Diabetic Retinopathy Model Research Articles

Bioinformatics analysis of immune infiltration in human diabetic retinopathy and identification of immune-related hub genes and their ceRNA networks

Diabetic retinopathy (DR) is the most common microvascular complication in diabetic patients, and recent studies have shown that immune regulatory mechanisms are closely associated with retinal damage in DR. Therefore, this study focused on exploring immune cells and immune-related genes (IRGs) in DR and gaining insight into the ceRNA mechanisms by which IRGs regulate DR progression. Four datasets from human DR model retinal tissues were obtained from the Gene Expression Omnibus (GEO) database. R software was first used to identify differentially expressed mRNAs (DE-mRNAs) in the dataset GSE160306-mRNAs, then the distribution of immune cells in the gene matrix was analyzed by xCell and ImmuCellAI, ImmPort and InnateDB database were used to obtain immune-related hub genes (IRHGs) in the DR, and finally the STRING online tool and Cytoscape to construct the immune-related ceRNA network. The datasets GSE102485, GSE160308 and GSE160306-lncRNAs were used to validate the results of the ceRNA network further. The results of immune cell infiltration analysis showed that macrophages are important immune cells in DR; immune-related gene screening showed that FCGR2B is an IRHG in DR, and 2 immune-related ceRNA networks of IRHG were obtained: DDN-AS1/miR-10a-5p/FCGR2B and LINC01515/miR-10a-5p/FCGR2B. Our study suggests that infiltration of immune cells, especially the immune role of macrophages, is an important component of DR progression; the immune-related hub gene FCGR2B and its ceRNA network may be a key regulatory network for DR progression. The discovery of key immune cells, IRHG and ceRNA networks in this study may provide new prospects for early intervention and targeted treatment of DR.

6635 A Call for High-Value Diabetic Retinopathy Screening and the Introduction of a Novel Predictive Model

Abstract Disclosure: P.J. Williams: None. C. Kim: None. K. Ziadeh: None. E. White: None. C.S. Chen-Milhone: None. L.D. Lugo: None. K.B. Ellis: None. M.S. Juzych: None. Introduction: Diabetic retinopathy (DR) remains one of the leading causes of preventable blindness in middle-aged and older populations. Late-stage DR is often visually symptomatic; however, early DR is generally asymptomatic and requires screening. For type 2 diabetes mellitus (T2DM), the American Diabetes Association and the National Institute of Diabetes and Digestive and Kidney Diseases recommend an initial dilated eye exam with follow-up every 1-3 years, or annually if diabetic retinopathy is detected. Despite these guidelines, providers and insurers in the United States routinely recommend annual screening regardless of other factors. Methods: A cross-sectional analysis with prospective data verification was done for new diabetic eye exam referrals from August 2021 through July 2022 to address screening discrepancies. Established patients and referrals unrelated to hyperglycemia were excluded. Data collection included duration of diabetes, last hemoglobin A1c (HbA1c), maximum HbA1c, age, race, gender, BMI, relevant medication use, and imaging, as well as the presence of hypertension, hypercholesterolemia, diabetic peripheral neuropathy (DPN), diabetic nephropathy, diabetic amputation, and DR and data was verified against objective records up to 2 years later. Logistical multivariate analysis was conducted by a trained statistician. Results: 474 consecutive patients were referred over 1 year. Among all referrals, 2.11% (10/474) of patients were prediabetic. Among the patients with diabetes, 5.39% (25/464) were type 1 and 94.6% (439/464) were type 2. Among patients with T2DM, the average age was 57.64±11.52 (range = 25-98) and females made up 52.16% (229/439). The average diabetes duration among T2DM patients with DR was 19.39±8.45 years (range = 11-51) compared to 6.12±5.77 years (range = 0-31 years) without DR. Maximum lifetime HbA1c (p=0.0016), duration of diabetes (p<0.0001), and presence of DPN (p = 0.0047) were the strongest predictors of DR in patients with T2DM. A predictive model for DR was created utilizing these variables and hypercholesterolemia (p=0.048) with a predictive accuracy of 89.5% (chi-squared value (4)=121.451, p<0.001, Nagelkerke R2=57%). Sensitivity and specificity measured 92.31% and 87.99% respectively. The area under the ROC curve measured 0.952 (CI 0.925 to 0.972, p<0.0001). Conclusion: Duration of diabetes, maximum HbA1c, and DPN are strongly associated with DR among patients with T2DM and should be tracked to guide screening. Among T2DM patients with a well-documented recent onset of diabetes, maximum HbA1c less than 8, and benign initial eye exam, annual diabetic eye exams are unlikely to reveal DR in the first 10 years after onset. Based on our data, annual diabetic screening exams are unnecessary for many patients, and upper limits of screening intervals should be considered in low-risk patients to increase high-value care. Presentation: 6/2/2024

