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NADPH氧化酶4抑制剂对贝伐单抗诱导人RPE细胞上皮-间质转化的抑制作用
谢超慧
郝相慧
杨玲玲
徐海峰
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20210407-00234
Inhibitory effects of NADPH oxidase 4 inhibitor on the epithelial-mesenchymal transition of human RPE cells induced by bevacizumab
Xie Chaohui
Hao Xianghui
Yang Lingling
Xu Haifeng
Authors Info & Affiliations
Xie Chaohui
Medical College, Qingdao University, Qingdao 266071, China
Hao Xianghui
Eye Institute of Shandong First Medical University, Qingdao Eye Hospital of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
Yang Lingling
Eye Institute of Shandong First Medical University, Qingdao Eye Hospital of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
Xu Haifeng
Eye Institute of Shandong First Medical University, Qingdao Eye Hospital of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, China
·
DOI： 10.3760/cma.j.cn115989-20210407-00234
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摘要

目的观察还原型烟酰胺腺嘌呤二核苷酸氧化酶4(NOX4)抑制剂对贝伐单抗诱导人视网膜色素上皮(RPE)细胞发生上皮-间质转化(EMT)的影响。

方法将人RPE细胞系ARPE-19细胞分为空白对照组、贝伐单抗组、贝伐单抗+VAS2870组和贝伐单抗+GKT137831组，其中空白对照组不做任何处理，贝伐单抗组、贝伐单抗+VAS2870组和贝伐单抗+GKT137831组培养基中分别加入0.25 g/L贝伐单抗、0.25 g/L贝伐单抗+3 μmol/L VAS2870、0.25 g/L贝伐单抗+20 μmol/L GKT137831培养72 h，VAS2870和GKT137831均为NOX4抑制剂。采用实时荧光定量PCR和Western blot法测定NOX4和EMT标志物纤维连接蛋白(FN)、波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)及紧密连接相关蛋白闭锁小带蛋白-1(ZO-1)mRNA和蛋白表达水平；采用细胞免疫荧光染色法验证NOX4和EMT标志物蛋白在各组细胞中的表达情况。

结果实时荧光定量PCR和Western blot检测结果显示，空白对照组、贝伐单抗组、贝伐单抗+VAS2870组和贝伐单抗+GKT137831组细胞中FN、Vimentin、α-SMA、ZO-1和NOX4 mRNA和蛋白相对表达量总体比较差异均有统计学意义(mRNA： F＝97.07、195.40、722.40、38.56、70.81，均 P<0.001；蛋白： F＝23.09、64.58、58.19、26.97、63.19，均 P<0.001)，其中贝伐单抗组FN、Vimentin、α-SMA和NOX4 mRNA和蛋白相对表达量明显高于空白对照组，ZO-1 mRNA和蛋白相对表达量明显低于空白对照组，差异均有统计学意义(均 P<0.05)。贝伐单抗+VAS2870组和贝伐单抗+GKT137831组FN、Vimentin、α-SMA和NOX4 mRNA和蛋白相对表达量明显低于贝伐单抗组，ZO-1 mRNA和蛋白相对表达量明显高于贝伐单抗组，差异均有统计学意义(均 P<0.05)。免疫荧光染色结果显示，贝伐单抗组FN、Vimentin、α-SMA荧光强度强于空白对照组，ZO-1荧光强度弱于空白对照组；贝伐单抗+VAS2870组和贝伐单抗+GKT137831组FN、Vimentin、α-SMA荧光强度弱于贝伐单抗组，ZO-1荧光强度强于贝伐单抗组。

结论NOX4参与了贝伐单抗诱导RPE细胞EMT的发生，NOX4抑制剂可减轻贝伐单抗诱导人RPE细胞的EMT程度。

贝伐单抗;视网膜色素上皮细胞;上皮-间质转化;还原型烟酰胺腺嘌呤二核苷酸氧化酶-4;抑制剂
ABSTRACT

ObjectiveTo observe the influence of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4 inhibitors on epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (RPE) cells induced by bevacizumab.

MethodsThe cultured ARPE-19 cells were divided into blank control group, bevacizumab group, bevacizumab+ VAS2870 group and bevacizumab+ GKT137831 group.Cells were cultured with 0.25 g/L bevacizumab, 0.25 g/L bevacizumab plus 3 μmol/L VAS2870 (a NOX4 inhibitor), 0.25 g/L bevaczumab plus 20 μmol/L GKT137831 (a NOX4 inhibitor) for 72 hours according to grouping.No intervention was administered to the blank control group.The mRNA and protein expression levels of NOX4 and EMT markers including fibronectin (FN), vimentin, α-smooth muscle actin (α-SMA) and tight junction related protein zonula occludens-1 (ZO-1) were measured by real-time PCR and Western blot assay, and the expression levels in different intervention groups were compared.The expressions of NOX4 and EMT markers were verified by immunofluorescence staining.

