CD200R promotes high glucose-induced oxidative stress and damage in human retinal pigment epithelial cells by activating the mTOR signaling pathway.

Diabetic retinopathy (DR) is established as the primary cause of visual impairment and preventable blindness, posing significant social and economic burdens on healthcare systems worldwide. Oxidative stress has been identified as a major contributor to DR, yet the precise role of the transmembrane glycoprotein CD200R in this context remains elusive. We studied human retinal pigment epithelia ARPE-19 cells to investigate the role of CD200R in high-glucose (HG) induced oxidative stress. Under HG conditions, we found a significant increase in CD200R expression in a time-dependent pattern. Conversely, knockdown of CD200R effectively alleviated oxidative stress and restored cell viability in HG-treated ARPE-19 cells, a phenomenon corroborated by the addition of a reactive oxygen species (ROS) scavenger. Exploration of the AKT/mTOR signaling pathway confirmed its mediating role regarding CD200R knockdown suppression of the expression of key proteins induced by HG conditions. Additionally, we found that the inhibition of mTOR signaling with Rapamycin effectively countered HG-induced oxidative stress in ARPE-19 cells, suggesting a promising therapeutic target against oxidative stress in the context of DR. This study establishes the crucial role of CD200R in HG-induced oxidative stress and identifies potential therapeutic avenues for the treatment of DR.

Bone morphogenetic protein-6 suppresses TGF-ß2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

AIM: To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-ß2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). METHODS: Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-ß2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-ß2+empty plasmid group, BMP-6 overexpression group, and TGF-ß2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. RESULTS: Compared with the control group, the migration of RPE cells in the TGF-ß2 group was significantly enhanced. TGF-ß2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-ß2 and prevented TGF-ß2 from affecting EMT-related biomarkers (P<0.05). CONCLUSION: BMP-6 prevents the EMT in RPE cells induced by TGF-ß2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Melatonin antagonizes oxidative stress-induced apoptosis in retinal ganglion cells through activating the thioredoxin-1 pathway.

This study aimed to explore the role of melatonin in oxidative stress-induced injury on retinal ganglion cells and the underlying mechanisms. The immortalized RGC-5 cells were treated with H2O2 to induce oxidative injury. Cell viability was measured by Cell Counting Kit-8, and apoptosis was determined by flow cytometry and western blot assays. Reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels were examined to evaluate oxidative stress levels. In addition, Thioredoxin-1 (Trx1) was silenced in RGC-5 cells using small interfering RNA followed by signaling pathway examination to explore the underlying mechanisms of melatonin in alleviating oxidative injury. Melatonin pre-treatment significantly alleviated H2O2-induced apoptosis in RGC-5 cells. Melatonin also markedly reversed the upregulation of cleaved-caspase 3, cleaved-caspase 9, and Bax expression and downregulation of Bcl-2 expression induced by H2O2. Further analyses presented that melatonin significantly attenuated the increase of ROS, LDH, and MDA levels in RGC-5 cells after H2O2 treatment. Melatonin also abolished the downregulated expression of Superoxide dismutase type 1, Trx1, and Thioredoxin reductase 1, and the reduced activity of thioredoxin reductase in RGC-5 cells after H2O2 treatment. Notably, Trx1 knockdown significantly mitigated the protective effect of melatonin in alleviating H2O2-induced apoptosis and oxidative stress, while administration of compound C, a common inhibitor of c-Jun N-terminal kinase (JNK) signaling, partially reversed the effect of Trx1 silencing, thereby ameliorating the apoptosis and oxidative injury induced by H2O2 in RGC-5 cells. Melatonin could significantly alleviate oxidative stress-induced injury of retinal ganglion cells via modulating Trx1-mediated JNK signaling pathway.

