Diabetic retinopathy (DR) is one of the most common and harmful microvascular complications of diabetes, and it is also a common eye disease that causes blindness (1). Most patients progress into DR after 20 years of diabetes (2, 3), and about half of the patients with untreated proliferative retinopathy will go blind within 5 years (4). It imposes a heavy economic burden on the families, health systems and societies. Previous studies have shown that oxidative stress, endoplasmic reticulum stress, apoptosis and autophagy (5–8) can induce mild and chronic retinal inflammation in retinal tissues (9), resulting in retinal vessel hyperpermeability (10), retinal angiogenesis and retinal neuron injury (11).

Autophagy is a process in which autophagy engulfs its own cytoplasmic proteins or organelles and encapsulates them into vesicles, fuses with lysosomes to form autophagy lysosomes, and degrades the contents of autophagy. It meets the demand of cellular metabolic needs and is involved in the renewal of some organelles. It is highly conserved in evolution and crucial for the degradation and circulation of cellular substances (12). Autophagy disorders may have fatal consequences to the cells and result in some ocular diseases (13), such as age-associated macular degeneration (AMD), glaucoma and other eye diseases (14–21). For example, glucosamine (GlcN) can induce autophagy to reduce photoreceptor outer segment (POS)-derived lipofuscin-like autofluorescence (LLAF) in retinal pigment epithelial (RPE) cells through the AMPK-mTOR pathway, which provides a novel insight for AMD (19). In the glaucoma model, the neurosteroid allopregnanolone (AlloP) can reduce the apoptosis of retinal ganglion cells (RGC) by activating autophagy (21).

Recently, the role of autophagy in DR has been gradually uncovered. Damage of outer blood-retina barrier due to diabetes is key to the pathogenesis of diabetic macular edema. It leads to decrease of visual acuity in patients with DR. RPE cells are the main components of the outer blood-retina barrier. It has been demonstrated that the outer blood-retina barrier was destroyed in diabetes by modulation of autophagy in RPE cells. However, the underlying mechanism of autophagy in devastating RPE cells under diabetes stress is still unclear. In this study, we analyzed the previously published dataset containing samples from DR and non-diabetic individuals to identify the differentially expressed genes (DEGs) related to DR. It was further analyzed to figure out the correlation between the differentially expressed autophagy-related genes in DR. Then, functional enrichment and protein-protein interaction (PPI) network analysis were used to clarify the interaction and biological function of these genes. 10 hub genes were identified by PPI network analysis, and it was further verified in the DR model by quantitative real-time polymerase chain reaction (qRT-PCR) in vitro. We found that the expression of MAPK3 was consistent with the results of bioinformatics analysis of mRNA chip in the DR model, suggesting it was involved in the development of DR by regulating autophagy ( Figure 1 ).

DR is a chronic progressive complication of patients with diabetes, which is an important cause of blindness in patients with diabetes (1, 25, 26). The pathogenesis of DR is complex. Current studies have shown that a variety of metabolic pathways are involved in the formation of DR, such as the increase of polyol pathway, the activation of protein kinase C, oxidative stress and endoplasmic reticulum stress (5, 6). The abnormality of these pathways can not only cause microvascular complications such as the destruction of blood-retinal barrier (27, 28), but also lead to neurodegeneration and neuroinflammation (29). However, the exact pathogenic mechanism of DR has not been fully elucidated. Accumulating evidence show that autophagy, as the main catabolic pathway for the degradation and recycling of damaged proteins or organelles, may be involved in the pathogenesis of DR. Long-term hyperglycemia can cause autophagy disorder by inhibiting mTOR, resulting in the loss of retinal ganglion cells (30). In addition, low glucose (15mM) could induce mitochondrial autophagy in RPE cells, while under the stimulation of HG (50mM) or hydrogen peroxide, ROS could mediate the inactivation of mitochondrial autophagy-related proteins PINK1 and Parkin, and inhibited the occurrence of mitochondrial autophagy, indicating that glucose affected the occurrence of mitochondrial autophagy in RPE cells in a dose-dependent manner (31). Other studies have shown that knockout of high mobility group box1 (HMGB1) gene in RPE cells in the early stage of DR could save lysosome membrane permeabilization (LMP) through cathepsin B (CTSB)-dependent pathway. It restored the degradation ability of autophagy and thus protected RPE cells from apoptosis (32). The above results show that many forms of autophagy participate in the occurrence and development of DR, but its specific mechanism remains unclear. Further studies are required to broaden our knowledge of autophagy in the pathogenesis of DR.

