[1]张楚,陈倩雯,周文杰,等.miR-22-3p对蓝光暴露下大鼠视网膜神经节细胞的保护作用及其机制[J].眼科新进展,2022,42(10):780-785.[doi:10.13389/j.cnki.rao.2022.0160]
　ZHANG Chu,CHEN Qianwen,ZHOU Wenjie,et al.Protective effect and mechanism of miR-22-3p on rat retinal ganglion cells exposed to blue light[J].Recent Advances in Ophthalmology,2022,42(10):780-785.[doi:10.13389/j.cnki.rao.2022.0160]
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miR-22-3p对蓝光暴露下大鼠视网膜神经节细胞的保护作用及其机制/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年10期
页码:
780-785
栏目:
实验研究
出版日期:
2022-10-05

文章信息/Info

Title:
Protective effect and mechanism of miR-22-3p on rat retinal ganglion cells exposed to blue light
作者:
张楚; 陈倩雯; 周文杰; 李征亚; 俞永珍; 邓启凤; 邹玉平
510010　广东省广州市，中国人民解放军南部战区总医院眼科
Author(s):
ZHANG Chu; CHEN Qianwen; ZHOU Wenjie; LI Zhengya; YU Yongzhen; DENG Qifeng; ZOU Yuping
Department of Ophthalmology,General Hospital of the Southern Theater Command of the Chinese People’s Liberation Army,Guangzhou 510010,Guangdong Province,China
关键词:
miR-22-3p; 视网膜神经节细胞; 蓝光; 细胞凋亡
Keywords:
miR-22-3p;  retinal ganglion cells;  blue light;  apoptosis
分类号:
R774.6
DOI:
10.13389/j.cnki.rao.2022.0160
文献标志码:
A
摘要:
目的　探讨miR-22-3p对蓝光暴露下的大鼠视网膜神经节细胞（RGC）的保护作用及机制。方法　取36只清洁级SD大鼠, 将12只SD大鼠随机分为2组：对照组与蓝光暴露组，每组6只。对照组采用正常的12 h明暗循环饲养；蓝光暴露组大鼠先进行暗适应24 h，随后采用复方托吡卡胺散瞳，将大鼠暴露在蓝光（光照强度1500 lux）下2 h；另取24只大鼠随机分为4组：对照组、蓝光暴露组、AAV-miR22组、AAV-miR22&PTEN组，每组6只。对照组采用正常的12 h明暗循环饲养，其余3组均接受蓝光暴露处理，AAV-miR22组注射1 μL含 2.5×109 vg(基因组拷贝数)的AAV-7m8-miR-22-3p，AAV-miR22&PTEN组注射1 μL含2.5×109 vg的AAV-7m8-miR-22-3p&PTEN，对照组与蓝光暴露组向大鼠玻璃体内注射1 μL生理盐水，注射结束使用氧氟沙星眼膏预防感染。取大鼠眼球组织行HE染色，在光学显微镜下观察检测神经节细胞层（GCL）、GCL至外核层 (ONL)的厚度，其中包含有内丛状层（IPL）、内核层(INL)、外丛状层（OPL）；采用NeuN免疫荧光组织化学染色标记RGC，计数视网膜上NeuN标记的阳性细胞数；TUNEL染色检测细胞凋亡；采用Lipofectamine 2000转染试剂盒转染阴性对照、miR-22-3p mimic、miR-22-3p inhibitor至RGC-5细胞，转染48 h后测定miR-22-3p表达量，转染成功即可采用Western blot检测PTEN蛋白表达情况；采用实时荧光定量PCR检测miR-22-3p表达，Western blot检测PTEN、p-Akt、Akt与Nrf2蛋白表达情况；双荧光素酶报告实验在酶标仪上测定荧光素酶活性。结果　HE染色结果证实：与对照组相比，蓝光暴露组大鼠视网膜组织萎缩，视网膜GCL、GCL至ONL厚度变薄（P<0.05）。另外，NeuN免疫荧光组织化学染色发现：与对照组相比，蓝光暴露组大鼠视网膜组织RGC数减少（P<0.05）。实时荧光定量PCR实验结果显示：与对照组相比，蓝光暴露组大鼠视网膜组织内miR-22-3p相对表达量降低（P<0.05）。Western blot检测结果显示：与对照组相比，蓝光暴露组大鼠视网膜组织内PTEN蛋白的表达水平升高，Nrf2蛋白的表达水平和p-Akt/Akt蛋白表达比值均降低（均为P<0.05）；miR-22-3p能够负向调控PTEN蛋白在RGC内的表达水平（P<0.05）。HE染色结果显示：过表达miR-22-3p能够缓解蓝光诱导的大鼠视网膜萎缩，并且能够提高大鼠视网膜GCL、GCL至ONL厚度。NeuN免疫荧光组织化学染色结果证实：过表达miR-22-3p能够通过靶向抑制PTEN表达提高蓝光暴露大鼠视网膜上RGC数。TUNEL实验结果证实：miR-22-3p能够通过抑制PTEN表达缓解蓝光诱导的大鼠视网膜RGC凋亡。结论　miR-22-3p能够通过靶向抑制PTEN的表达，激活PI3K/Akt/Nrf2通路，缓解蓝光诱导的大鼠RGC凋亡。
Abstract:
Objective　To investigate the effect of miR-22-3p on rat retinal ganglion cells (RGC) exposed to blue light and its mechanism. Methods　Thirty-six SD rats were selected, and 12 of them were randomly divided into the control group and blue light exposure group, with 6 rats in each group. Rats in the control group were fed under normal conditions for 24 h, while rats in the blue light exposure group were first exposed to dark light for 24 h and then exposed to blue light (1500 lux) for 2 h after mydriasis by compound tropicamide. The remaining 24 rats were randomly divided into the