Glycosides of Buyang Huanwu decoction inhibits inflammation associated with cerebral ischemia-reperfusion via the PINK1/Parkin mitophagy pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: A classic stroke formula is Buyang Huanwu Decoction (BYHWD), Glycosides are the pharmacological components found in BYHWD, which are utilized for the prevention and management of cerebral ischemia-reperfusion (CIR), as demonstrated in a previous study. Its neuroprotective properties are closely related to its ability to modulate inflammation, but its mechanism is as yet unclear. AIM OF THE STUDY: A research was undertaken to investigate the impact of glycosides on the inflammation of CIR through the PTEN-induced putative kinase-1 (PINK1)/Parkin mitophagy pathway. MATERIALS AND METHODS: Analyzing glycosides containing serum components was performed with ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Glycosides were applied to rat of Middle cerebral artery occlusion/reperfusion (MCAO/R) model and primary neural cell of Oxygen glucose deprivation/reperfusion (OGD/R) model. The neuroprotective effect and the regulation of mitophagy of glycosides were evaluated through neural damage and PINK1/Parkin mitophagy activation. Moreover, the assessment of the relationship between glycosides regulation of mitophagy and its anti-inflammatory effects subsequent to mitophagy blockade was conducted by examining neural damage, PINK1/Parkin mitophagy activation, and levels of pyroptosis. RESULTS: (1) It was observed that the administration of glycosides resulted in a decrease in neurological function scores, a reduction in cerebral infarction volume, an increase in mitochondrial autophagosome, and the maintenance of a high expression status of light chain 3 (LC3) II/LC3Ⅰ protein. Additionally, there was a significant inhibition of p62 protein expression and an enhancement of PINK1 and Parkin protein expression. Furthermore, it was found that the effect of glycosides at a dosage of 0.128 g · kg-1 was significantly superior to that of glycosides at a dosage of 0.064 g · kg-1. Notably, the neuroprotective effect and inhibition of pyroptosis protein of glycosides at a dosage of 0.128 g · kg-1 were attenuated when mitochondrial autophagy was blocked. (2) Glycosides repaired cellular morphological damage, enhanced cell survival, and reduced Lactate dehydrogenase (LDH) leakage, with glycosides (2.36 µg·mL-1 and 4.72 µg·mL-1) neuronal protection being the strongest. Glycosides (4.72 µg·mL-1) maintained LC3II/LC3Ⅰ protein high expression state, inhibited p62 protein expression, and promoted PINK1 and Parkin protein expression, which was stronger than glycosides (2.36 µg·mL-1). The blockade of mitophagy resulted in a reduction of neuroprotection and inhibition of pyroptosis protein exerted by glycosides. CONCLUSION: Glycosides demonstrate the ability to hinder inflammation through the activation of the PINK1/Parkin mitophagy pathway, thereby leading to subsequent neuroprotective effects on CIR.

Setanaxib mitigates oxidative damage following retinal ischemia-reperfusion via NOX1 and NOX4 inhibition in retinal ganglion cells.

Glaucoma, a prevalent cause of permanent visual impairment worldwide, is characterized by the progressive degeneration of retinal ganglion cells (RGCs). NADPH oxidase (NOX) 1 and NOX4 are pivotal nodes in various retinal diseases. Setanaxib, a potent and highly selective inhibitor of NOX1 and NOX4, can impede the progression of various diseases. This study investigated the efficacy of setanaxib in ameliorating retinal ischemia-reperfusion (I/R) injury and elucidated its underlying mechanisms. The model of retinal I/R induced by acute intraocular hypertension and the oxygen-glucose deprivation/reoxygenation (OGD/R) model of primary RGCs were established. By suppressing NOX1 and NOX4 expression in RGCs, setanaxib mitigated I/R-induced retinal neuronal loss, structural disruption, and dysfunction. Setanaxib reduced TUNEL-positive cells, upregulated Bcl-2, and inhibited Bax, Bad, and cleaved-caspase-3 overexpression after I/R injury in vitro and in vivo. Moreover, setanaxib also significantly reduced cellular senescence, as demonstrated by downregulating SA-ß-gal-positive and p16-INK4a expression. Furthermore, setanaxib significantly suppressed ROS production, Hif-1α and FOXO1 upregulation, and NRF2 downregulation in damaged RGCs. These findings highlight that the setanaxib effectively inhibited NOX1 and NOX4, thereby regulating ROS production and redox signal activation. This inhibition further prevents the activation of apoptosis and senescence related factors in RGCs, ultimately protecting them against retinal I/R injury. Consequently, setanaxib exhibits promising potential as a therapeutic intervention for glaucoma.

LncRNA AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cell through regulating microRNA-1199-5p/HSPA5 axis.

AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.

Upregulated CD200 in pre-retinal proliferative fibrovascular membranes of proliferative diabetic retinopathy patients and its correlation with vascular endothelial growth factor.

OBJECTIVE AND DESIGN: The objective was to determine the expression of CD200 in the pre-retinal proliferative fibrovascular membranes (PFVM) of patients with proliferative diabetic retinopathy (PDR) and to clarify its correlation with vascular endothelial growth factor (VEGF) and corresponding receptors. METHODS: PFVM samples were collected by vitrectomy from 14 patients with PDR, and 11 non-diabetic patients who accepted vitrectomy for idiopathic epiretinal membranes removal. The expression of CD200, VEGF,VEGF-R1 and VEGF-R2 was measured via qPCR and immunofluorescent staining. RESULTS: The mRNA level of CD200 was significantly higher in PDR patients than that in control patients. Meanwhile, CD200 and CD31 were found co-located and statistically associated in PFVM of PDR patients. The mRNA levels of VEGF, VEGF-R1 and VEGF-R2 were also significantly higher in PDR patients. Moreover, statistical association was found between CD200 and VEGF, VEGF-R1 in mRNA levels. But there was no significant correlationship between CD200 and VEGF-R2. CONCLUSIONS: These results suggest a significantly increased expression of CD200 in PFVM of patients with PDR and present a crucial association between CD200 and VEGF-involved pathway. It represents a potential therapy that interfering with CD200 may inhibit the VEFG expression and neovascular formation in PDR patients.

