Inhibitory effects of NADPH oxidase 4 inhibitor on the epithelial-mesenchymal transition of human RPE cells induced by bevacizumab

Auhtors: Xie Chaohui,  Hao Xianghui,  Yang Lingling,  Xu Haifeng
DOI: 10.3760/cma.j.cn115989-20210407-00234
Published 2022-06-10
Cite asChin J Exp Ophthalmol, 2022, 40(6): 507-513.

Abstract

Objective

To 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.

Methods

The 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.

Results

There 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.

Conclusions

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

Key words:

Bevacizumab; Retinal pigment epithelial cells; Epithelial-mesenchymal transition; NADPH oxidase 4; Inhibitor

Contributor Information

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

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