Oxidative Stress In ARPE-19 Cells Research Articles

Extracellular Vesicles Derived from Adipose-Derived Mesenchymal Stem Cells Alleviate Apoptosis and Oxidative Stress of Retinal Pigment Epithelial Cells Through Activation of Nrf2 Signaling Pathway.

Purpose: To examine the potential protective effects of adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASC-EVs) on ARPE-19 cells exposed to hydrogen peroxide (H2O2) stress and to evaluate their ability to delay retinal degeneration in Royal College of Surgeons (RCS) rats. Methods: ARPE-19 cells were pre-treated with ASC-EVs for 24 h, followed by exposure to 200 μM H2O2 for an additional 24 h. RCS rats received an intravitreal injection of phosphate-buffered saline in one eye and ASC-EVs in the other eye. Results: ASC-EV pretreatment significantly protected against H2O2 in the Cell Counting Kit-8 assay and was also effective in the lactate dehydrogenase-release assay. It notably reduced early apoptosis (Annexin V-fluorescein isothiocyanate/propidium iodide assay) and late apoptosis (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling assay), while significantly decreasing intracellular reactive oxygen species, glutathione levels, and superoxide dismutase activity. NFE2L2, HMOX1, and NQO1 mRNA levels, along with Nrf2, HO-1, and NQO1 protein levels, were significantly elevated with ASC-EV pretreatment. Compared with ARPE-19-derived EVs, 11 miRNAs were upregulated and 34 were downregulated in ASC-EVs. In RCS rats, intravitreal injections of ASC-EVs led to significant preservation of the outer nuclear layer and photoreceptor segments, along with increased nuclear Nrf2 expression and elevated HO-1 and NQO1 levels in the inner retina. Eyes that received intravitreal injections of ASC-EVs demonstrated significantly preserved electroretinography a- and b-wave amplitudes at 1 week post-injection, though this effect faded by 2 weeks. Conclusions: ASC-EVs mitigated apoptosis and oxidative stress in ARPE-19 cells subjected to H2O2 exposure and temporarily slowed retinal degeneration in RCS rats via Nrf2 pathway activation by miRNAs.

TIN2-mediated reduction of mitophagy induces RPE senescence under high glucose

The telomere-associated protein TIN2 localizes to both telomeres and mitochondria. Nevertheless, the impact of TIN2 on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. This research aims to examine the role of TIN2 in the senescence of RPE and its potential as a therapeutic target. Western blotting and immunofluorescence staining were utilized to identify TIN2 expression and mitophagy. RT-qPCR was employed to identify senescent associated secretory phenotype (SASP) in ARPE-19 cells infected with TIN2 overexpression. To examine mitochondria and the cellular senescence of RPE, TEM, SA-β-gal staining, and cell cycle analysis were used. The impact of TIN2 was examined using OCT and immunohistochemistry in mice. DHE staining and ZO-1 immunofluorescence were applied to detect RPE oxidative stress and tight junctions. Our research revealed that increased mitochondria-localized TIN2 aggravated the cellular senescence of RPE cells both in vivo and in vitro under hyperglycemia. TIN2 overexpression stimulated the mTOR signaling pathway in ARPE-19 cells and exacerbated the inhibition of mitophagy levels under high glucose, which can be remedied through the mTOR inhibitor, rapamycin. Knockdown of TIN2 significantly reduced senescence and mitochondrial oxidative stress in ARPE-19 cells under high glucose and restored retinal thickness and RPE cell tight junctions in DR mice. Our study indicates that increased mitochondria-localized TIN2 induced cellular senescence in RPE via compromised mitophagy and activated mTOR signaling. These results propose that targeting TIN2 could potentially serve as a therapeutic strategy in the treatment of DR.

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.

USP48 deubiquitination stabilizes SLC1A5 to inhibit retinal pigment epithelium cell inflammation, oxidative stress and ferroptosis in the progression of diabetic retinopathy.

