缺血性视网膜损伤致细胞凋亡的内质网应激机制研究

doi:10.3969/j.issn.1673-5765.2024.06.008

摘要/Abstract

摘要： 目的分析缺血对视网膜神经节细胞（retinal ganglion cells，RGCs）凋亡过程中内质网应激（endoplasmic reticulum stress，ERS）相关蛋白葡萄糖调节蛋白78（glucose regulated protein78，GRP78）、增强子结合蛋白同源蛋白（C/BEBP homologous protein，CHOP）、半胱氨酸天冬氨酸蛋白酶（cysteinyl aspartate specific proteinase，Caspase）-12表达的影响，探究缺血性视网膜损伤（ischemic retinal injury，IRI）致RGCs凋亡的分子水平发病机制，为预防和治疗IRI提供新的思路和方法。
方法应用前房高眼压法制作大鼠右眼IRI模型。健康雄性SD大鼠40只，按照缺血时间的不同分为正常对照组、IRI 30 min组、IRI 60 min组以及IRI 120 min组,每组各10只。应用苏木精-伊红（hematoxylin-eosin，HE）染色法观察不同分组中大鼠视网膜组织形态学变化；应用免疫组织化学（immunohistochemical，IHC）染色法观察不同分组中细胞凋亡相关因子B细胞原癌基因-2（B-cell lymphocyte/leukemia-2，Bcl-2）、Bcl-2关联X蛋白（Bcl-2 associated X protein，Bax）和Caspase-3的表达及定位情况；应用免疫荧光（immunofluorescence，IF）染色法观察不同分组中ERS相关蛋白GRP78、CHOP和Caspase-12的表达及定位情况。
结果 HE染色结果显示，与正常对照组相比，IRI各组可见视网膜水肿，视网膜内层厚度增加，视网膜细胞分布紊乱，RGCs数量减少，空泡组织增多。IHC染色结果显示，正常对照组视网膜中仅有少量Bax、Caspase-3蛋白表达，IRI各组中Bax、Caspase-3蛋白主要表达于神经节细胞层和内核层，与正常对照组相比，IRI各组视网膜中Bax、Caspase-3蛋白表达量明显增加，且IRI各组间差异也具有统计学意义。正常对照组视网膜神经节细胞层和内核层中可见大量Bcl-2蛋白表达，与正常对照组相比，IRI各组视网膜中Bcl-2蛋白表达量明显减少，且IRI各组间差异具有统计学意义。IF染色结果显示，正常对照组视网膜中GRP78、CHOP、Caspase-12阳性表达较少，与正常对照组相比，IRI各组视网膜神经节细胞层和内核层中GRP78、CHOP、Caspase-12阳性表达均显著增多，且差异有统计学意义。
结论 IRI可诱导视网膜发生细胞凋亡，其机制可能与ERS相关通路激活有关。

文章导读： 本文通过大鼠模型，探索了IRI后视网膜组织中与凋亡相关的介质的表达情况，对明确IRI后细胞凋亡的分子机制，发现临床诊疗可能的干预靶点具有一定的意义。

关键词: 缺血性损伤; 视网膜神经节细胞; 细胞凋亡; 内质网应激

Abstract: Objective To analyze the effects of ischemia on the expression of endoplasmic reticulum stress (ERS)-related proteins glucose regulated protein78 (GRP78), C/BEBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase (Caspase)-12 during the apoptotic process of retinal ganglion cells (RGCs), and to investigate the molecular level pathogenesis of ischemic retinal injury (IRI)-induced RGCs apoptosis, to provide a new idea and method for the prevention and treatment of IRI.
Methods The anterior chamber high intraocular pressure method was applied to produce a rat right eye IRI model. Forty healthy male SD rats were randomly divided into the normal control group, 30-min IRI group, 60-min IRI group, and 120-min IRI group according to the ischemic time, with 10 rats in each group. Hematoxylin-eosin (HE) staining was applied to observe the changes in retinal histomorphology in different groups of rats; immunohistochemical (IHC) staining was applied to observe the expression and localization of apoptosis-related factors such as B-cell lymphocyte/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), and Caspase-3 in different groups; immunofluorescence (IF) staining was applied to observe the expression and localization of ERS-related proteins such as GRP78, CHOP and Caspase-12 in other groups.
Results HE staining results showed that compared with the normal control group, retinal edema, increased thickness of the inner retinal layer, disturbed distribution of retinal cells, decreased number of RGCs, and increased vacuolar tissues were observed in the IRI groups. IHC staining results showed that only a small amount of Bax and Caspase-3 proteins were expressed in the retina of the normal control group, while Bax and Caspase-3 proteins were mainly expressed in the ganglion cell layer and the inner nuclear layer of the retina in the IRI groups. The expression of Bax and Caspase-3 proteins in the retina of the IRI groups was significantly higher than that of the normal control group, and the differences between the IRI groups were also statistically significant. A large amount of Bcl-2 protein was observed in the ganglion cell layer and inner nuclear layer of the retina in the normal control group. Compared to the normal control group, the expression of Bcl-2 protein in the retina of each IRI group was significantly reduced, and the differences between the IRI groups were also statistically significant. The results of IF staining showed that the positive expressions of GRP78, CHOP, and Caspase-12 in the retina of the normal control group were small, while their expressions in the ganglion cell layer and the inner nuclear layer in the retina of the IRI groups were significantly increased compared with the normal control group.
Conclusions IRI can induce apoptosis in the retinal cells, and its mechanism may be related to the activation of ERS-related pathways.

Key words: Ischemic injury; Retinal ganglion cell; Apoptosis; Endoplasmic reticulum stress

中图分类号:

于文瑄, 梁冰, 曹永亮. 缺血性视网膜损伤致细胞凋亡的内质网应激机制研究[J]. 中国卒中杂志, 2024, 19(6): 664-671.

YU Wenxuan, LIANG Bing, CAO Yongliang. Research on the Endoplasmic Reticulum Stress Mechanism of Apoptosis Induced by Ischemic Retinal Injury[J]. Chinese Journal of Stroke, 2024, 19(6): 664-671.

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