[1]李湘波,刘艳婷,张永红,等.Bax抑制因子1对紫外线B诱导下人晶状体上皮细胞凋亡的抑制作用[J].眼科新进展,2021,41(6):528-533.[doi:10.13389/j.cnki.rao.2021.0110]
　LI Xiangbo,LIU Yanting,ZHANG Yonghong,et al.Inhibitory effect of Bax inhibitor 1 on ultraviolet B-induced apoptosis of human lens epithelial cells[J].Recent Advances in Ophthalmology,2021,41(6):528-533.[doi:10.13389/j.cnki.rao.2021.0110]
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Bax抑制因子1对紫外线B诱导下人晶状体上皮细胞凋亡的抑制作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年6期
页码:
528-533
栏目:
实验研究
出版日期:
2021-06-05

文章信息/Info

Title:
Inhibitory effect of Bax inhibitor 1 on ultraviolet B-induced apoptosis of human lens epithelial cells
作者:
李湘波; 刘艳婷; 张永红; 蒋殊; 周克兵
421002　湖南省衡阳市，南华大学附属南华医院眼科
Author(s):
LI Xiangbo; LIU Yanting; ZHANG Yonghong; JIANG Shu; ZHOU Kebing
Department of Ophthalmology,Nanhua Hospital Affiliated to University of South China,Hengyang 421002,Hunan Province,China
关键词:
Bax抑制因子1; 紫外线B; 白内障; 内质网应激
Keywords:
Bax inhibitor 1;  ultraviolet B;  cataract;  endoplasmic reticulum stress
分类号:
R776
DOI:
10.13389/j.cnki.rao.2021.0110
文献标志码:
A
摘要:
目的　探讨Bax抑制因子1（BI-1）对紫外线B（UVB）诱导下人晶状体上皮细胞凋亡的抑制作用。方法　取对数生长期的人晶状体上皮细胞（HLEC）系SRA01/04细胞，将BI-1过表达质粒及其空载质粒转染至SRA01/04细胞中，分别记为BI-1组、Vector组，另取不做任何处理的SRA01/04细胞作为blank组。转染48 h后，采用实时荧光定量PCR和Western blot检测转染后各组细胞中BI-1 mRNA和蛋白表达水平。而后再根据实验需要将细胞分为6组：（1）Control组，SRA01/04细胞不经任何处理；（2）UVB组，采用紫外线（2.0 W·cm-2）处理SRA01/04细胞60 min；（3）Vector组，转染BI-1空载质粒的SRA01/04细胞；（4）BI-1组，转染BI-1过表达质粒的SRA01/04细胞；（5）Vector+UVB组，转染BI-1空载质粒的SRA01/04细胞，再经紫外线（2.0 W·cm-2）照射60 min；（6）BI-1+UVB组，转染BI-1过表达质粒的SRA01/04细胞，再经紫外线（2.0 W·cm-2）照射60 min。采用实时荧光定量PCR检测UVB照射后各组细胞中BI-1 mRNA表达水平，CCK-8法检测细胞增殖情况，流式细胞仪检测各组细胞中活性氧（ROS）含量，荧光探针法检测细胞内质网内Ca2+浓度，流式细胞仪检测细胞凋亡率，Western blot检测各组细胞中相关蛋白表达水平。结果　本研究中所培养的SRA01/04细胞在荧光显微镜下可观察到细胞内αA-晶状体蛋白呈红色荧光。随着UVB照射强度增加，SRA01/04细胞内BI-1 mRNA表达水平逐渐降低，本研究选择UVB照射强度为2.0 W·cm-2进行后续的实验。与blank组或Vector组相比，BI-1组细胞中BI-1 mRNA和蛋白表达水平均显著上升，差异均有统计学意义（均为P＜0.05）。与Control组比较，UVB组细胞增殖活性显著降低，细胞内ROS含量显著增加，细胞内质网内Ca2+浓度显著升高，细胞凋亡率显著升高，细胞内Cleaved-caspase-3、Bax蛋白表达水平显著上调，Bcl-2蛋白表达水平显著下调，细胞内GRP78、p-IRE1、p-JNK和Cleaved-caspase-12蛋白表达水平显著上调（均为P＜0.05）。BI-1+UVB组细胞增殖活性较UVB组显著增高，细胞内ROS含量较UVB组显著降低，细胞内质网内Ca2+浓度较UVB组显著下降；与UVB组比较，BI-1+UVB组细胞凋亡率显著下降，细胞内Cleaved-caspase-3、Bax蛋白表达水平显著下调，Bcl-2蛋白表达水平显著上调；BI-1+UVB组细胞内GRP78、p-IRE1、p-JNK和Cleaved-caspase-12蛋白表达水平较UVB组显著下调（均为P＜0.05）。结论　BI-1在UVB诱导的HLEC系SRA01/04细胞中表达显著下调，BI-1过表达可通过降低内质网内Ca2+浓度抑制内质网应激介导的IRE1-JNK信号通路的激活，进而抑制UVB诱导的SRA01/04细胞凋亡。
Abstract:
Objective　To investigate the inhibitory effect of Bax inhibitor 1 (BI-1) on the apoptosis of human lens epithelial cells induced by ultraviolet B (UVB).Methods　Human lens epithelial cells (HLECs) line SRA01/04 selected from logarithmic phase were transfected with BI-1 over expressed plasmid and its empty plasmid, which were respectively classified as BI-1 group and Vector group. SRA01/04 cells without any treatment were selected as the blank group. After transfection for 48 hours, the mRNA and protein expression levels of BI-1 in transfected cells of each group were detected by qRT-PCR and Western blot. The cells were then divided into six groups according to experimental needs: (1) Control group, SRA01/04 cells without treatment in any way; (2) UVB