Diabetic retinopathy is one of the complications of diabetes mellitus. The aim of this study was to explore the effects of ubiquitin-specific protease 48 (USP48) and its underlying mechanisms in the development of diabetic retinopathy. CCK-8 assay, EdU assay, and flow cytometry were used to measure the proliferative ability and the apoptotic rate of ARPE-19 cells, respectively. ELISA kits were utilized to assess the levels of inflammatory cytokines. The levels of Fe2+, ROS and MDA were detected using the corresponding biochemical kits. The protein expression of USP48 and SLC1A5 was examined through western blot. The mRNA level of SLC1A5 was determined using RT-qPCR. The interaction relationship between USP48 and SLC1A5 was evaluated using Co-IP assay. High glucose (HG) treatment significantly inhibited cell proliferation and elevated cell apoptosis, inflammation, ferroptosis and oxidative stress in ARPE-19 cells. HG treatment-caused cell damage was hindered by USP48 or SLC1A5 overexpression in ARPE-19 cells. Fer-1 treatment improved HG-caused cell damage in ARPE-19 cells, which was blocked by USP48 knockdown. Moreover, USP48 knockdown decreased SLC1A5 expression. SLC1A5 downregulation reversed the improvement effects of USP48 upregulation on cell damage in HG-treated ARPE-19 cells. USP48 overexpression deubiquitinated SLC1A5 to elevate cell proliferation and suppress cell apoptosis, inflammation, ferroptosis and oxidative stress in HG-triggered ARPE-19 cells, thereby inhibiting the progression of diabetic retinopathy.

PRGF Membrane with Tailored Optical Properties Preserves the Cytoprotective Effect of Plasma Rich in Growth Factors: In Vitro Model of Retinal Pigment Epithelial Cells

The present study evaluates the ability of a novel plasma rich in growth factors (PRGF) membrane with improved optical properties to reduce oxidative stress in retinal pigment epithelial cells (ARPE-19 cells) exposed to blue light. PRGF was obtained from three healthy donors and divided into four main groups: (i) PRGF membrane (M-PRGF), (ii) PRGF supernatant (S-PRGF), (iii) platelet-poor plasma (PPP) membrane diluted 50% with S-PRGF (M-PPP 50%), and (iv) M-PPP 50% supernatant (S-PPP 50%). ARPE-19 cells were exposed to blue light and then incubated with the different PRGF-derived formulations or control for 24 and 48 h under blue light exposure. Mitochondrial and cell viability, reactive oxygen species (ROS) production, and heme oxygenase-1 (HO-1) and ZO-1 expression were evaluated. Mitochondrial viability and cell survival were significantly increased after treatment with the different PRGF-derived formulations. ROS synthesis and HO-1 expression were significantly reduced after cell treatment with any of the PRGF-derived formulations. Furthermore, the different PRGF-derived formulations significantly increased ZO-1 expression in ARPE-19 exposed to blue light. The new PRGF membrane with improved optical properties and its supernatant (M-PPP 50% and S-PPP 50%) protected and reversed blue light-induced oxidative stress in ARPE-19 cells at levels like those of a natural PRGF membrane and its supernatant.

Polarized RPE Secretome Preserves Photoreceptors in Retinal Dystrophic RCS Rats.

Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions.

Repeated Topical Administration of 3 nm Cerium Oxide Nanoparticles Reverts Disease Atrophic Phenotype and Arrests Neovascular Degeneration in AMD Mouse Models.

