Rat Model Of Diabetic Retinopathy Research Articles

Garlic extract (Allium sativum L.) attenuates neurodegeneration and microvascular damage in rats with streptozotocin-induced diabetic retinopathy

Diabetic retinopathy (DR) is a severe complication of diabetes mellitus (DM). Despite numerous studies exploring the anti-diabetic properties of garlic in managing DM symptoms, its impact on DR remains limited. This study aimed to test the effectiveness of garlic in managing DR in a streptozotocin-induced DR rat model. Rats were divided into three groups: normal control (NC), diabetic control (DC), and diabetic group (DG) treated with aqueous garlic extract (GE). Histological examination revealed a significant reduction in retinal thickness in DC rats compared to that in DG rats, which showed restored retinal thickness similar to the NC group. Immunohistochemical analyses revealed significant improvements in retinal vascular density, main artery diameter, and blood vessel numbers in DG rats compared to DC rats. The findings suggest that GE protects against DR. Furthermore, garlic could be recommended as a beneficial dietary supplement for diabetic patients to manage and delay the progression of DR.

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.

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.

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.

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.

The impact of sestrin2 on reactive oxygen species in diabetic retinopathy.

Diabetic retinopathy (DR) is a significant complication of diabetes that often leads to blindness, impacting Müller cells, the primary retinal macroglia involved in DR pathogenesis. Reactive oxygen species (ROS) play a crucial role in the development of DR. The objective of this study was to investigate the involvement of sestrin2 in DR using a high-glucose (HG)-induced Müller cell model and assessing cell proliferation with 5-ethynyl-2-deoxyuridine (EdU) labeling. Following this, sestrin2 was upregulated in Müller cells to investigate its effects on ROS, tube formation, and inflammation both in vitro and in vivo, as well as its interaction with the nuclear factor erythroid2-related factor 2 (Nrf2) signaling pathway. The findings demonstrated a gradual increase in the number of EdU-positive cells over time, with a subsequent decrease after 72 h of exposure to high glucose levels. Additionally, the expression of sestrin2 exhibited a progressive increase over time, followed by a decrease at 72 h. The rh-sestrin2 treatment suppressed the injury of Müller cells, decreased ROS level, and inhibited the tube formation. Rh-sestrin2 treatment enhanced the expression of sestrin2, Nrf2, heme oxygenase-1 (HO-1), and glutamine synthetase (GS); however, the ML385 treatment reversed the protective effect of rh-sestrin2. Finally, we evaluated the effect of sestrin2 in a DR rat model. Sestrin2 overexpression treatment improved the pathological injury of retina and attenuated the oxidative damage and inflammatory reaction. Our results highlighted the inhibitory effect of sestrin2 in the damage of retina, thus presenting a novel therapeutic sight for DR.

IDF23-0554 Effect of two anti-VEGF antibodies on NRF2 expression in a rat model of diabetic retinopathy

IDF23-0554 Effect of two anti-VEGF antibodies on NRF2 expression in a rat model of diabetic retinopathy

Beneficial Effect of Sirolimus-Pretreated Mesenchymal Stem Cell Implantation on Diabetic Retinopathy in Rats.

Diabetic retinopathy (DR) is a vision-threatening complication that affects virtually all diabetic patients. Various treatments have been attempted, but they have many side effects and limitations. Alternatively, stem cell therapy is being actively researched, but it faces challenges due to a low cell survival rate. In this study, stem cells were pretreated with sirolimus, which is known to promote cell differentiation and enhance the survival rate. Additionally, the subconjunctival route was employed to reduce complications following intravitreal injections. Diabetes mellitus was induced by intraperitoneal injection of 55 mg/kg of streptozotocin (STZ), and DR was confirmed at 10 weeks after DM induction through electroretinogram (ERG). The rats were divided into four groups: intact control group (INT), diabetic retinopathy group (DR), DR group with subconjunctival MSC injection (DR-MSC), and DR group with subconjunctival sirolimus-pretreated MSC injection (DR-MSC-S). The effects of transplantation were evaluated using ERG and histological examinations. The ERG results showed that the DR-MSC-S group did not significantly differ from the INT in b-wave amplitude and exhibited significantly higher values than the DR-MSC and DR groups (p < 0.01). The flicker amplitude results showed that the DR-MSC and DR-MSC-S groups had significantly higher values than the DR group (p < 0.01). Histological examination revealed that the retinal layers were thinner in the DR-induced groups compared to the INT group, with the DR-MSC-S group showing the thickest retinal layers among them. Subconjunctival injection of sirolimus-pretreated MSCs can enhance retinal function and mitigate histological changes in the STZ-induced DR rat model.

