTT法检测发现，30 μmol·L-1的青蒿素对细胞增殖作用最强 （均为P<0.01）。青蒿素预保护组与过氧化氢组相比，细胞增殖率升高，凋亡细胞数显著减少，差异均有统计学意义（均为P<0.05）。线粒体膜电位染色显示，青蒿素预保护组与过氧化氢组相比绿色染色减少，红色染色增多，说明青蒿素预保护组凋亡细胞数减少。与对照组相比，青蒿素组ARPE-19细胞中SOD含量显著增加（P<0.05）；与过氧化氢组相比，青蒿素预保护组细胞内SOD含量明显增高（P<0.05），说明青蒿素能增加细胞内抗氧化物质，减少细胞凋亡。与对照组相比，青蒿素组ROS阳性细胞减少（P<0.05）；与过氧化氢组相比，青蒿素预保护组ROS阳性细胞亦明显减少（P<0.05），说明青蒿素可减少过氧化氢诱导的活性氧物质积聚。通过Western blot检测发现，青蒿素能促进ARPE-19细胞内AKT的磷酸化，过氧化氢组ARPE-19细胞内Caspase-3、PARP、 Keap1相对表达升高，Bcl-2、Nrf2、HO-1降低；青蒿素预保护组ARPE-19细胞内Caspase-3、PARP、 Keap1相对表达降低，Bcl-2、Nrf2、HO-1升高，差异均有统计学意义（均为P<0.05）。结论　青蒿素通过调节Nrf2/keap1信号通路来减小过氧化氢诱导的ARPE-19细胞的氧化应激损伤。
Abstract:
Objective　To investigate the inhibitory effect of Artemisinin on oxidative stress injury induced by hydrogen peroxide in human retinal pigment epithelium （RPE） cells.Methods　ARPE-19 cells were seeded in 96-well plates with 7×103 cells in per well.After cells adhering the wall，different concentrations of Artemisinin were added to the cells.Then the best effective concentration of the drug was selected for cells proliferation.In this study，ARPE-19 cells were divided into four groups，and they were the control group，the hydrogen peroxide group，the Artemisinin group and the Artemisinin pre-protection group.The control group did not undergo any treatment，and the hydrogen peroxide group was treated with 100 μmol·L-1 hydrogen peroxide for 24 hours，and the Artemisinin group was treated with Artemisinin 30 μmol·L-1 for 24 hours after hydrogen peroxide intervention，while the Artemisinin pre-protection group was treated with 100 μmol·L-1 hydrogen peroxide for 24 hours after pre-treatment with 30 μmol·L-1 Artemisinin for 24 hours.The MTT assays were used to select the best concentration of Artemisinin in ARPE-19 cells.The status of cells apoptosis were observed by the Hoechst33342 staining and the detection of mitochondrial membrane potential.The content of superoxide dismutase （SOD） in cells and the numbers of reactive oxygen species （ROS） positive cells were detected.Western blot was used to examine the expression levels of related-proteins.Results　The MTT assay showed that the strongest proliferation concentration of Artemisinin in the cells was 30 μmol·L-1 （all P<0.01）.The rate of cell proliferation was higher in the Artemisinin pre-protection group than in the hydrogen peroxide group，but the number of apoptotic cells was less in Artemisinin pre-protection group than in the hydrogen peroxide group，and the differences were statistically significant （all P<0.05）.The staining of mitochondrial membrane potential showed that the green staining in the Artemisinin pre-protection group was less than that in the hydrogen peroxide group；meanwhile，the red staining in the Artemisinin pre-protection group was more than that in the hydrogen peroxide group，indicating that the number of apoptotic cells decreased in the Artemisinin pre-protection group.Compared with the control group，the content of SOD in ARPE-19 cells significantly increased in the Artemisinin group （P<0.05）.Similarly，the content of SOD in the Artemisinin pre-protection group was much more than that in the hydrogen peroxide group （P<0.05）.These proved that Artemisinin could enhance the content of intracellular antioxidant and reduce apoptosis.The ROS positive cells in the Artemisinin group were fewer than those in the control group （P<0.05）.At the same time，the ROS positive cells in Artemisinin pre-protection group were also fewer than that in the hydrogen peroxide group （P<0.05）.These testified that Artemisinin could reduce the accumulation of reactive oxygen species induced by hydrogen peroxide.Western blot analysis showed that Artemisinin could promote AKT phosphorylated action in ARPE-19 cells.The relative expression levels of Caspase-3，PARP and Keap1 proteins in the hydrogen peroxide group significantly increased，and the relative expression levels of Bcl-2，Nrf2 and HO-1 proteins in the hydrogen peroxide group significantly decreased.On the contrary，the relative expression of Caspase-3，PARP and Keap1 proteins in the Artemisinin pre-protection group were down-regulated when compared with the hydrogen peroxide group，and relative expression of Bcl-2，Nrf2 and HO-1 proteins in the Artemisinin pre-protection group were up-regulated when compared with the hydrogen peroxide group，and the differences were statistically significant （all P<0.05）.Conclusion　Artemisinin could reduce the oxidative stress injury induced by hydrogen peroxide in ARPE-19 cells through regulating the Nrf2/Keap1 signaling pathway.