Increased oxidative stress in the retina and retinal pigment epithelium is implicated in age-related macular degeneration (AMD). Antioxidant cerium oxide nanoparticles (CeO2NPs) have been used to treat degenerative retinal pathologies in animal models, although their delivery route is not ideal for chronic patient treatment. In this work, we prepared a formulation for ocular topical delivery that contains small (3 nm), nonaggregated biocompatible CeO2NPs. In vitro results indicate the biocompatible and protective character of the CeO2NPs, reducing oxidative stress in ARPE19 cells and inhibiting neovascularization related to pathological angiogenesis in both HUVEC and in in vitro models of neovascular growth. In the in vivo experiments, we observed the capacity of CeO2NPs to reach the retina after topical delivery and a subsequent reversion of the altered retinal transcriptome of the retinal degenerative mouse model DKOrd8 toward that of healthy control mice, together with signs of decreased inflammation and arrest of degeneration. Furthermore, CeO2NP eye drops' treatment reduced laser-induced choroidal neovascular lesions in mice by lowering VEGF and increasing PEDF levels. These results indicate that CeO2NP eye drops are a beneficial antioxidant and neuroprotective treatment for both dry and wet forms of AMD disease.

Decorin Protects Retinal Pigment Epithelium Cells from Oxidative Stress and Apoptosis via AMPK-mTOR-Regulated Autophagy.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss among the elderly worldwide with unidentified pathogenesis and limited therapeutic options. Oxidative stress-induced damage to the retinal pigment epithelium (RPE) is central in the development and progression of AMD. Decorin (DCN), a small leucine-rich proteoglycan, possesses powerful antifibrotic, anti-inflammatory, and antiangiogenic properties. DCN has also been reported to serve a cytoprotective role in various cell types, but its protective effects against H2O2-induced oxidative stress and apoptosis in ARPE-19 cells remain unclear. In this study, we showed that DCN significantly attenuated the increase in cell viability loss, apoptosis rate, and reactive oxygen species (ROS) levels in ARPE-19 cells induced by H2O2. Furthermore, DCN activated the AMPK/mTOR pathway to promote autophagy while genetic inhibition of autophagy-related gene 5 (ATG5) hindered autophagic process and diminished the protective role of DCN against oxidative stress in ARPE-19 cells. Collectively, these results suggest that DCN could protect RPE cells from H2O2-induced oxidative stress and apoptosis via autophagy promotion, thus providing the therapeutic potential for AMD prevention and treatment.

Lutein activates downstream signaling pathways of unfolded protein response in hyperglycemic ARPE-19 cells

We have earlier demonstrated that lutein effectively prevents hyperglycemia generated sustained oxidative stress in ARPE-19 cells by activating Nrf2 (nuclear factor erythroid 2–related factor 2) signaling. Since evidence portrays an intricate connection between ER (endoplasmic reticulum) stress and hyperglycemia-mediated oxidative stress, we aimed to explore the protective mechanism of lutein on hyperglycemia-induced ER stress in ARPE-19 cells. To determine the effect of lutein, we probed three major downstream branches of unfolded protein response (UPR) signaling pathways using western blot, immunofluorescent and RT-PCR techniques. The data showed a reduction (38%) in protein expression of an imperative ER chaperon, BiP (binding immunoglobulin protein), in glucose-treated ARPE-19 cells. At the same time, lutein pretreatment blocked this glucose-mediated effect, leading to a significant increase in BiP expression. Lutein promoted the phosphorylation of IRE1 (inositol requiring enzyme 1) and subsequent splicing of XBP1 (X-box binding protein 1), leading to enhanced nuclear translocation. Likewise, lutein activated the expression and translocation of transcription factors, ATF6 (activating transcription factor 6) and ATF4 (activating transcription factor 4) suppressed by hyperglycemia. Lutein also increased CHOP (C/EBP-homologous protein) levels in ARPE-19 cultured under high glucose conditions. The mRNA expression study showed that lutein pretreatment upregulates downstream UPR genes HRD1 (ERAD-associated E3 ubiquitin-protein ligase HRD1), p58IPK (protein kinase inhibitor p58) compared to high glucose treatment alone. From our study, it is clear that lutein show protection against hyperglycemia-mediated ER stress in ARPE-19 cells by activating IRE1-XBP1, ATF6, and ATF4 pathways and their downstream activators. Thus, lutein may have the pharmacological potential for protection against widespread disease conditions of ER stress.