Neuritin alleviates Diabetic Retinopathy by Regulating Endoplasmic Reticulum Stress in Rats.

Neuritin, a small-molecule neurotrophic factor, maintains neuronal cell activity, inhibits apoptosis, promotes process growth, and regulates neural progenitor cell differentiation, migration, and synaptic maturation. Neuritin helps retinal ganglion cells (RGCs) survive optic nerve injury in rats and regenerate axons. However, the role of Neuritin in Diabetic retinopathy (DR) is unclear. This study is intended to investigate the effect and mechanism of Neuritin in DR. For this purpose, we established DR rat models and injected Neuritin into them. This study provides a potential treatment for diabetic retinopathy. The rat model of DR was established by streptozotocin (STZ) injection, and the effect of Neuritin on DR was detected by intravitreal injection. Histological analysis was performed by H&E and TUNEL methods. The mRNA and protein expressions of endoplasmic reticulum stress (ERS) pathway-related transcription factors were detected by qRT-PCR and western blot. The blood-retinal barrier (BRB) function was assessed using the patch-clamp technique and Evans blue leakage assay. Neuritin significantly improved the retinal structure, restrained the apoptosis of retinal cells, and protected the normal function of BRB in DR model rats. Mechanistically, Neuritin may function by inhibiting the expression of GRP78, ASK1, Caspase-12, VEGF, and so on. Our results indicate that Neuritin alleviates retinal damage in DR rats via the inactive endoplasmic reticulum pathway. Our study provides a potential treatment for DR.

Therapeutic potential of histamine H4 receptor antagonist as a preventive treatment for diabetic retinopathy in mice

Diabetic retinopathy (DR) is a prevalent complication of diabetes, often resulting in vision loss and blindness. Existing treatments primarily aim to control blood sugar levels and inhibit angiogenesis. However, current therapies for DR, such as anti-VEGF and laser photocoagulation, are frequently invasive, and can cause adverse side effects. Consequently, there is a critical need for new preventive therapeutics to address DR more effectively. This study aimed to examine the therapeutic potential of a histamine H4 receptor (HRH4) antagonist as a preventive treatment for DR in mice. A mouse model of DR was established by intraperitoneally injecting 200 mg/kg of streptozotocin (STZ). Immune cell infiltration into the retina of mice with STZ-induced diabetes was measured using fluorescence-activated cell sorting (FACS) 12 weeks after STZ injection. The preventive effects of the HRH4 antagonist on inflammation and pathological retinal vessel leakage were determined in a mouse model of DR. Infiltration of HRH4-expressing macrophages increased in the retina of mice with STZ-induced DR. The HRH4 antagonist prevented macrophage infiltration and retinal vascular leakage to prevent STZ-induced DR in mice without causing any retinal toxicity. The infiltration of macrophages increased in the retina of mice with STZ-induced diabetes through HRH4, indicating that HRH4 is potentially a novel preventative therapeutic target in DR. These findings suggest that targeting HRH4 is a promising strategy for the prevention and treatment of DR.

Polysaccharides from Ostrea rivularis alleviate type II diabetes induced-retinopathy and VGEF165-induced angiogenesis via PI3K/AKT signaling pathway

The purpose of this study was to investigate the role of polysaccharides from Ostrea rivularis Gloud (ORPs) in the progression of diabetic retinopathy (DR) and its anti-angiogenic effect on endothelial cell. Transgenic db/db mice with DR model were used to evaluate the protective effect of ORPs on retinal damage. It was found that ORPs could down-regulated levels of random blood glucose and fasting insulin, and further ameliorate retinal structure abnormalities as well as vascular network structure. Moreover, ORPs could reduce the expression of VEGF in retinal tissue and lessen pathological angiogenesis, thus slowing the progression of DR. In vitro, the proliferation, migration and tube formation of VGEF165-induced EA.hy926 cells were inhibited with ORPs administration. Furthermore, the expression of related proteins in the PI3K/AKT pathway and angiogenesis related factors were improved after ORPs intervention. Overall, these findings suggested that ORPs could effectively control the development of DR, and inhibit VGEF165-induced EA.hy926 cells proliferation, migration and tube formation, which effects might work through blocking the activation of PI3K/AKT signaling pathway.

