RGC-5 Cells Research Articles

Inflammation and apoptosis, two key events induced by hyperglycemia mediated reactive nitrogen species in RGC-5 cells

Nitrosative stress plays a critical role in retinal injury in high glucose (HG) environment of eye, but the mechanisms remain poorly understood. Here we tested the hypothesis that HG induced reactive nitrogen species (RNS) production acts as a key functional mediator of antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis. Our findings illustrated that exposure of cultured RGC-5 cells to HG significantly disrupts the antioxidant defense mechanism and mitochondrial machineries by increasing the loss of mitochondrial membrane potential (ΔѰM) and elevating mitochondrial mass. Furthermore, we used biochemical tools to analyze the changes in metabolites, sulfur amino acids (SAAs) such as L-glutathione (GSH) and L-cysteine (Cys), in the presence of HG environment. These metabolic changes were followed by an increase in glycolytic flux that is phosphofructokinase-2 (PFK-2) activity. Moreover, HG exposure results in a significant disruption of protein carbonylation (PC) and lipid peroxidation (LPO), downregulation of OGG1 and increase in 8-OHdG accumulations in RGC-5 cells. In addition, our results demonstrated that HG environment coinciding with increased expression of inflammatory mediators, cell cycle deregulation, decreased in cell viability and expression of FoxOs, increased lysosomal content leading to apoptosis. Pre-treatment of selective inhibitors of RNS significantly reduced the HG-induced cell cycle deregulation and apoptosis in RGC-5 cells. Collectively, these results illustrated that accumulated RNS exacerbates the antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis induced by HG exposure in RGC-5 cells. Treatment of pharmacological inhibitors attenuated the HG induced in retinal cells.

Carboxymethylated chitosan alleviated oxidative stress injury in retinal ganglion cells via IncRNA-THOR/IGF2BP1 axis.

Glaucoma is an advanced nerve disorder described by the deterioration of axon and RGCs. CMCS has been previously used as an anti-apoptotic and anti-oxidant agent. The current study aimed to explore the protective impact of CMCS against H2O2-induced injury in glaucoma in vitro. The relative expression of lncRNA THOR and the protein expression of IGF2BP1 in H2O2-induced RGC-5 cells were detected by RT-PCR and western blot methods respectively. The cell viability was measured using MTT assay while apoptosis rate was measured by flow cytometry. Moreover, ROS level was measured using ROS assay kit. Furthermore, the relations between THOR and IGF2BP1 were determined by using RNA pull-down. The expression of THOR was reduced in H2O2-induced RGCs. Also, RGCs viability was inhibited while the level of ROS and cell apoptosis were enhanced. CMCS treatment considerably enhanced the expression of THOR and IGF2BP1 protein and cell viability but reduced ROS level and cell apoptosis. Moreover, IGF2BP1 protein was positively regulated by lncRNA THOR. CMCS protected the RGCs from oxidative stress via regulating lncRNA THOR/IGF2BP1. CMCS enhanced the cell viability and reduced the cell apoptosis and ROS level and protected RGCs from oxidative stress via lncRNATHOR/IGF2BP1 pathway, potentially suggesting a new therapeutic strategy for the treatment of glaucoma.

CS nerve growth factor regulates sevoflurane anesthesiainduced nerve injury in nerve cells

Purpose: Sevoflurane is the most commonly used anesthetic agent for surgery. However, it is associated with deficiency in learning and memory abilities. The study was aimed at investigating the role of nerve growth factor (NGF) in sevoflurane anesthesia-induced nerve injury.Methods: RT-qPCR assay was applied to measure expressions of NGF, miR-98-5p and other factors related to apoptosis. CCK-8 assay was used for detecting cell viability while luciferase reporter assay was employed to measure binding condition between miR-98-5p and NGF. Expressions of proteins in PI3K/AKT/mTOR signaling pathway was measured with western blot.Results: Sevoflurane reduced cell viability of RGC-5 cells, promoted apoptosis and reduced the expression of NGF. In sevoflurane-induced RGC-5 cells, over-expression of NGF promoted cell viability with reduced apoptosis. Also, there was reduction in the protein expression of PI3K/AKT/mTOR signaling pathway by sevoflurane, while up-regulation of NGF promoted the expressions of these proteins. In the presence of PI3K inhibitor, reduction cell viability was reduced but apoptosis increased. Luciferase reporter assay detected MiR-98-5p as the target gene of NGF and its overexpression restored high cell viability in the over-expressed NGF. The rate of apoptosis and expressions of proteins was also restored with up-regulation of miR-98-5p.Conclusion: Sevoflurane caused damage to nerve cells, while over-expression of NGF reduced the injury through PI3K/AKT/mTOR signaling pathway and suppression of miR-98-5p.
 Keywords: Nerve growth factor, Sevoflurane, Nerve injury, Anesthesia, miR-98-5p

Neuroprotective Role of GLP-1 Analog for Retinal Ganglion Cells via PINK1/Parkin-Mediated Mitophagy in Diabetic Retinopathy.

