杨延振; 庄宪丽; 李树杰; 张莹; 杨家干; 李路路

doi:10.3760/cma.j.cn115989-20210930-00543

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• 实验研究 •

雷珠单抗对脉络膜新生血管模型大鼠视网膜氧化应激的抑制作用及其机制

中华实验眼科杂志, 2023,41(1) : 22-28. DOI: 10.3760/cma.j.cn115989-20210930-00543

摘要

目的

研究雷珠单抗对脉络膜新生血管(CNV)模型大鼠视网膜氧化应激的作用及其机制。

方法

选取10周龄SPF级雄性SD大鼠60只，按照随机数字表法随机分为正常对照组、模型对照组、雷珠单抗组、转录因子核因子E2相关因子2(Nrf2)抑制剂(ML385)组、雷珠单抗+ML385组，每组12只。除正常对照组外，其余各组以氪激光诱导CNV模型。雷珠单抗组、ML385组和雷珠单抗+ML385组分别玻璃体腔内注射1 μl雷珠单抗、ML385、雷珠单抗+ML385，模型对照组、正常对照组分别给予玻璃体腔内注射等体积生理盐水。脉络膜铺片法检测CNV面积；苏木精－伊红染色观察视网膜病理学变化；实时荧光定量PCR及Western blot检测视网膜组织中Nrf2、超氧化物歧化酶(SOD)、醌氧化还原酶(NQO1)mRNA及蛋白表达水平。

结果

模型对照组、ML385组和雷珠单抗+ML385组CNV面积分别为(23.01±1.52)×10³、(30.23±2.01)×10³和(18.56±1.85)×10³ μm²，明显高于雷珠单抗组的(12.35±1.22)×10³ μm²，雷珠单抗+ML385组CNV面积小于模型对照组和ML385组，ML385组CNV面积大于模型对照组，差异均有统计学意义(均P<0.001)。苏木精－伊红染色结果显示，雷珠单抗组RPE－脉络膜－巩膜复合体结构损害程度较模型对照组轻；雷珠单抗+ML385组RPE－脉络膜－巩膜复合体结构损害程度较雷珠单抗组重，但较模型对照组轻；ML385组RPE－脉络膜－巩膜复合体结构损害较模型对照组严重。雷珠单抗组Nrf2、SOD和NQO1 mRNA及蛋白相对表达量均低于正常对照组，但高于模型对照组、ML385组和雷珠单抗+ML385组，且雷珠单抗+ML385组Nrf2、SOD和NQO1 mRNA及蛋白相对表达量高于模型对照组和ML385组，差异均有统计学意义(均P<0.05)。

结论

雷珠单抗可抑制氪激光诱导的CNV生长，减轻RPE氧化应激损伤，其机制与Nrf2/ARE通路的激活有关。

引用本文: 杨延振, 庄宪丽, 李树杰, 等. 雷珠单抗对脉络膜新生血管模型大鼠视网膜氧化应激的抑制作用及其机制 [J] . 中华实验眼科杂志, 2023, 41(1) : 22-28. DOI: 10.3760/cma.j.cn115989-20210930-00543.

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除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。

年龄相关性黄斑变性(age-related macular degeneration，AMD)是一种多发于老年人群的进行性视力损害性疾病，根据其病理表现不同可分为湿性AMD和干性AMD，其中湿性AMD预后较差，是眼科防治的研究重点^[1,2]。近年来研究发现，湿性AMD的病变可能与脉络膜新生血管(choroidal neovascularization，CNV)及视网膜色素上皮(retinal pigment epithelium，RPE)氧化应激反应有关^[3,4]。雷珠单抗为抗血管内皮生长因子药物，可有效抑制血管生成，已被美国食品药物管理局批准用于湿性AMD的治疗^[5]。近年来，De Cillà等^[6]研究发现，阿柏西普/雷珠单抗以剂量和时间依赖性方式增加RPE细胞一氧化氮的释放，并可通过刺激人脐血管内皮细胞释放旁分泌物，在生理和过氧化调节下使RPE细胞免受过氧化损伤，因此推测雷珠单抗对湿性AMD的治疗作用可能也与抗氧化应激有关。转录因子NF-E2相关因子2(nuclear factor erythroid 2-related factor 2，Nrf2)/抗氧化反应元件(antioxidant responsive element，ARE)通路在细胞抗氧化应激中发挥重要的作用，且被证实参与湿性AMD的发生^[7,8]。目前雷珠单抗的抗氧化应激机制尚待明确，关于雷珠单抗是否可通过Nrf2/ARE通路影响湿性AMD的病理变化仍需进一步探索。本研究拟通过氪激光诱导CNV模型，以雷珠单抗及Nrf2抑制剂ML385进行干预，探讨雷珠单抗对湿性AMD大鼠视网膜氧化应激的作用及其机制。