VEGF-B prevents chronic hyperglycemia-induced retinal vascular leakage by regulating the CDC42-ZO1/VE-cadherin pathway.

Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins invitro and invivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated invitro and invivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.

Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Pericyte loss via glutaredoxin2 downregulation aggravates diabetes-induced microvascular dysfunction

Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes.

Knockdown of KCNQ1OT1 Alleviates the Activation of NLRP3 Inflammasome Through miR-17-5p/TXNIP Axis in Retinal Müller Cells

Purpose Diabetic retinopathy (DR) is one of the most severe and common complications caused by diabetic mellites. Inhibiting NLRP3 inflammasome activation displays a crucial therapeutic value in DR. Studies have shown that KCNQ1OT1 plays a critical role in regulating NLRP3 inflammasome activation and participates in the pathogenesis of diabetic complications. The present study aims to explore the role, and the potential mechanism of KCNQ1OT1 in regulating the activation of NLRP3 inflammasome in DR. Methods qRT-PCR was used to detect the expression of KCNQ1OT1, miR-17-5p, TXNIP, NLRP3, ASC, caspase-1 and IL-1β. Western blot was performed to detect the expression of NLRP3, ASC, caspase-1, IL-1β and TXNIP. Immunohistochemistry and immunostaining were performed to detect the expression of caspase-1. The levels of the inflammatory cytokine IL-1β were determined by ELISA assay. FISH was used to detect the subcellular localisation of KCNQ1OT1. Bioinformatic analysis, luciferase reporter assay and in vitro studies were performed to elucidate the mechanism of KCNQ1OT1-mediated dysfunction. Results The expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were increased in experimental DR models. KCNQ1OT1 knockdown alleviated NLRP3 inflammasome-associated molecules expression. In addition, KCNQ1OT1 was found to be localized mainly in the cytoplasm of Müller cells and facilitated TXNIP expression by acting as a miR-17-5p sponge. KCNQ1OT1 promoted the activation of NLRP3 inflammasome through miR-17-5p/TXNIP axis. Conclusions In conclusion, it was found in this study that KCNQ1OT1 promoted the activation of NLRP3 inflammasome both in vitro and in vivo, which was mediated by miR-17-5p/TXNIP axis. KCNQ1OT1 might be an effective interference target for the prevention and treatment of DR.

Allicin Mitigates Diabetic Retinopathy in Rats by Activating Phosphatase and Tensin Homolog-induced Kinase 1/Parkin-mitophagy and Inhibiting Oxidative Stress-mediated NOD-like Receptor Family Pyrin Domain Containing 3 Inflammasome.

Diabetic retinopathy (DR) is one of the significant disabling outcomes of diabetes mellitus characterized by retinal microvascular damage, inflammation, and neuronal dysfunction. Allicin (Alc), a natural compound found in garlic, has garnered attention for its antioxidant and anti-inflammatory properties, positioning it as a potential therapeutic agent for DR. The aim of the present study was to investigate the therapeutic efficacy of Alc in DR management and elucidate its underlying mechanisms of action. We established a DR model in male Sprague-Dawley rats (n = 50, 200-250 g, 12 weeks old) using a high-fat diet for 8 weeks plus a low dose of streptozotocin administered at the start of the 4th week. The diabetic (Diab) animals were administered Alc (16 mg/kg/day, orally), either alone or in combination with mitochondrial division inhibitor-1 (Mdivi-1) as a mitophagy inhibitor, starting 28 days before tissue sampling. We evaluated histopathological changes, metabolic abnormalities associated with type 2 diabetes mellitus (T2DM), the expression of proteins regulating pyroptosis (NOD-like receptor family pyrin domain containing 3, cleaved-caspase 1, and gasdermin D-N terminal) and mitophagy (phosphatase and tensin homolog-induced kinase 1 [PINK1] and Parkin), as well as the levels of oxidative stress mediators and proinflammatory cytokines. Alc treatment effectively ameliorated histopathological changes and metabolic abnormalities associated with T2DM. It downregulated pyroptosis-related proteins, upregulated mitophagy-related proteins, reduced proinflammatory cytokine levels, and attenuated oxidative stress. Treatment with Mdivi-1 suppressed the beneficial effects of Alc. Our findings highlight the therapeutic potential of Alc in managing DR by targeting multiple pathophysiological pathways, including pyroptosis, inflammation, and oxidative stress. The observed antipyroptotic effects of Alc were partially mediated by the activation of the PINK1/parkin-mediated mitophagy pathway. Additional studies are necessary to thoroughly understand the therapeutic mechanisms of Alc and its viability as a treatment choice for DR.