GLP-1 analogs have been widely used to treat patients with type 2 diabetes in recent years and studies have found that GLP-1 analogs have multiple organ benefits. However, the role of GLP-1 analogs in diabetic retinopathy (DR), a common complication of diabetes mellitus (DM), remains controversial. Retinal ganglion cells (RGCs) are the only afferent neurons responsible for transmitting visual information to the visual center and are vulnerable in the early stage of DR. Protection of RGC is vital for visual function. The incretin glucagon-like peptide-1 (GLP-1), which is secreted by L-cells after food ingestion, could lower blood glucose level through stimulating the release of insulin. In the present study, we evaluated the effects of GLP-1 analog on RGCs both in vitro and in vivo. We established diabetic rat models in vivo and applied an RGC-5 cell line in vitro. The results showed that in high glucose conditions, GLP-1 analog alleviated the damage of RGCs. In addition, GLP-1 analog prevented mitophagy through the PINK1/Parkin pathway. Here we demonstrated the neuroprotective effect of GLP-1 analog, which may be beneficial for retinal function, and we further elucidated a novel mechanism in GLP-1 analog-regulated protection of the retina. These findings may expand the multi-organ benefits of GLP-1 analogs and provide new insights for the prevention of DR.

Resveratrol acts via the mitogen-activated protein kinase (MAPK) pathway to protect retinal ganglion cells from apoptosis induced by hydrogen peroxide

ABSTRACT The current study investigated the ability of resveratrol to protect RGC-5 retinal ganglion cells in culture against H2O2-induced apoptosis and the underlying mechanism of protection. RGC-5 cells were pre-exposed to resveratrol (5, 10, or 20 μM), followed by 200 μM H2O2. Cell viability and apoptosis were detected to assess the cell growth, and expression levels of apoptosis-related and MAPK cascade-associated proteins were determined using western blotting. Levels of reactive oxygen species and mitochondrial membrane potential were also tested, as well as the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GSH). At a concentration-dependent way, resveratrol reversed H2O2-induced increases in expressions of cleaved caspase-3 and cleaved caspase-9, production of ROS, loss of mitochondrial membrane potential and the expressions of p-p38, p-ERK, and p-JNK. It also promoted the activities of SOD, CAT, and GSH. Furthermore, the agonists of p38, ERK, and JNK partially weakened the protective effects of resveratrol against H2O2-induced apoptosis in RGC-5 cells. Thus, resveratrol can protect retinal ganglion cells against H2O2-induced apoptosis by suppressing MAPK cascades. The drug therefore shows potential for preventing glaucoma.

Ligustrazine induces viability, suppresses apoptosis and autophagy of retinal ganglion cells with ischemia/reperfusion injury through the PI3K/Akt/mTOR signaling pathway

ABSTRACT Ligustrazine, an alkaloid monomer extracted from Chuanxiong Rhizoma, has the function of protecting nerve cells. However, the effect and mechanism of ligustrazine on retinal ischemia/reperfusion (I/R) injury still need to be clarified. In our study, retinal ganglion cells (RGC-5) were used to establish a retinal I/R injury model by anaerobic cultivation. Cell viability, autophagy, and apoptosis were evaluated by cell counting kit 8 assay, transmission electron microscopy, and TUNEL staining after treatment with ligustrazine, PI3K inhibitor Ly294002, and/or mTOR inhibitor rapamycin, respectively. Besides, the levels of PI3K/Akt/mTOR pathway and autophagy-related proteins were determined by western blot. Moreover, one-way ANOVA was adopted for inter-group comparisons of measurement data. Our results demonstrated that low-concentration ligustrazine significantly enhanced cell viability and suppressed cell autophagy and apoptosis of RGC-5 cells after I/R injury, suggesting the protective effect of low-concentration ligustrazine on retinal I/R injury. Moreover, the alleviating effect of ligustrazine on RGC-5 with retinal I/R injury was mechanistically associated with the activation of the PI3K/Akt/mTOR pathway. In conclusion, low-concentration ligustrazine has a significant protective effect on RGC-5 cells with retinal I/R injury by activating the PI3K/Akt/mTOR pathway.