control group, blue light exposure group, AAV-miR22 group, and AAV-miR22 & PTEN group, with 6 rats in each group. Rats in the control group were fed under normal conditions for 24 h, while rats in the rest three groups were exposed to blue light. In addition, rats in the AAV-miR22 group were injected with 1 μL of AAV-7m8-miR-22-3p containing 2.5×109 vg (genome copy number), rats in the AAV-miR22 & PTEN group were injected with 1 μL of AAV-7m8-miR-22-3p & PTEN containing 2.5×109 vg, and rats in the control group and the blue light exposure group were intravitreally injected with 1 μL of normal saline. After injection, ofloxacin eye ointment was used to prevent infection. Eyeballs were taken to receive HE staining. The thickness of ganglion cell layer (GCL) and GCL to outer nuclear layer (ONL), including inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL), was measured under the optical microscope. RGCs were labeled with NeuN immunohistochemistry (IHC) staining to count the number of NeuN-labeled positive cells on the retina. TUNEL staining was performed to detect apoptosis. RGC-5 cells were transfected with negative control, miR-22-3p mimic, and miR-22-3p inhibitor using Lipofectamine 2000 transfection kit, and the expression of miR-22-3p was determined after 48 h. In the case of successful transfection, the PTEN protein expression was measured by Western blot. The miR-22-3p expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The PTEN, p-Akt, Akt and Nrf2 protein levels were assessed by Western blot. The luciferase activity was evaluated by the dual-luciferase reporter assay on a microplate reader. Results　HE staining results showed that compared with the control group, the rat retina in the blue light exposure group shrank, and the thickness of GCL and GCL to ONL decreased (P<0.05). NeuN IHC staining results showed that compared with the control group, the number of RGCs in the blue light exposure group decreased (P<0.05). RT-qPCR results showed that compared with the control group, the relative expression of retinal miR-22-3p in the blue light exposure group decreased (P<0.05). Western blot results showed that compared with the control group, the PTEN protein level in the retina increased under blue light exposure, while the Nrf2 protein level and the p-Akt/Akt ratio decreased (all P<0.05); miR-22-3p could negatively regulate the expression level of PTEN protein in RGCs (P<0.05). HE staining results showed that the overexpression of miR-22-3p could relieve retinal atrophy induced by blue light and increase the thickness of GCL and GCL to ONL. NeuN IHC staining results confirmed that the overexpression of miR-22-3p could increase the number of RGCs in the retina under blue light exposure by inhibiting the PTEN expression. TUNEL assay results confirmed that miR-22-3p could decrease the blue light-induced RGC apoptosis by inhibiting the PTEN expression. Conclusion　miR-22-3p reduces blue light-induced RGC apoptosis by inhibiting the PTEN expression and activating the PI3K/Akt/Nrf2 pathway.

