[1]陈云珍,张晶晶.自噬激活剂雷帕霉素对 A2E诱导视网膜色素上皮细胞自噬调节的影响[J].眼科新进展,2022,42(12):942-946.[doi:10.13389/j.cnki.rao.2022.0193]
　CHEN Yunzhen,ZHANG Jingjing.Regulation of autophagy activator rapamycin on the autophagy of retinal pigment epithelium cells induced by N-retinylidene-N-retinylethanolamine[J].Recent Advances in Ophthalmology,2022,42(12):942-946.[doi:10.13389/j.cnki.rao.2022.0193]
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自噬激活剂雷帕霉素对 A2E诱导视网膜色素上皮细胞自噬调节的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年12期
页码:
942-946
栏目:
实验研究
出版日期:
2022-12-05

文章信息/Info

Title:
Regulation of autophagy activator rapamycin on the autophagy of retinal pigment epithelium cells induced by N-retinylidene-N-retinylethanolamine
作者:
陈云珍; 张晶晶
102600　北京市，首都医科大学大兴教学医院眼科
Author(s):
CHEN Yunzhen; ZHANG Jingjing
Department of Ophthalmology,Daxing Teaching Hospital Affiliated to Capital Medical University,Beijing 102600,China
关键词:
N-亚视黄基-N-视黄基乙醇胺; 雷帕霉素; 视网膜色素上皮细胞; 自噬; 年龄相关性黄斑变性
Keywords:
N-retinylidene-N-retinylethanolamine;  rapamycin;  retinal pigment epithelium cells;  autophagy;  age-related macular degeneration
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2022.0193
文献标志码:
A
摘要:
目的　探讨自噬激活剂雷帕霉素对N-亚视黄基-N-视黄基乙醇胺（A2E）诱导视网膜色素上皮（RPE）细胞自噬调节的影响。方法　取人RPE细胞（ARPE-19细胞系）进行培养。将细胞分为4组，其中，对照组：使用DMEM-F12完全培养基孵育ARPE-19细胞；A2E组：使用含20 μmol·L-1 A2E的DMEM-F12完全培养基孵育ARPE-19细胞；雷帕霉素组：使用含100 nmol·L-1雷帕霉素的DMEM-F12完全培养基预处理细胞1 h后，再用DMEM-F12完全培养基孵育ARPE-19细胞；雷帕霉素联合A2E组：使用100 nmol·L-1雷帕霉素预处理细胞1 h后，再用含20 μmol·L-1 A2E的DMEM-F12完全培养基孵育ARPE-19细胞。使用CCK-8法检测各组细胞增殖情况，Procarta细胞因子分析试剂盒检测各组细胞培养上清液中细胞因子表达情况。透射电镜观察各组细胞超微结构，Western blot法检测各组细胞自噬相关蛋白Beclin-1和P62的表达，免疫荧光染色法检测各组细胞自噬标志物LC3蛋白的表达。结果　与对照组相比，A2E组细胞生存率显著下降（P<0.001），而雷帕霉素联合A2E组细胞生存率得到了部分恢复，与A2E组相比差异有统计学意义（P<0.01）。与A2E组相比，雷帕霉素联合A2E组中10种细胞因子含量均有所下降（均为P<0.01）。与对照组相比，雷帕霉素组细胞内出现散在自噬囊泡，A2E组细胞内出现较多的自噬囊泡，而雷帕霉素联合A2E组细胞内出现大量聚集的大体积的自噬囊泡。与对照组相比，A2E组中Beclin-1蛋白相对表达量显著升高（P<0.001），P62蛋白相对表达量显著降低（P<0.001）；与A2E组相比，雷帕霉素联合A2E组中Beclin-1蛋白相对表达量明显升高（P<0.01），而P62蛋白相对表达量明显降低（P<0.001）。雷帕霉素联合A2E组ARPE-19细胞内LC3蛋白的绿色荧光斑点大量聚集，与A2E组相比荧光强度增加，斑点体积更大，数量也更多。结论　雷帕霉素能够进一步激活A2E诱导的ARPE-19细胞自噬活性，一定程度上降低了A2E对RPE细胞造成的损伤，并抑制了ARPE-19细胞中炎症因子和血管生成因子的分泌。
Abstract:
Objective　To investigate the regulation action of autophagy activator rapamycin on the N-retinylidene-N-retinylethanolamine (A2E) induced autophagy of retinal pigment epithelium (RPE) cells. Methods　The adult RPE cells (ARPE-19 cell line) were cultured and divided into the control group (the ARPE-19 cells were cultured in DMEM-F12 complete medium), A2E group (the ARPE-19 cells were cultured in DMEM-F12 complete medium containing 20 μmol·L-1 A2E) , rapamycin group (the ARPE-19 cells were pre-treated in DMEM-F12 complete medium containing 100 nmol·L-1 rapamycin for 1 h and then cultured in normal DMEM-F12 complete medium), and rapamycin + A2E group (the ARPE-19 cells were pre-treated in DMEM-F12 complete medium containing 100 nmol·L-1 rapamycin for 1 h and then cultured in DMEM-F12 complete medium containing 20 μmol·L-1 A2E). Cell Counting Kit-8 was used to determine the proliferation of ARPE-19 cells in each group. Procarta Cytokine Assay Kit was used to measure the expression of cytokines in the cell culture supernatant of each group. The transmission electron microscope was used to observe the ultrastructure of ARPE-19 cells in each group. The expressions of autophagy-related proteins Beclin-1 and P62 were detected by Western blot, and the expression of autophagy marker protein LC3 was detected by immunofluorescence staining. Results　Compared with the control group, the survival rate of ARPE-19 cells in the A2E group decreased significantly (P<0.001), while the survival rate in the rapamycin + A2E group partially recovered compared to the A2E group (P<0.01). Compared with the A2E group, the levels of 10 cytokines decreased in the rapamycin + A2E group (all P<0.01). Compared with the control group, there were scattered autophagic vesicles in the ARPE-19 cells in the rapamycin group, more autophagic vesicles in the A2E group, and a large number of aggregated autophagic vesicles with large sizes in the rapamycin + A2E group. Compared with the control group, the expression of protein Beclin-1 in the A2E group was significantly increased (P<0.001), while the expression of P62 was significantly decreased (P<0.001). Compared with the A2E group, the expression of protein Beclin-1 in the rapamycin + A2E group was significantly increased (P<0.01), while the expression of P62 declined considerably (P<0.001). Compared with the A2E group, the green fluorescence spots of protein LC3 in ARPE-19 cells in the rapamycin + A2E group were clustered significantly, with higher fluorescent intensity, larger size and quantity. Conclusion　Rapamycin can further activate the A2E-induced ARPE-19 cell autophagy activity, reduce the injury caused by A2E to RPE cells, and inhibit the secretion of inflammatory factors and angiogenic factors in ARPE-19 cells.