Artemisinin Inhibits the Migration and Invasion in Uveal Melanoma via Inhibition of the PI3K/AKT/mTOR Signaling Pathway.

Uveal melanoma is the most common primary ocular neoplasm in adults, with many patients ending up developing liver metastasis and facing a significant reduction of their life expectancy due to the lack of efficient treatments. Artemisinin is an antimalarial drug that has been widely used in the clinic and whose anticancer properties have also been described. Its reported safety, affordability, and ability to reach the ocular tissues point that it has a potential therapeutic agent against uveal melanoma. In the present study, we found that a subantimalaria dosage of artemisinin significantly attenuated the migration and invasion potential of uveal melanoma cells, in a concentration-dependent manner. Assessment of the mechanisms underlying artemisinin anticancer action revealed that its use dramatically reduced the phosphorylation of PI3K, AKT, and mTOR in UM cells. Further inhibition of PI3K signaling, using LY294002, or of mTOR, by rapamycin, blocked the migration and invasion of UM cells similarly to artemisinin. In contrast, AKT or mTOR activator (Sc79 and MHY1485, respectively) attenuated the inhibitory effect of artemisinin on the migration and invasion abilities of UM cells, further validating that artemisinin's anticancer effect is likely to be mediated via inhibition of the PI3K/AKT/mTOR pathway. Artemisinin also induced mitochondrial membrane potential loss and apoptosis of UM cells, having no significant toxic effect on normal retinal neuronal cells RGC-5 and epithelial cells D407. These findings and the reported safety of artemisinin's clinical dosage strongly suggest the therapeutic potential of artemisinin in the prevention and treatment of uveal melanomas.

Emodin protected against retinal ischemia insulted neurons through the downregulation of protein overexpression of \u03b2-catenin and vascular endothelium factor

BackgroundEmodin has been proved to have an anti-ischemic effect on the brain, however little research has been done on its effect on vision-threatening retinal ischemia. Thus, an investigation was carried out into the hypothetical efficacy of emodin against retinal ischemia and the role of β-catenin/VEGF in its therapeutic mechanism.MethodsRetinal ischemia, followed by reperfusion (IR), was inducted by raising the intraocular pressure of a Wistar rat’s eye to 120 mmHg for 60 min. Additionally, pre-ischemic/post-ischemic intravitreous injections of emodin (4, 10 and 20 μM) or vehicle were carried out on the eye with retinal ischemia. MTT assay, electroretinograms, cresyl violet staining retinal thickness measurements, and fluorogold retrograde labelling of retinal ganglion cells (RGCs) as well as Western blotting were carried out.ResultsCultured RGC-5 cells subjected to oxygen glucose deprivation (OGD) were used to confirm the effective concentrations of emodin (administered 1 h pre-OGD, pre-OGD emodin). The most effective and significant (P = 0.04) dose of pre-OGD emodin was observed at 0.5 μM (cell viability: 47.52 ± 3.99%) as compared to pre-OGD vehicle treatment group (38.30 ± 2.51%). Furthermore, pre-ischemic intravitreous injection of 20 μM emodin (Emo20 + IR = 0.99 ± 0.18, P < 0.001) significantly attenuated the ischemia induced reduction in ERG b-wave amplitude, as compared to pre-ischemic intravitreous vehicle (Vehicle+IR = 0.04 ± 0.02). Post-ischemic intravitreous 20 μM emodin also significantly (P < 0.001) attenuated the ischemia associated b-wave reduction (IR + Em20 = 0.24 ± 0.09). Compared with pre-ischemic intravitreous vehicle (Vehicle+IR; whole retina thickness = 71.80 ± 1.08 μm; inner retina thickness = 20.97 ± 0.85 μm; RGC =2069.12 ± 212.82/0.17mm2), the significant (P < 0.001) protective effect was also present with pre-ischemic administration of emodin. This was shown by observing cresyl violet stained retinal thickness (Emo20 + IR: whole retina = 170.10 ± 0.10 μm; inner retina = 70.65 ± 2.06 μm) and retrograde fluorogold immunolabeled RGC density (4623.53 ± 179.48/0.17mm2). As compared to the normal control (the ratio of β-catenin/VEGF to β-actin was set as 1 in the Sham group), the β-catenin/VEGF protein level significantly (P < 0.001) increased after retinal ischemia and when pre-ischemic intravitreous vehicle (Vehicle+IR = 1.64 ± 0.14/7.67 ± 2.57) was carried out. However, these elevations were significantly (P = 0.02) attenuated by treatment with emodin 20 μM (Emo20 + IR = 1.00 ± 0.19/1.23 ± 0.44).ConclusionsThe present results suggest that emodin might protect against retinal ischemia insulted neurons such as RGCs by significantly downregulating the upregulation of β-catenin/VEGF protein that occurs during ischemia.