PLK-3-mediated phosphorylation of BAP1 prevents diabetic retinopathy

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus, and its main clinical manifestation is retinal vascular dysfunction. DR causes blindness and is a problem with significant global health implications. However, treating DR is still challenging. In this study, we aimed to explore the role of polo-like kinase-3 (PLK-3) and the potential regulatory mechanism in DR. Sprague-Dawley rats were injected intraperitoneally with streptozotocin (STZ, 60 mg/kg) to induce a rat model of DR, and rat retinal microvascular endothelial cells (RRMECs) were treated with high glucose (HG, 25 mmol/L glucose) to develop a cell model of DR. We found that PLK-3 was significantly downregulated in the retinal tissues of STZ-induced diabetic rats and HG-induced RRMECs. Lentivirus-mediated PLK-3 overexpression alleviated the histological damages in DR rats. After HG stimulation, cell proliferation, migration, and angiogenesis in RRMECs were inhibited after PLK-3 upregulation. By using label-free proteomics, we identified 82 differentially expressed proteins downstream of PLK-3, including BRCA1-associated protein 1 (BAP1), which was significantly upregulated in PLK-3-overexpressed RRMECs compared to control cells under the HG condition. In vivo and in vitro assays indicated that the forced expression of PLK-3 increased the phosphorylation of BAP1 at serine 592 and caspase-8 expression. Detailed evidence showed that BAP1-shRNA-mediated knockdown restored the cell function in HG-treated RRMECs when PLK-3 was overexpressed. Collectively, this study shows that PLK-3 alleviates retinal vascular dysfunction in DR by inhibiting the phosphorylation of BAP1. Thus, PLK-3 may develop as a promising target for the therapy of DR.

Role of Gpr124 in the Migration and Proliferation of Retinal Microvascular Endothelial Cells and Microangiopathies in Diabetic Retinopathy.

Retinal microangiopathies, such as neovascularization and preretinal and vitreous hemorrhages, are the primary pathological features of diabetic retinopathy (DR). These conditions can worsen visual impairment and may result in blindness. Furthermore, multiple metabolic pathways are associated with microangiopathy in DR. However, the specific underlying pathological mechanisms remain unclear. Several studies have demonstrated the important role of G protein-coupled receptor 124 (Gpr124) in cerebral vascular endothelial cells, but its effect on the retinal endothelium has not been elucidated. In this study, we found that Gpr124 is expressed in both pathological retinal fibrous vascular membranes of DR patients and retinal blood vessels of mice, with elevated protein expression specifically observed in the retinas of DR model mice. Furthermore, Gpr124 expression was elevated after high-glucose treatment of human retinal microvascular endothelial cells (HRMECs). Inhibition of Gpr124 expression affected the high glucose-induced proliferation, migration, and tube-forming ability of HRMECs. We concluded that Gpr124 expression was upregulated in DR and promoted HRMECs angiogenesis in a high-glucose environment. This finding may help to elucidate the pathogenesis of DR and provide a critical research basis for identifying effective treatments.

LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA.