Dapagliflozin ameliorated retinal vascular permeability in diabetic retinopathy rats by suppressing inflammatory factors

BackgroundDiabetic retinopathy is a common microvascular complication of diabetes and one of the major causes of blindness in the working-age population. Emerging evidence has elucidated that inflammation drives the key mechanism of diabetes-mediated retinal disturbance. As a new therapeutic drug targeting diabetes, whether dapagliflozin could improve vascular permeability from the perspective of anti-inflammatory effect need to be further explored. MethodsType 2 diabetic retinopathy rat model was established and confirmed by fundus fluorescein angiography (FFA). ELISA detected level of plasma inflammatory factors and C-peptide. HE staining, immunohistochemistry and western blot detected histopathology changes of retina, expression of retinal inflammatory factors and tight junction proteins. ResultsDapagliflozin exhibited hypoglycemic effect comparable to insulin, but did not affect body weight. By inhibiting expression of inflammatory factors (NLRP3, Caspase-1, IL-18, NF-κB) in diabetic retina and plasma, dapagliflozin reduced damage of retinal tight junction proteins and improved retinal vascular permeability. The anti-inflammatory effect of dapagliflozin was superior to insulin. ConclusionsDapagliflozin improved retinal vascular permeability by reducing diabetic retinal and plasma inflammatory factors. The anti-inflammatory mechanism of dapagliflozin is independent of hypoglycemic effect and superior to insulin.

EIF4A3-mediated circEHMT1 regulation in retinal microvascular endothelial dysfunction in diabetic retinopathy

Background and objectiveLiterature has reported that circular RNAs (circRNAs) are crucially associated with diabetic retinopathy (DR). Furthermore, circEHMT1 has been identified to maintain endothelial cell barrier function. This study aimed to investigate the mechanisms that regulate aberrant circEHMT1 expression and its role in the pathogenesis of DR. MethodsIn this study, retinal microvascular endothelial cells were exposed to a high glucose (HG) environment, and subsequently, tube formation and intercellular junction proteins were evaluated. Furthermore, the biological functions of circEHMT1 and its potential regulatory factor, eIF4A3, in microvascular endothelial cells under HG conditions were also assessed. In addition, the regulatory role of eIF4A3 on circEHMT1 expression was confirmed. Moreover, to elucidate the in vivo functions of eIF4A3 and circEHMT1, streptozotocin (STZ) was used to establish a DR model in rats. ResultsIt was revealed that HG condition decreased circEHMT1 and eIF4A3 expressions and reduced ZO-1, Claudin-5, and Occludin levels in retinal microvascular endothelial cells. Furthermore, it was observed that eIF4A3 could regulate the expression of circEHMT1. Overexpression of eIF4A3 or circEHMT1 under HG conditions improved endothelial cell injury and decreased tube-formation ability. Additionally, in the DR rat model, eIF4A3 overexpression restored circEHMT1 levels and ameliorated retinal vasculature changes. ConclusionAltogether, eIF4A3 regulates circEHMT1 expression, thereby affecting microvascular endothelial cell injury and tube formation. Further understanding the regulatory effect of eIF4A3 on circEHMT1 may provide novel therapeutic targets for DR.

Tacrolimus Improves Therapeutic Efficacy of Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Diabetic Retinopathy by Suppressing DRP1-Mediated Mitochondrial Fission.

