[1]石磊,王爱媛,归东梅,等.紫外线B照射在抑制人视网膜色素上皮细胞自噬中的作用[J].眼科新进展,2021,41(4):306-310.[doi:10.13389/j.cnki.rao.2021.0064]
　SHI Lei,WANG Aiyuan,GUI Dongmei,et al.Ultraviolet B exposure inhibits autophagy in retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2021,41(4):306-310.[doi:10.13389/j.cnki.rao.2021.0064]
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紫外线B照射在抑制人视网膜色素上皮细胞自噬中的作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年4期
页码:
306-310
栏目:
实验研究
出版日期:
2021-04-05

文章信息/Info

Title:
Ultraviolet B exposure inhibits autophagy in retinal pigment epithelial cells
作者:
石磊; 王爱媛; 归东梅; 杨宏伟
110004　辽宁省沈阳市，中国医科大学附属盛京医院眼科
Author(s):
SHI Lei; WANG Aiyuan; GUI Dongmei; YANG Hongwei
Department of Ophthalmology,Shengjing Hospital of China Medical University,Shenyang 110004,Liaoning Province,China
关键词:
紫外线B;  年龄相关性黄斑变性; 视网膜色素上皮细胞;  自噬
Keywords:
ultraviolet B;  age-related macular degeneration;  retinal pigment epithelial cells;  autophagy
分类号:
R774
DOI:
10.13389/j.cnki.rao.2021.0064
文献标志码:
A
摘要:
目的　探讨紫外线B(ultraviolet B,UVB)照射在抑制人视网膜色素上皮细胞株ARPE-19细胞自噬中的作用。方法　不同剂量UVB照射ARPE-19细胞后不同时间收集细胞，应用MTT法检测UVB照射对细胞存活的影响；Western blot检测细胞中自噬蛋白LC3的表达及LC3-II/LC3-I比率、Beclin-1蛋白表达水平；应用细胞自噬增强剂雷帕霉素（rapamycin，RAPA）和细胞自噬抑制剂氯化铵（NH4Cl）进一步验证UVB照射对于细胞自噬的调节；采用环氧霉素（epoxomicin，EPO）检测泛素-蛋白酶体系统(ubiquitin-proteasome system，UPS)对于UVB作用的影响。MTT法检测细胞自噬变化对于细胞生存的影响。结果　UVB照射组诱导细胞剂量依赖性的死亡，100 mJ·cm-2 UVB照射后24 h细胞出现明显死亡，和对照组相比差异具有统计学意义（P<0.05）；200 mJ·cm-2 UVB照射后24 h和对照组相比差异有显著统计学意义(P<0.01)；两种剂量的UVB照射后48 h细胞有一定程度的恢复。和对照组相比，UVB照射组ARPE-19细胞中LC3-II/LC3-I比率和Beclin-1蛋白表达水平均降低（均为P<0.05)。和UVB照射组相比，UVB+RAPA组ARPE-19细胞中明显升高了LC3-II/LC3-I比率，UVB+NH4Cl组进一步降低了LC3-II/LC3-I比率，RAPA和NH4Cl在一定程度上恢复了UVB照射降低的Beclin-1蛋白表达水平（均为P<0.05)。和对照组相比，EPO组明显增加了ARPE-19细胞中LC3-II/LC3-I比率、升高了Beclin-1蛋白表达水平（均为P<0.05)。与UVB照射组相比，UVB+EPO组ARPE-19细胞中LC3-II/LC3-I比率无明显改变(P>0.05)，而Beclin-1蛋白表达水平差异有统计学意义(P<0.05)。与对照组相比，RAPA组细胞死亡率增多，两组间差异有统计学意义（P<0.05）,NH4Cl组细胞无明显死亡，与UVB照射组相比，UVB+RAPA组和UVB+NH4Cl组细胞死亡率均无明显改变（均为P>0.05）。细胞自噬的改变不能逆转UVB诱导的ARPE-19细胞死亡。结论　UVB照射抑制了ARPE-19细胞自噬的启动及自噬流，UPS的改变不影响UVB对于自噬流的抑制。UVB照射可通过减少RPE细胞、损害细胞自噬能力增加RPE细胞变性，增加年龄相关性黄斑变性发生的风险。
Abstract:
Objective　To investigate the changes in autophagy in retinal pigment epithelial cell line ARPE-19 irradiated by ultraviolet B (UVB).Methods　The cells were collected at different time points after different doses of UVB irradiation and the effect of UVB on cell survival was detected by MTT methods. After UVB irradiation, the expression of autophagy protein LC3, the conversion ratio of LC3-I to LC3-II and the expression of Beclin-1 protein were detected by Western blot, and the regulation of UVB on autophagy was further verified by rapamicin (RAPA), an autophagy enhancer, and ammonium chloride (NH4Cl), an autophagy inhibitor. The effect of ubiqutin proteasome system (UPS) on UVB was detected by protease inhibitor epoxomicin (EPO). MTT methods were used to detect the changes of cell growth after UVB irradiation.Results　UVB induced cell dose-dependent death. Cells death occurred obviously at 24 hours after 100 mJ·cm-2 irradiation, with statistically significant compared with the control group (P<0.05). There was markedly statistical significance at 24 hours after 200 mJ·cm-2 irradiation compared with the control group (P<0.01). Cellular recovery was observed at 48 hours after 100 mJ·cm-2 irradiation and 200 mJ·cm-2 irradiation. UVB decreased the ratio of LC3-II/LC3-I and the expression level of Beclin-1 protein compared with the control group (both P<0.05). When compared with UVB group, RAPA group could reverse the ratio of LC3-II/LC3-I decreased by UVB, while NH4Cl enhanced the effect of UVB, and RAPA and NH4Cl restored the expression level of Beclin-1 protein decreased by UVB to a certain extent (all P<0.05). EPO increased the ratio of LC3-II/LC3-I and the level of Beclin-1 protein, but did not significantly change the ratio of LC3-II/LC3-I reduced by UVB（P>0.05）, and restored the inhibition of UVB on Beclin-1 to a certain extent (P<0.05). The death rate of ARPE-19 cells in RAPA group increased, which was statistically significant compared with the control group (P<0.05), but there was no significant cell death in NH4Cl group. Compared with the UVB group, there was no significant change in the mortality of the UVB+RAPA group and the UVB+NH4Cl group (both P>0.05). Changes in autophagy cannot reverse the death of ARPE-19 cells induced by UVB. Conclusion　UVB can inhibit the autophagy and autophagy flux in ARPE-19 cells. The change in UPS does not affect the autophagy flux suppressed by UVB, but it can restore the inhibitory effect of UVB on autophagy initiation to a certain extent. UVB can increase the degeneration of RPE cells by reducing RPE cells and damaging the autophagy ability of cells, which increase the risk of age-related macular degeneration.

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备注/Memo

备注/Memo:
辽宁省自然科学基金资助项目（编号：2019-ZD-0738）
更新日期/Last Update: 2021-04-05