This study sought to investigate the regulation of long noncoding RNA (lncRNA) XIST on the microRNA (miR)-101-3p/vascular endothelial growth factor A (VEGFA) axis in neovascularization in diabetic retinopathy (DR). Serum of patients with DR was extracted for the analysis of XIST, miR-101-3p, and VEGFA expression levels. High glucose (HG)-insulted HRMECs and DR model rats were treated with lentiviral vectors. MTT, transwell, and tube formation assays were performed to evaluate cell viability, migration, and angiogenesis, and ELISA was conducted to detect the levels of inflammatory cytokines. Dual-luciferase reporter, RIP, and RNA pull-down experiments were used to validate the relationships among XIST, miR-101-3p, and VEGFA. XIST and VEGFA were upregulated and miR-101-3p was downregulated in serum from patients with DR. XIST knockdown inhibited proliferation, migration, vessel tube formation, and inflammatory responsein HG-treated HRMECs, whereas the above effects were nullified by miR-101-3p inhibition or VEGFA overexpression. miR-101-3p could bind to XIST and VEGFA. XIST promoted DR development in rats by regulating the miR-101-3p/VEGFA axis. LncRNA XIST promotes VEGFA expression by downregulating miR-101-3p, thereby stimulating angiogenesis and inflammatory response in DR.

Role of Hsa_circ_0000880 in the Regulation of High Glucose-Induced Apoptosis of Retinal Microvascular Endothelial Cells.

Circular RNAs (circRNAs) have been verified to participate in multiple biological processes and disease progression. Yet, the role of circRNAs in the pathogenesis of diabetic retinopathy (DR) is still poorly understood and deserves further study. This study aimed to investigate the role of circRNAs in the regulation of high glucose (HG)-induced apoptosis of retinal microvascular endothelial cells (RMECs). Epiretinal membranes from patients with DR and nondiabetic patients with idiopathic macular epiretinal membrane were collected for this study. The circRNA microarrays were performed using high-throughput sequencing. Hierarchical clustering, functional enrichment, and network regulation analyses were used to analyze the data generated by high-throughput sequencing. Next, RMECs were subjected to HG (25mM) conditions to induce RMECs apoptosis in vitro. A series of experiments, such as Transwell, the Scratch wound, and tube formation, were conducted to explore the regulatory effect of circRNA on RMECs. Fluorescence in situ hybridization (FISH), immunofluorescence staining, and Western blot were used to study the mechanism underlying circRNA-mediated regulation. A total of 53 differentially expressed circRNAs were found in patients with DR. Among these, hsa_circ_0000880 was significantly upregulated in both the diabetic epiretinal membranes and in an in vitro DR model of HG-treated RMECs. Hsa_circ_0000880 knockout facilitated RMECs vitality and decreased the paracellular permeability of RMECs under hyperglycemia. More importantly, silencing of hsa_circ_0000880 significantly inhibited HG-induced ROS production and RMECs apoptosis. Hsa_circ_0000880 acted as an endogenous sponge for eukaryotic initiation factor 4A-III (EIF4A3). Knockout of hsa_circ_0000880 reversed HG-induced decrease in EIF4A3 protein level. Our findings suggest that hsa_circ_0000880 is a novel circRNA can induce RMECs apoptosis in response to HG conditions by sponging EIF4A3, offering an innovative treatment approach against DR. The circRNAs participate in the dysregulation of microvascular endothelial function induced by HG conditions, indicating a promising therapeutic target for DR.

Network Pharmacology and Molecular Docking to Explore the Mechanism of Compound Qilian Tablets in Treating Diabetic Retinopathy

Background: Diabetic Retinopathy (DR) is one of the common chronic complications of diabetes mellitus, which has developed into the leading cause of irreversible visual impairment in adults worldwide. The Compound Qilian Tablets (CQLT) were developed in China for the treatment and prevention of DR, but their mechanism of action is still unclear. Objective: In the present study, network pharmacology, molecular docking, and in vivo validation experiments were used to investigate the active components and molecular mechanisms of CQLT against DR. Methods: The active components and targets of CQLT were collected through the TCSMP database, and the targets of DR were obtained from GeneCards, OMIM, and Drugbank databases. We established a protein-protein interaction network using the STRING database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the Metascape database. Molecular docking using AutoDock Vina was performed to investigate the interactions between components of CQLT and core targets. Moreover, we selected ZDF rats to establish a DR model for the experimental studies. Results: 39 active components and 448 targets in CQLT were screened, among which 90 targets were shared with DR. KEGG pathway enrichment analysis identified 181 pathways. The molecular docking results demonstrated that the main active components had strong binding ability to the core targets. The results from animal experiments indicate that the mechanism of CQLT against DR is associated with inhibiting the retinal mTOR/HIF-1α/VEGF signaling pathway, alleviating the inflammatory response, suppressing retinal neovascularization, and protecting the function and morphology of the retina. Conclusion: The present study preliminarily explored the mechanism of CQLT in treating DR and demonstrated that CQLT exerts anti-DR effects through multiple components, multiple targets, and multiple pathways. These findings suggest that CQLT shows promise as a potential therapeutic agent for DR and could contribute to developing novel treatments.