Diabetic retinopathy (DR) is a leading cause of blindness in diabetic patients. Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are emerging as a promising new drug for degenerative disease associated with diabetes. Recent studies have shown that high glucose-increased excessive calcium levels are a major risk factor for mitochondrial reactive oxygen species (mtROS) accumulation and apoptosis. This study aimed to investigate the role of high glucose-induced NFATC1 signaling in mitochondrial oxidative stress-stimulated apoptosis and the effect of tacrolimus on the therapeutic efficacy of subconjunctival transplantation of UCB-MSCs in a DR rat model. High glucose increased mtROS and cleaved caspase-9 expression in UCB-MSCs. High glucose conditions increased O-GlcNAcylated protein expression and nuclear translocation of NFATC1. Tacrolimus pretreatment recovered high glucose-induced mtROS levels and apoptosis. In the DR rat model, subconjunctival transplantation of tacrolimus-pretreated MSCs improved retinal vessel formation, retinal function, and uveitis. In high glucose conditions, tacrolimus pretreatment reduced protein and mRNA expression levels of DRP1 and inhibited mitochondrial fission. In conclusion, we demonstrated that high glucose-induced O-GlcNAcylation activates NFATC1 signaling, which is important for DRP1-mediated mitochondrial fission and mitochondrial apoptosis. Finally, we proposed NFATC1 suppression by tacrolimus as a promising therapeutic strategy to improve the therapeutic efficacy of UCB-MSC transplantation for DR treatment.

Effects of Gold Nanoparticles Phytoreduced with Rutin in an Early Rat Model of Diabetic Retinopathy and Cataracts.

Diabetic retinopathy (DR) and cataracts (CA) have an early onset in diabetes mellitus (DM) due to the redox imbalance and inflammation triggered by hyperglycaemia. Plant-based therapies are characterised by low tissue bioavailability. The study aimed to investigate the effect of gold nanoparticles phytoreduced with Rutin (AuNPsR), as a possible solution. Insulin, Rutin, and AuNPsR were administered to an early, six-week rat model of DR and CA. Oxidative stress (MDA, CAT, SOD) was assessed in serum and eye homogenates, and inflammatory cytokines (IL-1 beta, IL-6, TNF alpha) were quantified in ocular tissues. Eye fundus of retinal arterioles, transmission electron microscopy (TEM) of lenses, and histopathology of retinas were also performed. DM was linked to constricted retinal arterioles, reduced endogen antioxidants, and eye inflammation. Histologically, retinal wall thickness decreased. TEM showed increased lens opacity and fibre disorganisation. Rutin improved retinal arteriolar diameter, while reducing oxidative stress and inflammation. Retinas were moderately oedematous. Lens structure was preserved on TEM. Insulin restored retinal arteriolar diameter, while increasing MDA, and amplifying TEM lens opacity. The best outcomes were obtained for AuNPsR, as it improved fundus appearance of retinal arterioles, decreased MDA and increased antioxidant capacity. Retinal edema and disorganisation in lens fibres were still present.

The hsa_circ_0004805/hsa_miR-149-5p/TGFB2 axis plays critical roles in the pathophysiology of diabetic retinopathy in vitro and in vivo

The aim of this study was to investigate the mechanism underlying the role of a recently identified hsa_circ_0004805/hsa_miR-149-5p/transforming growth factor beta 2 (TGFB2) axis in the progression of diabetic retinopathy (DR). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis suggested that hsa_circ_0004805 was highly expressed in aqueous humor samples of patients with DR, whereas hsa_miR-149-5p showed the opposite trend. Meanwhile, the results of a dual-luciferase reporter assay indicated that hsa_miR-149-5p directly interacted with both hsa_circ_0004805 and TGFB2. Using a variety of assays (Cell Counting Kit-8, EdU-labeling, Transwell, flow cytometric, wound healing, tube formation assays), we found that the overexpression of hsa_circ_0004805 significantly downregulated the level of hsa_miR-149-5p and promoted DNA synthesis, proliferation, migration, and tube formation in human retinal microvascular epithelial cells (hRECs) cultivated in a high-glucose environment. In contrast, hsa_miR-149-5p mimics inhibited DNA synthesis, proliferation, migration, and tube formation in hRECs by reducing the expression of its downstream target TGFB2 as well as the levels of phosphorylated SMAD2; however, these effects were reversed by the overexpression of hsa_circ_0004805. In a streptozotocin-induced Sprague-Dawley rat model of DR, retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis were evident, which could be reversed by vitreous microinjection of rat miR-149-5p mimics (rno-miR-149-5p agomir). Combined, our findings indicated that, under hyperglycemia, the hsa_circ_0004805/hsa_miR-149-5p/TGFB2 axis plays a critical role in the retinal pathophysiology associated with the development of DR, and has potential as a therapeutic target in the treatment of this condition.