Quercetin protects retina external barrier from oxidative stress injury by promoting autophagy

Purpose: This study aimed to investigate the protective effects of quercetin on the tight junction proteins of human retinal pigment epithelial cells (ARPE-19 cells) suffering from oxidative stress injury and explore the possible mechanism. Methods: H2O2 (300 μM) was used to establish an oxidative stress model of ARPE-19 cells. ARPE-19 cells were pretreated with different concentrations (0–80 μM) of quercetin before H2O2 exposure. The expression and distribution of tight junction proteins and autophagy-related proteins were detected by Western blot and immunostaining. ARPE-19 cells were pretreated with 5 mM 3-methyladenine (3-MA). Results: The cell viability weakened in the H2O2 group compared with the control group. However, it was preserved after pretreatment with quercetin. It was observed that the expression levels of occludin, claudin-1 were decreased in the H2O2 group. Quercetin treatment significantly enhanced the expression levels of them as compared to the H2O2 group. H2O2 alone strongly decreased the Zonula occludens protein 1 (ZO-1) expression in the cytomembrane. Quercetin supplementation enhanced the accumulation of ZO-1 in ARPE-19 cells. The expression levels of Beclin-1 and Microtubule associated protein light chain 3 II (LC-3II) increased, and that of P62 decreased in the quercetin protection group. The appearance of LC-3II, which examined by immunofluorescence experiments, enhanced in the quercetin protection group as compared with the control group. The expression levels of beclin-1 and LC-3II increased, and that of P62 increased in the autophagy-inhibited group compared with the quercetin protection group. The levels of occludin and claudin-1 also decreased. Conclusion: Quercetin prevents the loss of tight junction proteins by upregulating autophagy after oxidative stress in ARPE-19 cells.

Protective effects of PARP1-inhibitory compound in dry age-related macular degeneration

Poly (ADP-ribose) polymerase 1 (PARP1)-dependent cell death in the retinal pigment epithelium (RPE) is implicated in dry age-related macular degeneration (AMD). Although PARP1 inhibitors are available for treating dry AMD, their delivery route is not ideal for patients. The aim of this study was to test the efficacy of a novel PARP1-inhibitory compound (PIC) in vitro and in vivo. This study presents PIC, a novel small molecule, with superior efficacy to PARP1 inhibitors in the market. PIC demonstrated a distinctive inhibitory profile against PARP isotypes than the FDA-approved PARP1 inhibitors. PIC inhibited PARP1 activation at an IC50 of 0.41 ± 0.15 nM in an enzyme-based assay in vitro and at IC50 and EC50 in ARPE-19 cells of 0.11 ± 0.02 nM and 0.22 ± 0.02 nM, respectively, upon H2O2 insult. PIC also moderated mitochondrial fission and depolarization and maintained cellular energy levels under oxidative stress in ARPE-19 cells. Furthermore, PIC demonstrated good corneal penetration in a rat model, presenting PIC as a promising candidate for eye drop therapeutics for dry AMD. When PIC was administered as an eye drop formulation, RPE morphology was preserved, maintaining the thickness of the outer nuclear layers under sodium iodate (SI) treatment in rats. In SI-treated rabbits, eye drop administration of PIC also retained the structural and functional integrity when analyzed using funduscopy and electroretinogram. Collectively, our data portray PIC as an attractive treatment measure for dry AMD.

Chitosan-sodium alginate-fatty acid nanocarrier system: Lutein bioavailability, absorption pharmacokinetics in diabetic rat and protection of retinal cells against H2O2 induced oxidative stress in vitro

Aiming to enhance therapeutic efficiency of lutein, lutein loaded chitosan-sodium alginate (CS-SA) based nanocarrier system (LNCs) were prepared and evaluated for lutein bioavailability and pharmacokinetics in diabetic rats in comparison to micellar lutein (control). Further, cytotoxicity, cellular uptake and protective activity against H2O2 induced oxidative stress in ARPE-19 cells were studied. Results revealed that LNCs displayed maximal lutein AUC in plasma, liver and eye respectively in normal (3.1, 2.7 and 5.2 folds) and diabetic (7.3, 3.4 and 2.8 folds) rats. Lutein from LNCs exhibited a higher half-life time, mean residence time and slow clearance from the plasma, indicating prolonged circulation compared to control. In ARPE-19 cells, pre-treatment with LNCs (10 μM) have significantly attenuated H2O2 induced cell death, intracellular ROS and mitochondrial membrane potential compared to control. In conclusion, LNCs improve the lutein bioavailability in conditions like diabetes, diabetic retinopathy and cataract to curtail oxidative stress in retinal cells.

PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress.

The molecular mechanism underlying autophagy impairment in the retinal pigment epithelium (RPE) in dry age-related macular degeneration (AMD) is not yet clear. Based on the causative role of poly(ADP-ribose) polymerase 1 (PARP1) in RPE necrosis, this study examined whether PARP1 is involved in the autophagy impairment observed during dry AMD pathogenesis. We found that autophagy was downregulated following H2O2-induced PARP1 activation in ARPE-19 cells and olaparib, PARP1 inhibitor, preserved the autophagy process upon H2O2 exposure in ARPE-19 cells. These findings imply that PARP1 participates in the autophagy impairment upon oxidative stress in ARPE-19 cells. Furthermore, PARP1 inhibited autolysosome formation but did not affect autophagosome formation in H2O2-exposed ARPE-19 cells, demonstrating that PARP1 is responsible for impairment of late-stage autophagy in particular. Because PARP1 consumes NAD+ while exerting its catalytic activity, we investigated whether PARP1 impedes autophagy mediated by sirtuin1 (SIRT1), which uses NAD+ as its cofactor. A NAD+ precursor restored autophagy and protected mitochondria in ARPE-19 cells by preserving SIRT1 activity upon H2O2. Moreover, olaparib failed to restore autophagy in SIRT1-depleted ARPE-19 cells, indicating that PARP1 inhibits autophagy through SIRT1 inhibition. Next, we further examined whether PARP1-induced autophagy impairment occurs in the retinas of dry AMD model mice. Histological analyses revealed that olaparib treatment protected mouse retinas against sodium iodate (SI) insult, but not in retinas cotreated with SI and wortmannin, an autophagy inhibitor. Collectively, our data demonstrate that PARP1-dependent inhibition of SIRT1 activity impedes autophagic survival of RPE cells, leading to retinal degeneration during dry AMD pathogenesis.

Resvega Alleviates Hydroquinone-Induced Oxidative Stress in ARPE-19 Cells.

Retinal pigment epithelial (RPE) cells maintain homeostasis at the retina and they are under continuous oxidative stress. Cigarette smoke is a prominent environmental risk factor for age-related macular degeneration (AMD), which further increases the oxidant load in retinal tissues. In this study, we measured oxidative stress and inflammatory markers upon cigarette smoke-derived hydroquinone exposure on human ARPE-19 cells. In addition, we studied the effects of commercial Resvega product on hydroquinone-induced oxidative stress. Previously, it was observed that Resvega induces autophagy during impaired protein clearance in ARPE-19 cells, for which it has the potential to alleviate pro-inflammatory pathways. Cell viability was determined while using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the cytokine levels were measured using the enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production were measured using the 2′,7′-dichlorofluorescin diacetate (H2DCFDA) probe. Hydroquinone compromised the cell viability and increased ROS production in ARPE-19 cells. Resvega significantly improved cell viability upon hydroquinone exposure and reduced the release of interleukin (IL)-8 and monocytic chemoattractant protein (MCP)-1 from RPE cells. Resvega, N-acetyl-cysteine (NAC) and aminopyrrolidine-2,4-dicarboxylic acid (APDC) alleviated hydroquinone-induced ROS production in RPE cells. Collectively, our results indicate that hydroquinone induces cytotoxicity and increases oxidative stress through NADPH oxidase activity in RPE cells, and resveratrol-containing Resvega products prevent those adverse effects.