WNT-inhibitory factor 1-mediated glycolysis protects photoreceptor cells in diabetic retinopathy

BackgroundIn diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR.MethodTwelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting.ResultTypical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability.ConclusionsWIF1 exerts an inhibitory effect on the Wnt/β-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.Graphical

Transthyretin-Regulated Diabetic Retinopathy Through the VEGFA/PI3K/AKT Pathway.

Transthyretin (TTR) plays a regulatory role in a variety of diabetes-related diseases. The objective of this work was to probe whether TTR affects diabetic retinopathy (DR) through the VEGFA/PI3K/AKT pathway. High glucose (HG, 25mM) was used to treat human retinal microvascular endothelial cells (hRMECs) and C57BL/6J mice were intraperitoneally injected with STZ (50mg/kg) to construct a DR model. In vitro, the effect of TTR on DR was evaluated by measuring hRMEC proliferation, migration, and angiogenesis. The changes in retinal tissue were observed by hematoxylin and eosin staining in vivo. ELISA, immunohistochemistry, and immunofluorescence staining were used to measure VEGFA or CD31 levels. The levels of all proteins were evaluated through Western blot. The increase of proliferation, migration, and angiogenesis and decrease of apoptosis in hRMECs caused by HG were notably reversed by TTR. TTR greatly impeded HG-raised VEGFA, PI3K p-p85, and p-AKT in hRMECs. Inhibition of TTR further exacerbated the effect of HG-induced hRMECs. Inhibition of VEGFA reversed the effect of HG-induced hRMECs. VEGFA neutralized the function of TTR on cell proliferation, apoptosis, migration, and angiogenesis in HG-triggered hRMECs. It was further confirmed in vivo that TTR can alleviate the occurrence of DR in diabetic mice models. TTR significantly restrained the progression of DR via molecular modulation of the VEGFA/PI3K/AKT axis.

Compound Danshen dripping pills prevent early diabetic retinopathy: roles of vascular protection and neuroprotection.

Introduction: Diabetic retinopathy (DR) represents a major cause of adult blindness, and early discovery has led to significant increase in the number of patients with DR. The drugs currently used for treatment, such as ranibizumab, mainly focus on the middle and late periods of DR, and thus do not meet the clinical need. Here, the potential mechanisms by which compound Danshen Dripping Pills (CDDP) might protect against early DR were investigated. Methods: Db/db mice were used to establish a DR model. The initial weights and HbA1c levels of the mice were monitored, and retinal pathology was assessed by hematoxylin-eosin (HE) staining. The vascular permeability of the retina and thickness of each retinal layer were measured, and electroretinogram were performed together with fundus fluorescein angiography and optical coherence tomography. The levels of inflammatory factors were examined in retinal tissue, as well as those of intercellular adhesion molecule 1 (ICAM-1), IL-6, and monocyte chemoattractant protein 1 (MCP-1) in the serum using ELISA. Immunohistochemistry was used to evaluate levels of vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), and Bclassociated X protein (Bax). Retinal cell injury and apoptosis were examined by TdT-mediated dUTP Nick End Labeling (TUNEL) assays. Results: The data showed that CDDP significantly improved cellular disarrangement. Imaging data indicated that CDDP could reduce vascular permeability and the amplitude of oscillatory potentials (OPs), and restore the thickness of the ganglion cell layer. Moreover, CDDP reduced the expression levels of inflammatory factors in both the retina and serum. Conclusion: These findings strongly suggest that CDDP prevents early DR through vascular and neuroprotection.

MiR-26a-5p Attenuates Oxidative Stress and Inflammation in Diabetic Retinopathy through the USP14/NF-κB Signaling Pathway.