Astaxanthin inhibits oxidative stress and apoptosis in diabetic retinopathy

BackgroundThe pathophysiology of diabetic retinopathy (DR) is thought to be influenced by oxidative stress. Astaxanthin (ASX) is a natural product with antioxidant effect, but it is not clear whether its mechanism of inhibiting the development of DR is related to anti-oxidation. MethodsRats were intraperitoneally injected with streptozotocin (60 mg/kg) to create DR rat models followed by ASX (20 mg/kg) for 45 days. Retinal tissue was examined by Hematoxylin and Eosin staining. By using Enzyme-linked immunosorbent assay (ELISA), 2,7-Dichlorodrhydrofluorescein diace (DCFH-DA) probes, immunohistochemistry and western blot, it was feasible to evaluate the contents of inflammation-related factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and macrophage inhibitory cytokine-1 (MIC-1)), oxidative stress-related indicators (glutathione (GSH), malonic dialdehyde (MDA), glutathione peroxidase (GPx), reactive oxygen species (ROS) and Total antioxidant capacity (T-AOC)), antioxidant enzymes (hemoxgenase-1(HO-1) and Quinone Oxidoreductase 1 (NQO1)), and apoptosis-related proteins (Bcl-2, Bcl2 Associated X Protein (BAX), and cleaved-caspase-3). Additionally, antioxidant proteins downstream of the nuclear factor E2 related factors (Nrf-2) pathway, expression levels of Nrf2/ Kelch-like ECH-associated protein 1(Keap 1) pathway-associated proteins, and nuclear and cytoplasmic levels of Nrf2 were assessed using immunohistochemistry, western blot, or quantitative real-time polymerase chain reaction (qRT-PCR). ResultsASX alleviated retinal tissue damage by increasing overall retina thickness and ganglion cell layer (GCL) cell numbers and exerted the anti-inflammatory, anti-oxidative stress, and anti-apoptosis effects in DR rats. Additionally, ASX could inhibit the expression of Keap1, promote the transport of Nrf2 from cytoplasm to nucleus and facilitate the expressions of HO-1, NQO1, γ-glutamylcysteine synthetase, (γ-GCS) and GPx. ConclusionASX exerted antioxidant effects through Nrf2/keap1 pathway, thereby alleviating apoptosis, inflammation, and oxidative stress in retinal tissues of DR rats.

Hyper-reflective dots in optical coherence tomography imaging and inflammation markers in diabetic retinopathy.

The aim of this study is to correlate small dot hyper-reflective foci (HRF) observed in spectral domain optical coherence tomography (SD-OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD-OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba-1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba-1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia-mediated inflammatory component of progressive diseases showing HRF.