group, in which SRA01/04 cells were treated with ultraviolet radiation (2.0 W·cm-2) for 60 minutes; (3) Vector group, in which SRA01/04 cells were transfected with BI-1 empty plasmid; (4) BI-1 group, in which SRA01/04 cells were transfected with BI-1 overexpressing plasmid; (5) Vector+UVB group, in which SRA01/04 cells were transfected with BI-1empty plasmid and irradiated with ultraviolet light (2.0 W·cm-2) for 60 minutes; (6) BI-1+UVB group, in which SRA01/04 cells were transfected with BI-1overexpressing plasmid and irradiated with ultraviolet light (2.0 W·cm-2) for 60 minutes. The expression level of BI-1 mRNA in the cells irradiated with ultraviolet light was detected by qRT-PCR, the cell proliferation was detected by CCK-8, the content of reactive oxygen species (ROS) was detected by flow cytometry, the concentration of Ca2+ in endoplasmic reticulum was detected by fluorescence probe method, and the apoptosis rate was detected by flow cytometry. The expression levels of related proteins in cells were detected by Western blot.Results　The cultured SRA01/04 cells in this study showed red fluorescence of αA-lens protein under fluorescence microscope. With the increase of UVB irradiation intensity, the expression level of BI-1 mRNA in SRA01/04 cells gradually decreased. UVB irradiation intensity of 2.0 W·cm-2 was selected for subsequent experiments in this study. Compared with blank group or Vector group, the mRNA and protein expression levels of BI-1 in BI-1 group were significantly increased, and the differences were statistical significance (all P<0.05). Compared with Control group, the cell proliferation activity of UVB group was significantly decreased, the intracellular content of ROS, Ca2+ concentration, the cell apoptosis rate were significantly increased, the protein expression levels of Cleaved caspase-3 and Bax were significantly up-regulated, and Bcl-2 protein expression level was significantly down-regulated, and the protein expression levels of GRP78, p-IRE1, p-JNK and Cleaved caspase-12 were significantly up-regulated (all P<0.05). Compared with the UVB group, the proliferation activity of cells in the BI-1+UVB group was significantly enhanced, the intracellular ROS content and the intracellular Ca2+ concentration were significantly decreased. Compared with the UVB group, the apoptosis rate in BI-1+UVB group was significantly decreased, the protein expression levels of Cleaved caspase-3 and Bax were significantly down-regulated, and the protein expression level of Bcl-2 was significantly up-regulated, while the protein expression levels of GRP78, p-IRE1, p-JNK and Cleaved caspase-12 were significantly down-regulated (all P<0.05).Conclusion　The expression of BI-1 was significantly down-regulated in UVB induced SRA01/04 cells. Overexpression of BI-1 inhibited the activation of IEE1-JNK signaling pathway mediated by endoplasmic reticulum stress by reducing the concentration of Ca2+ in endoplasmic reticulum, thereby inhibiting the apoptosis of UVB induced SRA01/04 cells.

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备注/Memo

备注/Memo:
湖南省临床医疗技术创新引导计划（编号：2017SK50220）
更新日期/Last Update: 2021-06-05