Diabetic retinopathy (DR) is an ocular disease caused by diabetes and may lead to vision impairment and even blindness. Oxidative stress and inflammation are two key pathogenic factors of DR. Recently, regulatory roles of different microRNAs (miRNAs) in DR have been widely verified. miR-26a-5p has been confirmed to be a potential biomarker of DR. Nevertheless, the specific functions of miR-26a-5p in DR are still unclear. Primary cultured mouse retinal Müller cells in exposure to high glucose (HG) were used to establish an in vitro DR model. Müller cells were identified via morphology observation under phase contrast microscope and fluorescence staining for glutamine synthetase. The in vivo animal models for DR were constructed using streptozotocin-induced diabetic C57BL/6 mice. Western blotting was performed to quantify cytochrome c protein level in the cytoplasm and mitochondria of Müller cells and to measure protein levels of glial fibrillary acidic protein (GFAP), ubiquitin-specific peptidase 14 (USP14), as well as factors associated with NF-κB signaling (p-IκBα, IκBα, p-p65, and p65) in Müller cells or murine retinal tissues. ROS production was detected by CM-H2DCFDA staining, and the concentration of oxidative stress markers (MDA, SOD, and CAT) was estimated by using corresponding commercial kits. Quantification of mRNA expression was conducted by RT-qPCR analysis. The concentration of proinflammatory factors (TNF-α, IL-1β, and IL-6) was evaluated by ELISA. Hematoxylin-eosin staining for murine retinal tissues was performed for histopathological analysis. Immunofluorescence staining was conducted to determine NF-κB p65 nuclear translocation in Müller cells. Furthermore, the interaction between miR-26a-5p and USP14 was verified via the luciferase reporter assays. HG stimulation contributed to Müller cell dysfunction by inducing inflammation, oxidative injury, and mitochondrial damage to Müller cells. miR-26a-5p was downregulated in Müller cells under HG condition, and overexpression of miR-26a-5p relieved HG-induced Müller cell dysfunction. Moreover, miR-26a-5p targeted USP14 and inversely regulated USP14 expression. Additionally, HG-evoked activation of NF-κB signaling was suppressed by USP14 knockdown or miR-26a-5p upregulation. Rescue assays showed that the protective impact of miR-26a-5p upregulation against HG-induced Müller cell dysfunction was reversed by USP14 overexpression. Furthermore, USP14 upregulation and activation of NF-κB signaling in the retinas of DR mice were detected in animal experiments. Injection with miR-26a-5p agomir improved retinal histopathological injury and weakened the concentration of proinflammatory cytokines and oxidative stress markers in the retinas of DR mice. miR-26a-5p inhibits oxidative stress and inflammation in DR progression by targeting USP14 and inactivating the NF-κB signaling pathway.

Sestrin2 ameliorates diabetic retinopathy by regulating autophagy and ferroptosis.

Diabetic retinopathy (DR) is a serious microvascular complication of diabetes. The aim of this study was to explore the effect of Sestrin2 on DR through the regulation of autophagy and ferroptosis levels and its mechanism. In vitro and in vivo DR models were established by high glucose (HG) and streptozotocin (STZ) induction of ARPE-19 human retinal pigment epithelial cells and C57BL/6 mice, respectively. In this study, we demonstrated that after HG treatment, the activity of ARPE-19 cells was decreased, the apoptosis rate was increased, endoplasmic reticulum (ER) stress was activated, autophagy levels were decreased, and ferroptosis levels were increased. Overexpression of Sestrin2 enhanced cell viability, reduced apoptosis and ferroptosis, and enhanced autophagy. However, the effect of overexpression of Sestrin2 was attenuated after the addition of the STAT3 phosphorylation activator Colivelin TFA (C-TFA), the mTOR pathway activator MHY1485 or the autophagy inhibitor 3-methyladenine (3-MA). In addition, the effect of Sestrin2 knockdown on cells was opposite to the effect of overexpression of Sestrin2, while the effect of Sestrin2 knockdown was attenuated after treatment with the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Animal experiments also confirmed the results of cell experiments and attenuated the effects of overexpression of Sestrin2 after injection of the ferroptosis activators erastin or 3-MA. Our study revealed that Sestrin2 inhibits ferroptosis by inhibiting STAT3 phosphorylation and ER stress and promoting autophagy levels, thereby alleviating DR.