Cystic Fibrosis Transmembrane Conductance Regulator Attenuates Oxidative Stress-Induced Injury in Diabetic Retinopathy Rats

Purpose To investigate the effects of cystic fibrosis transmembrane conductance regulator (CFTR) on oxidative stress-induced injury of diabetic retinopathy (DR) rats. Methods DR rat model was constructed treated with Ad-CFTR. Hematoxylin and Eosin (HE) staining was applied for testing the thickness of each layer of retinal tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine levels of serum inflammatory cytokines and contents of oxidative stress related genes in rats. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining was used to detect retinal cell apoptosis, and western blotting to measure the expression of MAPK/NF-κB pathway-related proteins in retinal tissues. Results Our experiment revealed the remarkable decrease of CFTR protein in retinal tissues of DR rats. DR rats had decreased body weight and increased blood glucose level, with decreased thickness of total retinal thickness (TRT), outer nuclear layer and outer plexiform layer (ONL + OPL), inner nuclear layer (INL), and inner plexiform layer (IPL). Besides, DR rats were apparently up-regulated in the expression of pro-inflammatory cytokines, with increased malondial dehyde (MDA), p-ERK1/2/ERK1/2 and p-JNK1/2/JNK1/2 expressions, decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in retinal tissues, as well as up-regulated p65 protein in nucleus and down-regulated p65 protein in cytoplasm. DR rats treated with Ad-CFTR were effectively improved regarding the above parameters except body weight and blood glucose. Conclusions CFTR can inhibit MAPK/NF-κB signaling pathway to ameliorate inflammatory response and oxidative stress-induced injury of DR rats, thereby reducing retinal cell apoptosis and playing a protective role in retina.

SRY-box transcription factor 9 modulates Müller cell gliosis in diabetic retinopathy by upregulating TXNIP transcription.

Diabetic retinopathy (DR), a common complication of diabetes, involves excessive proliferation and inflammation of Muller cells and ultimately leads to vision loss and blindness. SRY-box transcription factor 9 (SOX9) has been reported to be highly expressed in Müller cells in light-induced retinal damage rats, but the functional role of SOX9 in DR remains unclear. To explore this issue, the DR rat model was successfully constructed via injection with streptozotocin (65 mg/kg) and the retinal thicknesses and blood glucose levels were evaluated. Müller cells were treated with 25 mmol/l glucose to create a cell model in vitro. The results indicated that SOX9 expression was significantly increased in DR rat retinas and in Müller cells stimulated with a high glucose (HG) concentration. HG treatment promoted the proliferation and migration capabilities of Müller cells, whereas SOX9 knockdown reversed those behaviors. Moreover, SOX9 knockdown provided protection against an HG-induced inflammatory response, as evidenced by reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in serum and decreased NLRP3 inflammasome activation. Notably, SOX9 acted as a transcription factor that positively regulated thioredoxin-interacting protein (TXNIP), a positive regulator of Müller cells gliosis under HG conditions. A dual-luciferase assay demonstrated that SOX9 could enhance TXNIP expression at the transcriptional level through binding to the promoter of TXNIP. Moreover, TXNIP overexpression restored the effects caused by SOX9 silencing. In conclusion, these findings demonstrate that SOX9 may accelerate the progression of DR by promoting glial cell proliferation, metastasis, and inflammation, which involves the transcriptional regulation of TXNIP, providing new theoretical fundamentals for DR therapy.

GRP75 Modulates Endoplasmic Reticulum-Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER-mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca2+) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER-mitochondria coupling was increased through activation of the IP3R1-GRP75-VDAC1 axis, which transferred Ca2+ from the ER to the mitochondria. Elevated mitochondrial Ca2+ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER-mitochondria coupling via the IP3R1-GRP75-VDAC1 axis and accelerated Ca2+-dependent cell apoptosis. Our results demonstrated that inhibition of ER-mitochondrial coupling, including inhibition of GRP75 or Ca2+ overload, may be a potential therapeutic target in DR.

VEGF promotes diabetic retinopathy by upregulating the PKC/ET/NF-κB/ICAM-1 signaling pathway.