CTRP3 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Diabetic retinopathy (DR) is one of the most common diabetic complications and remains the leading cause of vision loss among adults. C1q/TNF-related protein 3 (CTRP3) is a member of CTRP family that has been found to be involved in the progression of diabetes mellitus and diabetic complications. However, the role of CTRP3 in DR has not been fully understood. In the present study, the results showed that CTRP3 expression was significantly decreased in DR patients compared with controls. In vitro investigations proved that overexpression of CTRP3 improved cell viability of ARPE-19 cells in response to high glucose (HG) stimulation. CTRP3 also attenuated HG-induced oxidative stress in ARPE-19 cells with decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and increased superoxide dismutase (SOD) activity. Apoptotic rate was significantly decreased in CTRP3 overexpressing ARPE-19 cells. Besides, bcl-2 expression was increased, while bax expression was decreased by CTRP3 overexpression. Moreover, overexpression of CTRP3 enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) pathway in HG-stimulated ARPE-19 cells, and Nrf2 knockdown reversed CTRP3-mediated oxidative stress and apoptosis. These findings suggested that CTRP3 attenuated HG-stimulated oxidative stress and apoptosis in ARPE-19 cells, which were mediated by activation of Nrf2/HO-1 pathway.

Involvement of microRNA-34a in Age-Related Susceptibility to Oxidative Stress in ARPE-19 Cells by Targeting the Silent Mating Type Information Regulation 2 Homolog 1/p66shc Pathway: Implications for Age-Related Macular Degeneration.

The aging retinal pigment epithelium and oxidative stress, mediated by reactive oxygen species (ROS) accumulation, have been implicated in the mechanisms of age-related macular degeneration (AMD). The expression level of the adapter protein p66shc, a key protein that regulates cellular oxidative stress, is relatively low under normal conditions because of the effects of silent mating type information regulation 2 homolog 1 (SIRT1) on the binding of fully deacetylated histone H3’ to the p66shc promoter region, thus inhibiting p66shc transcription and expression. The equilibrium between SIRT1 and p66shc is disrupted in the presence of various stresses, including AMD. As a major target gene, SIRT1 is regulated by microRNA-34a (miR-34a), and overexpression of miR-34a results in significant inhibition of post-transcriptional expression of SIRT1. Furthermore, our recent studies demonstrated that miR-34a is significantly upregulated, accompanied by reduced tolerance to oxidative stress in hydrogen peroxide-induced prematurely senescent ARPE-19 cells. Moreover, the expression of SIRT1 is decreased, whereas that of p66shc is increased in these cells. Accordingly, miR-34a may play a key role in age-related susceptibility to oxidative stress in ARPE-19 cells by targeting the SIRT1/p66shc pathway, leading to AMD. In this review article, we discuss the functions of miR-34a in modulating the SIRT1/p66shc pathway in age-related conditions, including AMD.

Scutellarin protects human retinal pigment epithelial cells against hydrogen peroxide (H2O2)-induced oxidative damage

BackgroundProliferative vitreoretinopathy (PVR) is a severe blinding complication of retinal detachment surgery. Increasing evidence demonstrate that PVR is associated with oxidative stress. Scutellarin is a natural flavone compound that has been reported to have anti-oxidative activity. However, the effect of scutellarin on PVR remains unknown. In the current study, we assessed the effect of scutellarin on hydrogen peroxide (H2O2)-induced oxidative injury in human retinal pigment epithelium cells (ARPE-19).MethodsARPE-19 cells were pretreated with different concentrations of scutellarin for 2 h, and then challenged with H2O2 (1 mM) for 24 h. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) and glutathione (GSH) activity were measured to assess the level of oxidative stress. Flow cytometry was performed to detect the apoptosis rate of ARPE-19 cells. Expression levels of bcl-2, bax, cleaved-caspase-3, p-JAK2, JAK2, p-STAT3, and STAT3 were measured using western blot.ResultsOur results revealed that scutellarin improved the cell viability of H2O2-induced ARPE-19 cells. Scutellarin alleviated the H2O2-induced oxidative stress in ARPE-19 cells, which was illustrated by reduced levels of ROS and MDA, accompanied by increased SOD activity and GSH level. The increased apoptosis rate of ARPE-19 cells caused by H2O2 induction was significantly decreased after scutellarin treatment. H2O2 treatment resulted in significant increase in bax expression and decrease in bcl-2 expression, while the changes in the expressions of bax and bcl-2 were reversed by scutellarin treatment. In addition, scutellarin promoted the activation of JAK2/STAT3 signaling pathway in H2O2-induced ARPE-19 cells. Suppression of JAK2/STAT3 signaling pathway abolished the protective effects of scutellarin on H2O2-induced ARPE-19 cells.ConclusionThese findings suggested that scutellarin was capable for alleviating H2O2-induced oxidative damage in ARPE-19 cells, which might be ascribed to the activation of JAK2/STAT3 signaling pathway.