Gadd45α affects retinal ganglion cell injury in chronic ocular hypertension rats by regulating p38MAPK pathway.

To investigate the impact and the mechanism of Gadd45α mediating p38MAPK pathway on the retinal ganglion cells (RGCs) injury in chronic ocular hypertension (COH) rats. COH model in rats were established and intraocular pressure (IOP) was tested. Retrograde labeling was used for counting RGCs and TUNEL staining was performed for RGCs apoptosis. Western Blotting was conducted to examine the expression of Gadd45α and p38MAPK pathway. Besides, RGC-5 cells cultured in vitro were treated with H2O2. Cell viability was detected by CCK-8, ROS level tested by DCFH-DA assay, and cell apoptosis examined by flow cytometry. COH rats had increased expression of Gadd45α and p-p38/p38 protein 1-4weeks after surgery. Rats in COH group enhanced obviously in IOP, RGC apoptosis rate and the protein expression of Gadd45α, p-p38/p38, Bax/Bcl-2 and cleaved caspase-3, but declined appreciably in RGC counting. However, the above indicators of COH rats were effectively improved by Gadd45α shRNA treatment. Additionally, RGC-5 cells in H2O2 group reduced in cell viability and went up in ROS level and apoptosis rate. The H2O2-induced RGC-5 cells treated with Gadd45α shRNA were improved apparently in those indicators, and cells treated with pcDNA Gadd45α showed an opposite trend. Moreover, p38 MAPK inhibitor SB203580 can effectively reverse the damage of pcDNA Gadd45α from H2O2-induced RGC-5 cells. Silencing Gadd45α can reduce the RGC damage in COH rats by inhibiting p38MAPK pathway and such a protective role may be associated with the suppression of RGC apoptosis and the mitigation of oxidative stress.

Synergistic protection of RGCs by olfactory ensheathing cells and alpha-crystallin through regulation of the Akt/BAD Pathway

Our recent in vivo studies have shown that olfactory ensheathing cells (OECs) and α-crystallin can promote retinal ganglion cell (RGC) survival and axonal regeneration synergistically after optic nerve injury. However, the mechanism is still unknown. Here, we studied the synergistic effect and mechanism of OECs and α-crystallin on RGC survival after H2O2-induced oxidative damage and a crushing injury to the optic nerve in an adult rat model. After H2O2-induced oxidative damage, RGC-5 cells were treated with OECs, α-crystallin or a combination of OECs and α-crystallin. Apoptosis of RGC-5 cells was assessed by flow cytometry. Phosphorylated Akt, BAD, and cleaved-caspase3 were detected by Western blot after optic nerve injury in vivo and H2O2-induced RGC-5 oxidative damage in vitro. The results showed that OECs and α-crystallin could both independently inhibit RGC-5 apoptosis (P<0.01), increase the phosphorylation of both Akt and BAD, and decrease the activation of caspase-3 (P<0.01). However, the effect of the combination of both was more significant than either alone. These findings indicate that inhibition of superoxide damage to RGCs through regulation of the Akt/BAD pathway is one of the mechanisms by which OECs and α-crystallin promote optic nerve recovery after injury.