In this study, we identified 23 potential autophagy-related genes in DR for the first time through bioinformatics analysis. GO and KEGG enrichment analysis showed that these genes were closely related to regulation of autophagy, positive regulation of catabolic process, macroautophagy and other signal pathways. Next, we further identified 10 hub genes related to DR, including TSC1, RAF1, RB1, ITPR1, CDKN1B, MAPK1, FOXO3, DAPK1, MAPK3 and BCL2L1 by using PPI network and key module analysis. The function of these genes in the occurrence of DR has been extensively studied. For example, Ras/Raf-1/MEK/ERK signal cascade can promote the activation of MMP9, and Raf kinase can also interact with VEGF to promote the loss of retinal capillary cells, which eventually leads to the development of DR (33–36). Overexpression of Raf-1 Kinase Inhibitory Protein (RKIP) can prevent the occurrence of diabetic retinal neurodegeneration by inhibiting p38-MAPK pathway (37). Hu-zhang-qing-mai-yin (HZQMY) can regulate P38 and NF-κB pathways by targeting MAPK3 and inhibit the release of ROS under HG exposure in a dose-dependent manner, thus inhibiting the proliferation of human retinal capillary endothelial cells (HRCECs) and having a certain effect on DR (38). Studies have shown that these autophagy-related genes regulate autophagy activity in tumor, cerebral ischemic stroke and osteoporosis (39–42). However, the role of these genes in modulating autophagy in DR has not been fully explored.

In current study, HG treated ARPE-19 cells were used as DR model to testify the function of potential autophagy-related genes. It is due to following reasons. Firstly, the dysfunction and loss of RPE cells has been found in diabetic model. It is associated with macular edema arising from diabetes-induced disruption of outer blood-retinal barrier. Therefore, RPE cells have been widely utilized as in vitro model for DR research (22). Secondly, both autophagy associated markers and autolysosomes are obviously detected in HG treated RPE cells, indicating this in vitro DR model is suitable for autophagy investigation (23, 24). Among 10 predicted DR-related hub genes, we showed that only the expression of MAPK3 was consistent with that of bioinformatics analysis of mRNA chip. We speculate that because the bioinformatics results were originated from peripheral blood lymphocytes of DR patients and non-diabetic individuals, the differences in cell type and culture condition may change gene expression and provide contradicting results. MAPK3 has been demonstrated to modulate autophagy by regulating mTOR pathway and Beclin-1 expression (43). Further investigation is required to elucidate the way by which MAPK3 control autophagy in DR models and cells.

To sum up, 23 potential autophagy-related genes in DR were identified by bioinformatics analysis, 10 hub genes TSC1, RAF1, RB1, ITPR1, CDKN1B, MAPK1, FOXO3, DAPK1, MAPK3, BCL2L1 were identified by constructing PPI network and identifying key modules. MAPK3 was preliminarily identified by in vitro experiments, which may affect the occurrence and development of DR by regulating autophagy. In the future, further experiments are needed to investigate the regulatory role of MAPK3 in DR models in order to clarify its value as potential clinical biomarkers or therapeutic targets.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

NW analyzed the data. NW and LFW drafted the first draft. DL, QYZ, LXD, and XBX edited and provided comments to improve the manuscript. SQX designed this experiment and reviewed and revised the manuscripts. All authors contributed to the article and approved the final manuscript.

This study was financially supported by The National Natural Science Foundation of China (No.81974137 to SQX), The National Natural Science Foundation of China (No.82070966 to LD), The Natural Science Foundation of Hunan Province (No.2019JJ40507 to XS), and the Science and Technology Innovation Program of Hunan Province (No.2021RC3026 to LD).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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