参考文献/References:

［1］　ZOU X L,YU Y Z,YU H H,WANG G F,PI R B,XU Z,et al.Protective effects of lipoic acid-niacin dimers against blue light-induced oxidative damage to retinal pigment epithelium cells［J］.Int J Ophthalmol,2019,12(8):1262-1271.
［2］　DAI M,LIU Y,NIE X,ZHANG J,WANG Y,BEN J,et al.Expression of RBMX in the light-induced damage of rat retina in vivo［J］.Cell Mol Neurobiol,2015,35(4):463-471.
［3］　YANG P L,TSUJIMURA S,MATSUMOTO A,YAMASHITA W,YEH S L.Subjective time expansion with increased stimulation of intrinsically photosensitive retinal ganglion cells［J］.Sci Rep,2018,8(1):11693.
［4］　HATORI M,GRONFIER C,VAN GELDER R N,BERNSTEIN P S,CARRERAS J,PANDA S,et al.Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies［J］.NPJ Aging Mech Dis,2017,3:9.
［5］　ASAKAWA K,ISHIKAWA H.Why do melanopsin-containing retinal ganglion cells have the greatest sensitivity to blue light?［J］.Acta Ophthalmol,2015,93(4):e308-e309.
［6］　XU Y,YU S,SHU Q,YANG L,YANG C,WANG J,et al.Upregulation of CREM-1 relates to retinal ganglion cells apoptosis after light-induced damage in vivo［J］.J Mol Neurosci,2014,52(3):331-338.
［7］　SHU Q,XU Y,ZHUANG H,FAN J,SUN Z,ZHANG M,et al.Ras homolog enriched in the brain is linked to retinal ganglion cell apoptosis after light injury in rats［J］.J Mol Neurosci,2014,54(2):243-251.
［8］　OUYANG X L,CHEN B Y,YF X,WU Y D,GUO S J,DONG X Y,et al.Whole transcriptome analysis on blue light-induced eye damage［J］.Int J Ophthalmol,2020,13(8):1210-1222.
［9］　ZHENG Y,LIU J,CHEN P,LIN L,LUO Y,MA X,et al.Exosomal miR-22-3p from human umbilical cord blood-derived mesenchymal stem cells protects against lipopolysaccharid-induced acute lung injury［J］.Life Sci,2021,269:119004.
［10］　HU Z,LV X,CHEN L,GU X,QIAN H,FRANSISCA S,et al.Protective effects of microRNA-22-3p against retinal pigment epithelial inflammatory damage by targeting NLRP3 inflammasome［J］.J Cell Physiol,2019,234(10):18849-18857.
［11］　WANG X,WANG Y,KONG M,YANG J.MiR-22-3p suppresses sepsis-induced acute kidney injury by targeting PTEN［J］.Biosci Rep,2020,40(6):BSR20200527.
［12］　KRIGEL A,BERDUGO M,PICARD E,LEVY-BOUKRIS R,JAADANE I,JONET L,et al.Light-induced retinal damage using different light sources,protocols and rat strains reveals LED phototoxicity［J］.Neuroscience,2016,339:296-307.
［13］　NAKAMURA M,KUSE Y,TSURUMA K,SHIMAZAWA M,HARA H.The involvement of the oxidative stress in murine blue LED Light-Induced retinal damage model［J］.Biol Pharm Bull,2017,40(8):1219-1225.
［14］　李丹丹,邹秀兰,陈京霞,徐哲,俞永珍,周文杰,等.组织因子靶向肽对蓝光诱导的人视网膜色素上皮细胞损伤的保护作用［J］.中华实验眼科杂志,2017,35(7):603-609.
LI D D,ZOU X L,CHEN J X,XU Z,YU Y Z,ZHOU W J,et al.Protective effect of tissue factor targeting peptide on human retinal pigment epithelial cell injury induced by blue light［J］.Chin J Exp Ophthalmol,2017,35(7):603-609.
［15］　OSBORNE N N,NU＇EZ-LVAREZ C,OLMO-AGUADO O S.The effect of visual blue light on mitochondrial function associated with retinal ganglions cells［J］.Exp Eye Res,2014,128:8-14.
［16］　LIN C W,YANG C M,YANG C H.Protective effect of astaxanthin on blue light Light-Emitting Diode-Induced retinal cell damage via free radical scavenging and activation of PI3K/Akt/Nrf2 pathway in 661W cell model［J］.Mar Drugs,2020,18(8):387.
［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.
［18］　WEN Z,HOU W,WU W,ZHAO Y,DONG X,BAI X,et al.6’-O-Galloylpaeoniflorin attenuates cerebral ischemia Reperfusion-Induced neuroinflammation and oxidative stress via PI3K/Akt/Nrf2 activation［J］.Oxid Med Cell Longev,2018,2018:8678267.
［19］　SHEN Y,ZHAO H,WANG Z,GUAN W,KANG X,TAI X,et al.Silibinin declines blue light-induced apoptosis and inflammation through MEK/ERK/CREB of retinal ganglion cells［J］.Artif Cells Nanomed Biotechnol,2019,47(1):4059-4065.
［20］　NU＇EZ-LVAREZ C,OSBORNE N N.Blue light exacerbates and red light counteracts negative insults to retinal ganglion cells in situ and R28 cells in vitro［J］.Neurochem Int,2019,125:187-196.
［21］　LV J,XING Y.Down-regulation of Fra-2 alleviates light-induced retina damage by inhibiting the PARP-1/AIF pathway［J］.Int J Clin Exp Pathol,2018,11(8):4221-4229.
［22］　HUANG C,ZHANG P,WANG W,XU Y,WANG M,CHEN X,et al.Long-term blue light exposure induces RGC-5 cell death in vitro:involvement of mitochondria-dependent apoptosis,oxidative stress,and MAPK signaling pathways［J］.Apoptosis,2014,19(6):922-932.

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备注/Memo

备注/Memo:
广州市科技计划项目（编号：202002030413）
更新日期/Last Update: 2022-10-05