High glucose-induced ROS accumulation is a critical regulator of ERK1/2-Akt-tuberin-mTOR signalling in RGC-5 cells

Hyperglycemia and oxidative stress are the primary stressors that elicit mitochondria specific cell stress in diabetes. Here we hypothesized that elevated level of ROS in high glucose (HG) environment, trigger mitochondrial stress by damaging mitochondrial DNA (mtDNA), altering inflammatory mediators, and neurodegenerative markers via stress signalling pathway in retinal ganglion cells (RGC-5). Mechanistically, our findings illustrated that the HG environment increases the ROS production in retinal cells leading to the disruption of antioxidant defence mechanism, and altering mitochondrial machinery such as an increase in loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, and increase in mtDNA fragmentation. Furthermore, fragmented mtDNA escape from mitochondria into the cytosol, where it engaged with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN gene (STING) phosphorylation and activate interferon regulatory factor 3 (IRF3) via ERK1/2-Akt-tuberin-mTOR dependent pathways. Our results further indicate that siRNA-mediated gene silencing of tuberin suppresses the strong downregulation of tuberin-mTOR-IRF3 activation. HG environment resulted in activation of IRF3, coinciding with the increased expression of inflammatory mediators and neurodegenerative markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in RGC-5 cells significantly reduced the HG-induced IRF3 expression and declined the expression of neurodegenerative markers. Collectively, our results demonstrates that HG-induced over production of ROS, disrupts the antioxidant defence mechanism and mitochondrial dysfunction, leading to alterations of inflammatory mediators and neurodegenerative markers through the ERK1/2-Akt-tuberin-mTOR dependent signalling pathway in RGC-5 cells.

Downregulation of miR-145-5p elevates retinal ganglion cell survival to delay diabetic retinopathy progress by targeting FGF5.

Diabetic retinopathy (DR) is a leading cause of new-onset blindness. Recent studies showed that protecting retinal ganglion cells (RGCs) from high glucose-induced injury is a promising strategy for delaying DR. This study is to investigate the role of miR-145-5p in high glucose-induced RGC injury. Here, RGCs were randomly divided into low glucose and high glucose groups. PCR assay showed miR-145-5p was significantly upregulated in high glucose group. Transfection of miR-145-5p inhibitor decreased pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) levels, elevated cell viability and proliferation, as well as suppressed cell apoptosis by ELISA, MTT, EdU proliferation, colony formation and flow cytometry assays, respectively. Moreover, dual-luciferase reporter assay confirmed FGF5 as a target gene of miR-145-5p. FGF5 knockdown could partially reverse the protective effects of miR-145-5p on RGC-5 cells. In conclusion, our results demonstrated that inhibition of miR-145-5p might be a neuroprotective target for diabetes mellitus-related DR. Abbreviations: DR: diabetic retinopathy; RGCs: retinal ganglion cells; miR-145-5p: microRNA-145-5p; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; FGF: fibroblast growth factor; ATCC: American Type Culture Collection; WT: wild type; MUT: mutant type.

Protective effects of hypercapnic acidosis on Ischemia-reperfusion-induced retinal injury.

Ischemia–reperfusion (I/R) injury is associated with numerous retinal diseases, such as diabetic retinopathy, acute glaucoma, and other vascular retinopathies. Hypercapnic acidosis (HCA) has a protective effect on lung, myocardial, and central nervous system ischemic injury models. However, no study has evaluated its protective effects in an experimental retinal I/R injury model. In this study, retinal I/R injury was induced in Sprague Dawley rats by elevating the intraocular pressure to 110 mmHg for 60 minutes. HCA was induced before and after the injury. After 24 hours, the terminal dUTP nick end labeling assay was performed. Moreover, the ratios of cleaved caspase-3/total caspase-3, phosphorylated IκB/IκB, and phosphorylated p38 were measured through Western blotting. After 7 days, the rats’ aqueous humor was analyzed. In addition, electroretinography and retinal thickness measurement were performed in the rats. Moreover, the retinal neural cell line RGC-5 was exposed to 500 μM H2O2 for 24 hours to induce a sustained oxidative stress in vitro. The effects of HCA were evaluated by comparing oxidative stress, MAPK signals, NF-κB signals, survival rates, and apoptosis rates in the RGC-5 cells before and after H2O2 exposure. We further investigated whether the potent I/R-protective heat shock protein (HSP) 32 contribute to protective effects of HCA. Our results indicated that HCA has protective effects against retinal I/R injury both in vivo and in vitro, at multiple levels, including antiapoptotic, anti-inflammatory, antioxidative, and functional retinal cell protection. Further research clarifying the role of HCA in retinal I/R injury prevention and treatment is warranted.

LncRNA-MEG3 protects against ganglion cell dysplasia in congenital intestinal atresia through directly regulating miR-211-5p/GDNF axis

Background LncRNAs are known to take part in normal brain functions and nervous system diseases. Little evidence has pointed to the dysregulation of lncRNAs in congenital intestinal atresia. We aimed to investigate the underlying molecular mechanism of congenital intestinal atresia that involves in lncRNA-MEG3.Materials and methods The expressions of LncRNA-MEG3, miR-211-5p and GDNF were determined by the qRT-PCR and Western blot assay when appropriate. The results were verified in intestinal atresia Wistar rat model and bone marrow derived stem cell (BMSCs)-derived into intestinal ganglion cells. RNA immunoprecipitation and RNA pull-down assays were performed to analyze the regulatory mechanism between MEG3 and miR-211-5p. The effects of MEG3 on the cell proliferation and apoptosis of isolated intestinal ganglion cells were detected with an MTT assay and flow cytometry, respectively.Results The expression of MEG3 was detected to be declined in congenital intestinal atresia tissues at clinic and animal levels. MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and regulated GDNF expression in retinal ganglion cells (RGC-5 cells) via targeting miR-211-5p. Hypoxia induced the apoptosis of intestinal ganglion cells via MEG3/miR-211-5p/GDNF axis.Conclusion MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and inhibited the apoptosis of intestinal ganglion cells under the exposure of hypoxia to protect against CIA injury via directly regulating miR-211-5p/GDNF axis.

Expression of P2X7 receptor in RGC-5 cell line and selection of an effective siRNA sequence downregulating P2X7 receptor

Objective This study aims to investigate the gene expression of P2X7 receptor in retinal ganglion cell line (RGC-5), and to design and select the small interfering RNA (siRNA) which can specifically and effectively downregulate P2X7 receptor expression. Methods The mRNA and protein expressions of P2X7 receptor in RGC-5 cells were examined by real-time polymerase chain reaction (qRT-PCR), Western blot and Immunofluorescence, and compared with those of mouse retina and other tissues. Three P2X7 receptor-specific double stranded siRNAs (P2X7-siRNA-1-3) were designed and delivered into RGC-5 cells by riboFECT™CP. 60 hours after transfection, the expression level of P2X7 receptor was analyzed by qRT-PCR, Western blot and Immunofluorescence. Results The expression of P2X7 receptor was detected in both RGC-5 cell line and mouse retina, and the receptor was mainly expressed in the nucleus and cytoplasm of RGC-5 cells. P2X7-siRNA-3 can effectively down-regulate the mRNA and protein expression of P2X7 at the same time. Conclusions RGC-5 cell line can serve as a good model for studying the biological function of P2X7 receptor in retinal ganglion cells in vitro. We have managed to design and synthesize the siRNA pair that specifically and effectively lowering P2X7 receptor expression. The current study has laid the foundation for future investigations on treating glaucomatous retinopathy by downregulation of P2X7 receptor. Key words: Receptors, purinergic P2X7; Retinal ganglion cells; RNA, small interfering; In vitro

Transactive response DNA binding protein of 43/histone deacetylase 6 axis alleviates H 2 O 2 -induced retinal ganglion cells injury through inhibiting apoptosis and autophagy.

Oxidative damage is believed to contribute to the pathogenesis of diabetic retinopathy (DR). The current study aimed to detect the effects of transactive response DNA binding protein of 43 (TDP-43) on cell damage induced by hydrogen peroxide (H2 O2 ) in retinal ganglion cells (RGCs) and to investigate the molecular mechanisms involved in this process. We observed that TDP-43 was highly expressed in RGC-5 cells induced by H2 O2 , and that repression of TDP-43 obviously ameliorated H2 O2 -induced RGC-5 cell injury. In addition, loss of TDP-43 profoundly mitigated H2 O2 -triggered oxidative stress by decreasing the production of intracellular reactive oxygen species and the activity of oxidative stress indicator malondialdehyde, as well as enhancing the content of antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase to restore the antioxidant defense system. Moreover, suppression of TDP-43 obviously obstructed H2 O2 -induced apoptosis. Meanwhile, knockdown of TDP-43 attenuated the expression of the proapoptotic proteins Bax and Cytochrome c, elevated the anti-apoptotic protein Bcl-2, and suppressed the activation of caspase 3 in H2 O2 -induced RGC-5 cells. Moreover, elimination of TDP-43 inhibited H2 O2 -triggered autophagy, which appeared as decreased expression of LC3II/I and Beclin-1, along with p62 degradation. Importantly, silencing of TDP-43 diminished the expression of histone deacetylase 6 (HDAC6), and HDAC6 also abolished the inhibitory effect of TDP-43 inhibition on H2 O2 -induced apoptosis and autophagy. Collectively, our findings demonstrated that depletion of TDP-43 may protect RGC-5 cells against oxidative stress-mediated apoptosis and autophagy by suppressing its target HDAC6. Thus, the TDP-43/HDAC6 axis might be a promising strategy for the treatment of DR.