Anti-angiogenic and anti-inflammatory effects of CD200-CD200R1 axis in oxygen-induced retinopathy mice model.

OBJECTIVE: In this study, the expression changes and the potential effects of CD200 and its receptors during the process of retinal neovascularization (RNV) development had been detected, using a classic oxygen-induced retinopathy (OIR) mice model and CD200Fc (a CD200R1 agonist) intravitreal injection. MATERIALS AND METHODS: 7 day postnatal (P7) C57BL/6J mice were raised in hyperoxia incubators with 75±2% oxygen for 5 days, and returned to room air at P12. All animals were subdivided into three groups: normoxia control, OIR, and OIR+CD200Fc group. The mice of OIR+CD200Fc group were intravitreal injected with CD200Fc (2µg/µL, 0.5µL) at P12. Retinas and vitreous samples were harvested at P17. The expression and localization of CD200 and its receptors were analyzed by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and retinal whole-mount immunofluorescence. To investigate the effects of CD200Fc treatment, vascular endothelial growth factor (VEGF)-A, platelet-derived growth factor (PDGF)-BB, pro-inflammatory cytokines, NV area, and microglial activation were detected respectively. RESULTS: In OIR group, both protein and RNA levels of CD200 and CD200R1 were significantly up-regulated. The increased CD200 and CD200R1 were co-localized with Alex594-labeled Griffonia simplicifolia isolectin B4 (IB4) on vascular endothelial cells in NV area of OIR samples, and CD200R1 was co-expressed with ionized calcium-bind adapter molecule 1 (iba1) on microglia in OIR samples at the same time. CD200Fc intravitreal injection could significantly reduce the release of VEGF-A, PDGF-BB, and pro-inflammatory cytokines; shrink the NV area; and inhibit the activation of microglia in OIR mice. CONCLUSION: These findings suggested that the up-regulation of CD200 and CD200R1 was closely related to RNV development, and the antiangiogenic effects of CD200Fc in OIR model might be realized by inhibition of inflammatory response and microglia activation. The results may provide a new therapeutic target for RNV diseases.

Salubrinal attenuated retinal neovascularization by inhibiting CHOP-HIF1α-VEGF pathways.

Retinal neovascularization (RNV) related disease is the leading cause of irreversible blindness in the world. The aim of this study is to identify whether salubrinal could attenuate RNV by inhibiting CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP)- hypoxia inducible factors 1α (HIF1α) -vascular endothelial growth factor (VEGF) pathways in both mouse retinal microvascular endothelial cells (mRMECs) and oxygen-induced retinopathy (OIR) mouse model. After being treated with salubrinal (20µmol/L) or CHOP-siRNA, mRMECs were exposed to a hypoxia environment. OIR mice were intraperitoneally injected with salubrinal (0.5 mg/kg/day) from P12 to P17. With salubrinal or CHOP-siRNA treatment, the elevated CHOP protein and mRNA levels in hypoxia-induced mRMECs were significantly decreased. HIF1α-VEGF pathways were activated under hypoxia condition, then HIF1α protein was degraded and VEGF secretion was down-regulated after salubrinal or CHOP-siRNA treatment. In OIR mice, the areas of RNV were markedly decreased with salubrinal treatment. Moreover, elevated expressions of CHOP, HIF1α and VEGF in retinas of OIR mice were all reduced after salubrinal treatment. It suggested that salubrinal attenuated RNV in mRMECs and OIR mice by inhibiting CHOP-HIF1α-VEGF pathways and could be a potential therapeutic target for hypoxia-induced retinal microangiopathy.

Secretion of Down Syndrome Critical Region 1 Isoform 4 in Ischemic Retinal Ganglion Cells Displays Anti-Angiogenic Properties Via NFATc1-Dependent Pathway.

