NEUROD1 Promotes Apoptosis and Inflammation Response of Retinal Pigment Epithelial Cells and Participates in the Development of Diabetic Retinopathy

Abstract

Background: Diabetic retinopathy (DR) is a commonly encountered microvascular complication in diabetic patients. It is a serious public health issue because it threatens human vision. To delve deeper into the molecular regulation mechanisms underlying DR, the present study screened differentially expressed genes (DEGs) in DR using a bioinformatic approach, and then investigated the regulatory effects on DR of candidate genes in cultured retinal epithelial cells and DR model mice. Methods: We first searched the Gene Expression Omnibus (GEO) database using diabetic retinopathy as the keyword and selected the GSE102485 data set for analysis. In retinal pigment epithelial (RPE) cells, lentiviruses were employed to overexpress and interfere with the gene NEUROD1. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. DR mice were injected with overexpressed lentivirus and interfering lentivirus of NEUROD1. Finally, NEUROD1 was overexpressed and interfered with vascular endothelial cells (VECs) cultured in vitro. Quantitative polymerase chain reaction (qPCR) and western blot were applied to test the expressions of BCL2-associated X protein (BAX), BCL-2 homologous antagonist/killer (BAK), B-cell lymphoma 2 (BCL-2), Transforming growth factor beta (TGF-β), Tumor necrosis factor alpha (TNF-α), Interleukin 1 beta (IL-1β), and Interleukin 6 (IL-6). An immunofluorescence assay was applied to test NEUROD1 protein expression levels and cell apoptosis. Hematoxylin and eosin (HE) staining was applied to detect the condition of the retina in DR mice. An enzyme linked immunosorbent assay (ELISA) was used to detect the expressions of TNF-α, IL-1β, IL-6 and TGF-β in mouse serum. Results: The analysis revealed that the NEUROD1 gene was highly expressed in the DR group. When NEUROD1 was overexpressed, RPE cell viability was markedly reduced (p < 0.01) and apoptosis of RPE cells was increased. In contrast, after interference with NEUROD1, the viability of RPE cells was substantially increased (p < 0.05) and RPE cell apoptosis was decreased. After overexpression of NEUROD1, the expressions of BAX, BAK, TNF-α, IL-1β, and IL-6 increased markedly, whereas the expressions of the anti-apoptotic proteins BCL-2 and TGF-β decreased greatly. After overexpression of NEUROD1, the levels of blood glucose, total cholesterol, and triglyceride of mice were further increased. Interference of NEUROD1 aggravated the morphology of retinal blood vessels and the infiltration of inflammatory cells. Interference of NEUROD1 impaired the ability of VECs to adhere to the wall, causing cell aggregation and floating, thereby affecting cell viability. In the control group and the NEUROD1 overexpression group, VECs could grow and adhere to the wall and form primary blood vessels. Conclusions: The current study demonstrated that NEUROD1 inhibited the proliferation of RPE cells, promoted apoptosis and inflammation in RPE cells, promoted blood vessel formation, and exacerbated the development of DR. This research may establish a foundation for diagnosing and treating DR in a clinical setting.