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells.

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2(E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.

Down-regulated miR-187 promotes oxidative stress-induced retinal cell apoptosis through P2X7 receptor

Several microRNAs (miRNAs) expressed in the retina were confirmed to involve in retinal cell apoptosis, which was closely linked with the development of retinal diseases. Our previous studies have confirmed a vital role of miR-187 in retinal cells apoptosis. The aim of this study was to further elucidate the precise role of miR-187 and its probable mechanisms in RGC-5 cells apoptosis. The cellular oxidative stress status was assessed by reactive oxygen species (ROS) production and malondialdehyde (MDA) level. Our results showed that the elevated pressure, glutamate and H2O2-induced oxidative stress in RGC-5 cells was accompanied by a decrease in miR-187 expression and an increase in P2X7R expression. However, overexpression of miR-187 reversed this activation of oxidative stress in RGC-5 cells. Moreover, we also revealed that miR-187 inhibited the oxidative stress-induced apoptosis of RGC-5 cells through negative regulating P2X7R, probably through interacting with the 3′UTR of P2X7R. Finally, we confirmed that the forced miR-187 expression alleviated oxidative stress injury in retina tissues of rat models with chronic ocular hypertension. Our data demonstrated that miR-187/P2X7R signaling was involved in retinal cell apoptosis, at least in part, through activating oxidative stress.

Cytotoxic effect of interleukin-8 in retinal ganglion cells and its possible mechanisms

To investigate the effect of interleukin-8 (IL-8) on neural retinal ganglion cells (RGCs) and whether it can be alleviated by G31P. RGC-5 cells were exposed to IL-8 with or without its specific receptor antagonist G31P for 24h, and the cell viability was assessed by Cell Counting Kit 8 (CCK-8). Apoptosis was measured by examining nuclear morphology and quantifying with flow cytometry. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were used to investigate the expression of apoptosis-related genes. CCK-8 assay showed that IL-8 significantly inhibits the viability of RGC-5 cells in a dose-dependent manner. Cell apoptosis assays exhibited higher apoptotic rate in IL-8 treatment group compared to control group. We further found that IL-8 could promote Bax and caspase-3 expressions, but decrease the level of Bcl-2 in the aspect of mRNA and protein. However, pre-treatment with G31P partly attenuated these effects in RGC-5 cells (P<0.05). These results indicate that anti-proliferation effects of IL-8 through induction of cell apoptosis regulated by Bcl-2, Bax and caspase-3 expressions, can be ameliorated by G31P.

Intraocular miR-211 exacerbates pressure-induced cell death in retinal ganglion cells via direct repression of FRS2 signaling

MicroRNAs (miRNAs) are emerging as important regulators of neurodegenerative diseases. However, research into ocular intracellular miRNAs, though possessing great potential to uncover novel and valuable therapeutic targets for glaucoma, is only at an early stage. Here we show that expression levels of miR-211 were significantly induced in aqueous humor (AH) samples from patients with glaucoma when compared to normal AH samples. Notably, oxidative stress-elicited miR-211 potentiated high pressure-induced retinal ganglion cells (RGCs) death by impairing ERK activation but enhancing P38 activation, an effect mediated by direct downregulation of fibroblast growth factor receptor substrate 2 (Frs2) signaling pathway in a sequence complementarity-dependent fashion. In support of the concept of a fundamental connection between miR-211 upregulation and augmented pressure-induced cell death in RGCs, we showed that ectopic expression of the exogenous FRS2 was sufficient to neutralize the miR-211-induced decrease in the cell viability and significantly improved miR-211-inhibited cell survival, in high pressure-challenged RGC-5 cells. Together, our findings show that miR-211 negatively modulates cell survival programs upon high pressure challenge by regulating Frs2 signaling, and they define roles for miR-211/Frs2 cascade in a regulatory feedback loop that mediates the pathogenesis of glaucoma.