参考文献/References:

［1］　SINGH D B，GUPTA M K，SINGH D V，SINGH S K，MISRA K.Docking and in silico ADMET studies of noraristeromycin，curcumin and its derivatives with plasmodium falciparum SAH hydrolase：a molecular drug target against malaria［J］.Interdiscip Sci，2013，5（1）：1-12.
［2］　KHAN K N，El-ASRAG M E，KU C A，HOLDER G E，MCKIBBIN M，ARNO G，et al.Specific alleles of CLN7/MFSD8，a protein that localizes to photoreceptor synaptic terminals，cause a spectrum of nonsyndromic retinal dystrophy［J］.Invest Ophthalmol Vis Sci，2017，58 （7）：2906-2914.
［3］　MOHAN R，SIVAK J，ASHTON P，RUSSO L A，PHAM B Q，KASHARA N，et al.Curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor-2，including expression of matrix metalloproteinase gelatinase B［J］.J Biol Chem，2000，275（14）：10405-10412.
［4］　CHANG C W，CHEN Y S，TSAY Y G，HAN C L，CHEN Y J，YANG C C，et al.ROS-independent ER stress-mediated NRF2 activation promotes warburg effect to maintain stemness-associated properties of cancer-initiating cells［J］.Cell Death Dis，2018，9（2）：194.
［5］　CHONG C M，ZHENG W.Artemisinin protects human retinal pigment epithelial cells from hydrogen peroxide-induced oxidative damage through activation of ERK/CREB signaling［J］.Redox Biol，2016，9（10）：50-56
［6］　LI L，DU G，WANG D，ZHOU J，JIANG G，JIANG H.Overexpression of heme oxygenase-1 in mesenchymal stem cells augments their protection on retinal cells in vitro and attenuates retinal ischemia/reperfusion injury in vivo against oxidative stress［J］.Stem Cells Int，2017，2017：4985323.
［7］　LIAO Y J，BAI H Y，LI Z H，ZOU J，CHEN J W，ZHENG F，et al.Longikaurin A，a natural ent-kaurane，induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells［J］.Cell Death Dis，2014，5：e1137.
［8］　BABU E，ANANTH S，VEERANAN-KARMEGAM R，COOTHA-NKANDASWAMY V，SMITH S B，BOETTGER T，et al.Transport via SLC5A8 （SMCT1） is obligatory for-oxothiazolidine-4-carboxylate to enhance glutathione production in retinal pigment epithelial cells［J］.Invest Ophthalmol Vis Sci，2011，52（8）：5749-5757.
［9］　LOPEZ-LUPPO M，CATITA J，RAMOS D，NAVARRO M，CARRETRO A，MENDES-JORGE L，et al.Cellular senescence is associated with human retinal microaneurysm formation during aging［J］.Invest Ophthalmol Vis Sci，2017，58（7）：2832-2842.
［10］　MA X，LIN W，LIN Z，HAO M，GAO X，ZHANG Y，et al.Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways［J］.Peptides，2017，6（92）：1-8.
［11］　WEIGEL A L，HANDA J T，HJELMELAND L M.Microarray analysis of H2O2-，HNE-，or tBH-treated ARPE-19 cells［J］.Free Radical Biol Med，2002，33（10）：1419-1432.
［12］　KOZLOWSKI M R.The ARPE-19 cell line：mortality status and utility in macular degeneration research［J］.Curr Eye Res，2015，40（5）：501-509.
［13］　PANAGIS L，ZHAO X，GE Y，REN L，MITTAG T W，DANIAS J.Gene expression changes in areas of focal loss of retinal ganglion cells in the retina of DBA/2J mice［J］.Invest Ophthalmol Vis Sci，2010，51（4）：2024-2034.
［14］　YOKOYAMA Y，MARUYAMA K，YAMAYOTO K，OMODAKA K，YASUDA M，HIMORI N，et al.The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage［J］.Biochem Biophys Res Commun，2014，451（4）：510-515.
［15］　ZHENG Y，LIU Y，GE J，WANG X，LIU L，BU Z，et al.Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase，SOD-1，and HO-1 expression［J］.Mol Vis，2010，16：1467-1474.
［16］　MATSUNAGA D，SREEKUMAR P G，ISHIKAWA K，TERASAKI H，BARRON E，COHEN P，et al.Human in protects rpe cells from endoplasmic reticulum stress-induced apoptosis by upregulation of mitochondrial glutathione［J］.PLoS One，2016，11（10）：1-22.
［17］　KORIYAMA Y，NAKAYAMA Y，MATSUGO S，KATO S.Protective effect of lipoic acid against oxidative stress is mediated by Keap1/Nrf2-dependent heme oxygenase-1 induction in the RGC-5 cellline［J］.Brain Res，2013，1499：145-157.

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备注/Memo

备注/Memo:
国家自然科学基金资助（编号：31571184）；辽宁省科技厅-锦州医科大学联合基金项目（编号：20170540382）
更新日期/Last Update: 2019-04-15