ResultsThere were statistically significant differences in the relative mRNA and protein expression levels of FN, vimentin, α-SMA, ZO-1 and NOX4 among blank control group, bevacizumab group, bevacizumab+ VAS2870 group and bevacizumab+ GKT137831 group (mRNA: F=97.07, 195.40, 722.40, 38.56, 70.81; all at P<0.001.Protein: F=23.09, 64.58, 58.19, 26.97, 63.19; all at P<0.001). The relative mRNA and protein expression levels of FN, vimentin, α-SMA and NOX4 were significantly higher and the relative mRNA and protein expression level of ZO-1 was significantly lower in bevacizumab group than those in blank control group (all at P<0.05). The relative mRNA and protein expression levels of FN, vimentin, α-SMA and NOX4 were significantly lower and the relative mRNA and protein expression levels of ZO-1 were significantly higher in bevacizumab+ VAS2870 and bevacizumab+ GKT137831 groups than those in bevacizumab group (all at P<0.05). The immunofluorescence intensity of FN, vimentin and α-SMA was stronger and the immunofluorescence intensity of ZO-1 was weaker in bevacizumab group than blank control group.The immunofluorescence intensity of FN, vimentin and α-SMA were weaker and the immunofluorescence intensity of ZO-1 was stronger in bevacizumab+ VAS2870 group and bevacizumab+ GKT137831 group than those in bevacizumab group.

ConclusionsNOX4 is involved in the bevacizumab-induced EMT of human RPE cells, the degree of which can be reduced by NOX4 inhibitors.

Bevacizumab;Retinal pigment epithelial cells;Epithelial-mesenchymal transition;NADPH oxidase 4;Inhibitor
Xu Haifeng, Email: mocdef.6ab21fhxhc
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引用本文

谢超慧,郝相慧,杨玲玲,等. NADPH氧化酶4抑制剂对贝伐单抗诱导人RPE细胞上皮-间质转化的抑制作用[J]. 中华实验眼科杂志,2022,40(06)：507-513.

DOI:10.3760/cma.j.cn115989-20210407-00234

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目前，眼底新生血管性疾病的一线治疗方法是玻璃体腔内注射抗血管内皮生长因子(vascular endothelial growth factor，VEGF)药物 [ 1 ]。然而，随着抗VEGF制剂临床应用的日益广泛及随访时间的延长，发现抗VEGF治疗后无论是脉络膜还是视网膜新生血管均可发生严重纤维化，尤其是视网膜新生血管纤维化，短期内即可造成牵拉性视网膜脱离 [ 2 ]，脉络膜新生血管纤维化也严重影响视功能的恢复 [ 3 ]。因此，在抗VEGF治疗的同时如何减轻新生血管纤维化引起了广泛关注 [ 4 ]。本课题组前期研究证实，贝伐单抗对纤维化发生过程有调控作用，抗VEGF处理后纤维化及炎性因子的表达水平发生变化 [ 5 , 6 ]。有研究表明，抗VEGF处理后，RPE细胞中活性氧簇(reactive oxygen species，ROS)水平也明显升高，抗氧化功能降低，ROS水平升高与细胞纤维化发生直接相关 [ 7 , 8 ]。ROS在视网膜色素上皮(retinal pigment epithelium，RPE)细胞的上皮-间质转化(epithelial-mesenchymal transition，EMT)中起到促进作用 [ 9 ]。机体细胞中产生ROS的一个主要来源是还原型烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide phosphate，NADPH)氧化酶介导的氧化应激反应 [ 10 , 11 ]。NADPH氧化酶(NADPH oxidase，NOX)表达于血管内皮和血管平滑肌等多种细胞内，是血管系统ROS的主要来源。本课题组前期研究发现NOX4的过度表达使ROS水平升高，而抑制NOX4活性仅降低了ROS产生的基础水平，这表明NOX4并非刺激内皮细胞产生ROS的主要酶 [ 12 ]。已有研究发现，缺氧可引起细胞中NOX高表达，也可诱导肾小管上皮细胞纤维化 [ 13 ]。抑制NOX表达可抑制血管内皮细胞增生 [ 14 , 15 ]。这些发现提示NOX4的表达在细胞纤维化中发挥重要作用。EMT的特征之一在于诱导上皮基因的丢失以及多种细胞外基质蛋白的表达 [ 16 , 17 ]。闭锁小带蛋白-1(zonula occludens-1，ZO-1)是细胞紧密连接重要组成蛋白之一，其穿梭于质膜和细胞核或细胞质之间，参与信号传导途径。EMT发生期间，ZO-1分子受到破坏，其参与细胞骨架构成和维持细胞极性的特性减弱，细胞发生形态学变化。纤维连接蛋白(fibronectin，FN)作为一种高分子量糖蛋白，主要以3种形式存在，其中1种是由成纤维细胞和早期间充质细胞分泌合成，并在EMT过程中生成量增加 [ 18 ]。波形蛋白(Vimentin)是中间丝的其中1种蛋白质，其与微管及肌动蛋白微细丝组成细胞骨架，可锚定和支撑间充质细胞胞质内的细胞器，可作为EMT的标志物。与此机制相似的因子还有α-平滑肌肌动蛋白(α-smooth muscle actin，α-SMA)。NOX在促进视网膜血管纤维化中有潜在作用 [ 18 ]，已有研究证实NOX可调节VEGF的表达 [ 19 ]，然而至今尚无NOX调控RPE细胞发生EMT的直接证据。本研究拟探讨NOX4抑制剂对抗VEGF治疗导致的人RPE细胞发生EMT的影响。
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备注信息
A
徐海峰，Email： mocdef.6ab21fhxhc
B

徐海峰：参与选题和研究设计、研究过程指导、数据和资料分析、文章主要内容修改和定稿；谢超慧：参与研究设计和实验实施、收集和分析数据、论文撰写及修改和定稿；郝相慧：参与研究实施、收集和分析数据；杨玲玲：实验指导，对文章知识性内容的审阅和智力性内容的修改

C
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国家科技重大专项基金项目 (2017ZX09304010)
国家自然科学基金项目 (81670828)
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