参考文献/References:

［1］　BLASIAK J,PAWLOWSKA E,SOBCZUK A,SZCZEPANSKA J,KAARNIRANTA K.The aging stress response and its implication for AMD pathogenesis［J］.Int J Mol Sci,2020,21(22):8840.
［2］　YOU Q S,XU L,YANG H,LI Y B,WANG S,WANG J D,et al.Five-year incidence of age-related macular degeneration: the Beijing eye study［J］.Ophthalmology,2012,119:2519-2525.
［3］　SAADAT K A,MURAKAMI Y,TAN X,NOMURA Y,YASUKAWA T,OKADA E,et al.Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E［J］.FEBS Open Bio,2014,4:1007-1014.
［4］　BRONIEC A,PAWLAK A,SAMA T,WIELGUS A,ROBERTS J E,LAND E J,et al.Spectroscopic properties and reactivity of free radical forms of A2E［J］.Free Radic Biol Med,2005,38(8):1037-1046.
［5］　KURZ T,KARLSSON M,BRUNK U T,NILSSON S E,FRENNESSON C.ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron［J］.Autophagy,2009,5(4):494-501.
［6］　RYHANEN T,HYTTINEN J M,KOPITZ J,RILLA K,KUUSISTO E,MANNERMAA E,et al.Crosstalk between Hsp70 molecular chaperone,lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells［J］.J Cell Mol Med,2009,13(9B):3616-3631.
［7］　NAKAHIRA K,HASPEL J A,RATHINAM V A,LEE S J,DOLINAY T,LAM H C,et al.Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome［J］.Nat Immunol,2011,12(3):222-230.
［8］　KAARNIRANTA K,SALMINEN A,HAAPASALO A,SOININEN H,HILTUNEN M.Age-related macular degeneration (AMD): Alzheimer’s disease in the eye? ［J］.J Alzheimers Dis,2011,24(4):615-631.
［9］　LEE S J,KIM H P,JIN Y,CHOI A M,RYTER S W.Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis［J］.Autophagy,2011,7(8):829-839.
［10］　JUNG C H,RO S H,CAO J,OTTO N M,KIM D H.mTOR regulation of autophagy［J］.FEBS Lett,2010,584(7):1287-1295.
［11］　ZARBIN M A.Current concepts in the pathogenesis of age-related macular degeneration［J］.Arch Ophthalmol,2004,122(4):598-614.
［12］　KAARNIRANTA K,SINHA D,BLASIAK J,KAUPPINEN A,VEREB Z,SALMINEN A,et al.Autophagy and heterophagy dysregulation leads to retinal pigment epithelium dysfunction and development of age-related macular degeneration［J］.Autophagy,2013,9(7):973-984.
［13］　YAO J,TAO Z F,LI C P,LI X M,CAO G F,JIANG Q,et al.Regulation of autophagy by high glucose in human retinal pigment epithelium［J］.Cell Physiol Biochem,2014,33(1):107-116.
［14］　LI C P,YAO J,TAO Z F,LI X M,JIANG Q,YAN B.Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: a potential role for reducing UVB light-induced retinal damage［J］.Biochem Biophys Res Commun,2013,438(4):739-745.
［15］　KROHNE T U,STRATMANN N K,KOPITZ J,HOLZ F G.Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells［J］.Exp Eye Res,2010,90(3):465-471.
［16］　张晶晶，陈云珍，黄旅珍，陈莉．脂褐素成分A2E诱导人视网膜色素上皮细胞自噬及损伤的研究［J］.眼科新进展,2021,41(7)：608-614.
ZHANG J J,CHEN Y Z,HUANG L Z,CHEN L.Research on autophagy and injury of human retinal pigment epithelial cells induced by lipofuscincomponent A2E［J］.Rec Adv Ophthalmol,2021,41(7)：608-614.
［17］　JEFFERIES H B,FUMAGALLI S,DENNIS P B,REINHARD C,PEARSON R B,THOMAS G.Rapamycin suppresses 5’TOP mRNA translation through inhibition of p70s6k［J］.EMBO J,1997,16(12):3693-3704.
［18］　PANWALKAR A,VERSTOVSEK S,GILES F J.Mammalian target of rapamycin inhibition as therapy for hematologic malignancies［J］.Cancer,2004,100(4):657-666.
［19］　BJEDOV I,PARTRIDGE L.A longer and healthier life with TOR down-regulation: genetics and drugs［J］.Biochem Soc Trans,2011,39(2):460-465.
［20］　PANDYA K J,DAHLBERG S,HIDALGO M,COHEN R B,LEE M W,SCHILLER J H,et al.A randomized,phase II trial of two dose levels of temsirolimus (CCI-779) in patients with extensive-stage small-cell lung cancer who have responding or stable disease after induction chemotherapy: a trial of the Eastern Cooperative Oncology Group (E1500) ［J］.J Thorac Oncol,2007,2(11):1036-1041.
［21］　FECHNER G,CLASSEN K,SCHMIDT D,HAUSER S,MULLER S C.Rapamycin inhibits in vitro growth and release of angiogenetic factors in human bladder cancer［J］.Urology,2009,73(3):665-669.
［22］　KINKADE C W,CASTILLO-MARTIN M,PUZIO-KUTER A,YAN J,FOSTER T H,GAO H,et al.Targeting AKT/mTOR and ERK MAPK signaling inhibits hormone-refractory prostate cancer in a preclinical mouse model［J］.J Clin Invest,2008,118(9):3051-3064.
［23］　SHEN S,WANG X M,L H,SHI Y,XIAO L W.PADI4 mediates autophagy and participates in the role of ganoderic acid A monomers in delaying the senescence of Alzheimer’s cells through the Akt /mTOR pathway［J］.Biosci Biotechnol Biochem,2021,85(8): 1818-1829.
［24］　PRATIWI Y S,LESMANA R,GOENAWAN H,SYLVIANA N,SETIAWAN I,TARAWAN V M,et al.Nutmeg extract increases skeletal muscle mass in aging rats partly via IGF1-AKT-mTOR pathway and inhibition of autophagy［J］.Evid Based Complement Alternat Med,2018,2018: 2810840.

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备注/Memo

备注/Memo:
首都医科大学大兴教学医院院级课题（编号：4202142405）；中国博士后科学基金面上项目（编号：2015M570900）
更新日期/Last Update: 2022-12-05