Diabetic retinopathy (DR) is a common microvascular complication in patients with diabetes mellitus. DR is caused by chronic hyperglycemia and is characterized by progressive loss of vision because of damage to the retinal microvasculature. In this study, we investigated the regulatory role and clinical significance of the vascular endothelial growth factor (VEGF)/protein kinase C (PKC)/endothelin (ET)/nuclear factor-κB (NF-κB)/intercellular adhesion molecule 1 (ICAM-1) signaling pathway in DR using a rat model. Intraperitoneal injections of the VEGF agonist, streptozotocin (STZ) were used to generate the DR model rats. DR rats treated with the VEGF inhibitor (DR+VEGF inhibitor) were used to study the specific effects of VEGF on DR pathology and the underlying mechanisms. DR and DR+VEGF agonist rats were injected with the PKCβ2 inhibitor, GF109203X to determine the therapeutic potential of blocking the VEGF/PKC/ET/NF-κB/ICAM-1 signaling pathway. The body weights and blood glucose levels of the rats in all groups were evaluated at 16 weeks. DR-related retinal histopathology was analyzed by hematoxylin and eosin staining. ELISA assay was used to estimate the PKC activity in the retinal tissues. Western blotting and RT-qPCR assays were used to analyze the expression levels of PKC-β2, VEGF, ETs, NF-κB, and ICAM-1 in the retinal tissues. Immunohistochemistry was used to analyze VEGF and ICAM-1 expression in the rat retinal tissues. Our results showed that VEGF, ICAM-1, PKCβ2, ET, and NF-κB expression levels as well as PKC activity were significantly increased in the retinal tissues of the DR and DR+VEGF agonist rat groups compared to the control and DR+VEGF inhibitor rat groups. DR and DR+VEGF agonist rats showed significantly lower body weight and significantly higher retinal histopathology scores and blood glucose levels compared to the control and DR+VEGF inhibitor group rats. However, treatment of DR and DR+VEGF agonist rats with GF109203X partially alleviated DR pathology by inhibiting the VEGF/ PKC/ET/NF-κB/ICAM-1 signaling pathway. In summary, our data demonstrated that inhibition of the VEGF/ PKC/ET/NF-κB/ICAM-1 signaling pathway significantly alleviated DR-related pathology in the rat model. Therefore, VEGF/PKC/ET/NF-κB/ICAM-1 signaling axis is a promising therapeutic target for DR.

Ferrostatin-1 alleviates tissue and cell damage in diabetic retinopathy by improving the antioxidant capacity of the Xc--GPX4 system

Diabetic retinopathy (DR) is a common microvascular complication leading to a high blindness rate among patients with diabetes. Ferroptosis is a type of cell death caused by the accumulation of iron-dependent lipid peroxides. Studies have shown that ferroptosis plays an important role in DR. The rat model of DR was constructed and treated with Ferrostatin-1 (Ferr-1). Haematoxylin and eosin (HE) were used to detect the degree of retinopathy. Oxidative stress levels were detected by ELISA. Perl’s staining was used to detect iron deposition in retinal tissues. Ferritin levels were measured by ELISA. The expression of GPX4 was detected by immunohistochemistry (IHC). GSH/GSSG kit was used to detect the content and proportion of reduced/oxidized glutathione. Western blot was used to detect the expression of ferroptosis-related proteins. TUNEL assay was used to detect cell apoptosis. The expression of GSDMD was detected by fluorescence in situ hybridization (FISH). Western blot was used to detect the expression of apoptosis and pyroptosis-related proteins. Then, high glucose (HG)-induced retinal epithelial cell line ARPE-19 was treated by Erastin (ferroptosis activator) and Ferr-1. CCK-8, ELISA, western blot, flow cytometry, and immunofluorescence (IF) staining were used to detect oxidative stress levels, ferroptosis and cell damage. The mechanism was further explored by adding ferroptosis agonist Erastin. In vitro and in vivo results showed that oxidative stress was increased in DR model, resulting in ferroptosis and tissue or cell damage. After administration of Ferr-1, the antioxidant capacity was improved, ferroptosis levels were reduced and tissue or cell damage was alleviated. In vitro results showed that Ferr-1 reversed the impacts of Erastin on oxidative stress, ferroptosis, and cell damage in HG-induced ARPE-19 cells. Ferr-1 alleviated tissue and cell damage by improving the antioxidant capacity of the Xc--GPX4 system.