Down syndrome candidate region 1 (DSCR1) has two differentially regulated isoforms (DSCR1-1 and DSCR1-4) and is reported to play a role in a number of physiological processes, such as the inhibition of cardiac hypertrophy, attenuation of angiogenesis and carcinogenesis, and protection against neuronal death. However, the function of DSCR1 in the retina is still not clear. Therefore, we analyzed the expression and location of DSCR1 in the retina of neonatal mice with oxygen-induced retinopathy (OIR), and studied its effects on angiogenesis. The neonatal C57BL/6J mice were exposed to 75 % O2 for 5 days from postnatal day 7 (P7) to P12. At P12, the mice were returned to 21 % O2 at room air. The primary retinal ganglion cells (RGCs) were exposed to hypoxia (93 % N2, 5 % CO2, and 2 % O2) at 37 °C for 36 h. And then the mouse retinal microvascular endothelial cells (mRMECs) were treated with 25 ng/mL vascular endothelial growth factor (VEGF) or culture medium conditioned by hypoxic RGCs alone, hypoxic RGCs treated with DSCR1-4-siRNA (siDSCR1-4) or hypoxic RGCs treated with siDSCR1-4 and 200 ng/mL cyclosporin A (CsA), and then primed with VEGF (25 ng/mL). The expression of DSCR1-4 increased strongly at P16 after OIR. There was no change in messenger RNA (mRNA) expression of DSCR1-1 at P16 after OIR. The increased DSCR1 was mainly located in the RGCs of avascular retina. In addition, DSCR1-4 expression was increased in primary RGCs after hypoxia exposure. There was no change in mRNA expression of DSCR1-1 in primary RGCs after hypoxia exposure. Moreover, DSCR1-4 produced by hypoxic RGCs showed anti-angiogenic properties, with decreased cell proliferation, migration, tube formation, and inflammatory cytokines production. These properties were due to inhibited nuclear factor of activated T cell (NFATc) 1 dephosphorylation and translocation into nuclear in VEGF-treated mRMECs. Using siRNA-mediated knockdown of DSCR1-4 and NFATc1 inhibitor (Cs A) further demonstrated the inhibitory effect of DSCR1-4 on angiogenic properties in VEGF-induced mRMECs, and this effect was NFATc1-dependent. This report describes a novel effect of DSCR1-4 in the aspect of anti-angiogenesis, suggesting potential therapeutic strategies for proliferative retinopathies.

Increased sCD200 Levels in Vitreous of Patients With Proliferative Diabetic Retinopathy and Its Correlation With VEGF and Proinflammatory Cytokines.

PURPOSE: The purpose of this study was to determine the levels of sCD200 expression in the vitreous of proliferative diabetic retinopathy (PDR) patients and to clarify its correlation with different vitreoretinal conditions, VEGF and its receptors, and proinflammatory cytokines. METHODS: The expression of sCD200, VEGF and its receptors, and other proinflammatory cytokines were examined by using ELISA. Clinical stratification was performed on patients with different vitreoretinal conditions for correlation analysis. RESULTS: The vitreous levels of sCD200 were significantly higher in the PDR group (182.2 ± 17.63 pg/mL) compared with those in the control group (56.86 ± 6.573 pg/mL; P < 0.0001). The venous blood levels of sCD200 were 26.71 ± 4.32 pg/mL in the PDR group and 19.94 ± 3.87 pg/mL in the control group (P = 0.2614). The vitreous levels of sCD200 were significantly elevated in PDR patients with diabetic macular edema (DME; 266.9 ± 28.82 pg/mL) or traction retinal detachment (TRD; 256.9 ± 34.50 pg/mL) compared with the PDR group without DME (136.9 ± 15.13 pg/mL; P < 0.0001) or TRD (146.9 ± 15.97 pg/mL; P = 0.0024). The vitreous levels of CCL2, CXCL4, CXCL9, CXCL10, VEGF, sVEGFR-1, sVEGFR-2, IL-6, IL-8, IL-10, and IL-18 were also elevated significantly in the PDR group. Statistical association was found between sCD200 levels and VEGF (r = 0.6566, P < 0.0001), sVEGFR-1 (r = 0.5574, P = 0.006), sVEGFR-2 (r = 0.3605, P = 0.0362), CCL-2 (r = 0.6001, P = 0.0002), IL-6 (r = 0.5704, P = 0.0004), IL-8 (r = 0.3712, P = 0.0307), and IL-10 (r = 0.3618, P = 0.0355). CONCLUSIONS: Expression of sCD200 may contribute to retinal angiogenesis by interacting with VEGF-mediated inflammatory response and represents a potential therapeutic target for the patients with PDR.

The expression changes of myelin and lymphocyte protein (MAL) following optic nerve crush in adult rats retinal ganglion cells.

Myelin and lymphocyte protein (MAL), a component of compact myelin, is highly expressed in oligodendrocytes and Schwann cells. It has been reported that MAL may play a vital role in the process of neuronal apoptosis following acute spinal cord injury. However, acquaintance regarding its distribution and possible function in the retina is limited. Therefore, in a rodent model of optic nerve crush (ONC), the dynamic changes of MAL in retina was detected. The expression of MAL was mainly located in the retinal ganglion cells (RGCs) and was increased strongly after ONC. The peak of MAL expression appeared on the third day. In addition, there was a concomitant upregulation of active-caspase-3, which also co-localized with MAL in RGCs. Moreover, co-localization of MAL with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) was detected in RGCs after ONC. Collectively, all these results suggested that the upregulation of MAL might play an important role in the pathophysiology of RGCs after ONC.