贡献者信息

杨延振

滕州市中心人民医院眼科，滕州　277500

庄宪丽

滕州市中心人民医院眼科，滕州　277500

李树杰

滕州市中心人民医院眼科，滕州　277500

张莹

滕州市中心人民医院眼科，滕州　277500

杨家干

滕州市中心人民医院眼科，滕州　277500

李路路

滕州市中心人民医院眼科，滕州　277500

通信作者

李路路

滕州市中心人民医院眼科，滕州　277500

Email：liangzhuomengy@163.com

关键词

雷珠单抗; 黄斑变性; 视网膜; 氧化应激;

基金项目

山东省医药卫生科技发展计划项目（2018WSA05655）

作者声明

作者贡献声明

杨延振：酝酿和设计实验、实施研究、采集数据及分析数据；庄宪丽：实施研究、采集和分析数据；李树杰、张莹：起草并审阅文章；杨家干、李路路：统计分析、审阅文章并最终定稿

利益冲突

所有作者均声明不存在利益冲突

历史

出版日期：2023-01-10

收稿日期：2022-07-05

修回日期：2022-12-08

本文编辑

张宇

Experimental Science

Inhibitory effect of ranibizumab on retinal oxidative stress in a rat model of choroidal neovascularization and its mechanism

Yang Yanzhen, Zhuang Xianli, Li Shujie, Zhang Ying, Yang Jiagan, Li Lulu

Published 2023-01-10

Cite as Chin J Exp Ophthalmol, 2023, 41(1): 22-28. DOI: 10.3760/cma.j.cn115989-20210930-00543

Abstract

Objective

To study the effect of ranibizumab on retinal oxidative stress in a rat model of choroidal neovascularization (CNV) and its mechanism.

Methods

Sixty SPF male SD rats aged 10 weeks were randomly divided into normal control group, model control group, ranibizumab group, nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor (ML385) group, ranibizumab+ ML385 group, with 12 rats in each group according to a random number table.Except for the normal control group, the CNV model was established in the other four groups via krypton laser induction.According to grouping, the ranibizumab group, ML385 group and ranibizumab+ ML385 group were intravitreally injected with 1 μl of ranibizumab, ML385 and ranibizumab+ ML385, respectively.Model control group and normal control group received an intravitreal injection of normal saline of equal volume.The CNV area was measured through choroidal wholemounts.Pathological change of the retina was observed by hematoxylin and eosin staining.Expressions of Nrf2, superoxide dismutase (SOD) and quinone oxidoreductase 1 (NQO1) were detected using Western blot and real-time PCR.The use and care of animals complied with laboratory animal welfare guidelines.The study protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Tengzhou Central People's Hospital (No.JN.No20210214S1200430[121]).

Results

CNV areas of the model control group, ML385 group and ranibizumab+ ML385 group were (23.01±1.52)×10³, (30.23±2.01)×10³ and (18.56±1.85)×10³ μm², respectively, which were significantly higher than (12.35±1.22)×10³ μm² of ranibizumab group (all at P<0.001). The CNV area of ranibizumab+ ML385 group was smaller than that of model control group and ML385 group, and the CNV area of ML385 group was larger than that of model control group, showing statistically significant differences (all at P<0.001). Hematoxylin and eosin staining showed that the structural damage of the retinal pigment epithelium-choroid-sclera complex was slighter in ranibizumab group than model control group, severer in ranibizumab+ ML385 group than ranibizumab group but slighter than model control group, severer in ML385 group than model control group.The mRNA and protein expressions of Nrf2, SOD and NQO1 of ranibizumab group were lower than those of normal control group but higher than those of model control group, ML385 group and ranibizumab+ ML385 group, and the differences were statistically significant (all at P<0.05). The mRNA and protein expressions of Nrf2, SOD and NQO1 were higher in ranibizumab+ ML385 group than model control group and ML385 group, showing statistically significant differences (all at P<0.05).

Conclusions

Ranibizumab can inhibit the growth of CNV induced by krypton laser and reduce RPE damage caused by retinal oxidative stress.The mechanism is related to the activation of Nrf2/ARE pathway.

Key words:

Ranibizumab; Macular degeneration; Retina; Oxidative stress

Contributor Information

Yang Yanzhen