Protective effects of melatonin and memantine in human retinal pigment epithelium (ARPE-19) cells against 2-ethylpyridine-induced oxidative stress: implications for age-related macular degeneration

Purpose: To investigate the possible protective effects of melatonin and memantine (MMT) against 2-ethylpyridine (2-EP)-induced oxidative stress and mitochondrial dysfunction in human RPE (ARPE-19) cells in vitro.Materials and methods: The ARPE-19 cells were divided into seven groups. Oxidative stress was triggered by incubating the ARPE-19 cells with 30 μM of 2-EP for 24 h. Then, 200 μM of melatonin was administered over three days and 20 μM of MMT over six hours prior to the experiment. The effects of melatonin and MMT on the intracellular calcium release mechanism, reactive oxygen species production, caspase-3 and caspase-9 activities, as well as vascular endothelial growth factor levels were measured.Results: Melatonin and MMT were found to significantly decrease apoptosis levels. The intracellular calcium release was regulated by both melatonin and MMT. Further, melatonin and MMT significantly decreased both caspase-3 and caspase-9 activities, as well as pro-caspase and poly(ADP-ribose) polymerase expression, in ARPE-19 cells. Moreover, melatonin significantly increased the protective effect of MMT. The combination of melatonin and MMT significantly decreased 2-EP-induced oxidative toxicity and apoptosis by inhibiting the intracellular reactive oxygen species production and mitochondrial depolarization levels.Conclusions: These notable findings are the first to demonstrate the synergistic protective effects of melatonin and MMT against 2-EP-induced oxidative stress in ARPE-19 cells.

Combination of ginsenoside Rb1 and Rd protects the retina against bright light-induced degeneration

Photoreceptor degeneration is a central pathology of various retinal degenerative diseases which currently lack effective therapies. Antioxidant and anti-inflammatory activities are noted for Panax notoginsenoside saponins (PNS) and related saponin compound(s). However, the photoreceptor protective potentials of PNS or related saponin compound(s) remain unknown. The current study revealed that PNS protected against photoreceptor loss in bright light-exposed BALB/c mice. Combination of ginsenoside Rb1 and Rd, two major saponin compounds of PNS, recapitulated the retinal protection of PNS and attenuated retinal oxidative stress and inflammatory changes. Rb1 or Rd partially alleviated all-trans-Retinal-induced oxidative stress in ARPE19 cells. Rb1 or Rd suppressed lipopolysaccharides (LPS)-induced proinflammatory gene expression in ARPE19 and RAW264.7 cells. Rb1 or Rd also modulated the expression of proinflammatory microRNA, miR-155 and its direct target, anti-inflammatory SHIP1, in LPS-stimulated RAW264.7 cells. The retinal expression of miR-155 and SHIP1 was altered preceding extensive retinal damage, which was maintained at normal level by Rb1 and Rd combination. This work shows for the first time that altered expression of miR-155 and SHIP1 are involved in photoreceptor degeneration. Most importantly, novel retinal protective activities of combination of Rb1 and Rd justify further evaluation for the treatment of related retinal degenerative disorders.

Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells.