Protected ARPE-19 Cells Research Articles

Protective effect of ghrelin in oxidative stress-induced age-related macular degeneration in vitro and in vivo

Oxidative damage to human retinal pigment epithelial (RPE) cells is the main cause of age-related macular degeneration (AMD), in our previous work, we showed that ghrelin has an antioxidative effect on human lens epithelium (HLE) cells, however, the studies of using ghrelin in treating the degenerative diseases of the retina have rarely been reported. In this article, we assessed the effect of ghrelin on preventing oxidative stress induced by hydrogen peroxide (H2O2) in ARPE-19 cells and its mechanism. We observed that pretreatment with ghrelin protected ARPE-19 cells from H2O2-induced cell oxidative injuries and apoptosis responses. Furthermore, an oxidative stress-induced mouse model of AMD was established via injection of sodium iodate (NaIO3) to tail veins, and treatment with ghrelin preserved retinal function, and protected photoreceptors.

Oxya chinensis sinuosa (OC) Extracts Protects ARPE-19 Cells against Oxidative Stress via Activation of the Mitogen-Activated Protein Kinases (MAPKs)/ Nuclear Factor-κB (NF-κB) Pathway.

Oxya chinensis sinuosa (OC) is a well-known edible insect. Several researches on the health benefits of OC consumption have been performed to date; however, their effect on eye health remains largely unknown. This study aimed to assess the protective effects of OC extracts on the oxidative stress on the retinal pigment epithelium (RPE) cells. Oxidative damage has been identified as one of the key regulatory factors in age-related macular degeneration. H2O2-induced reactive oxygen species (ROS) production, a well-known oxidative stress factor, can cause cell death in retinal pigment epithelia cells. In this study, we found that three OC extracts effectively prevented H2O2-induced ROS production and subsequent death of ARPE-19 cells in a dose-dependent manner. In addition, the OC extracts inhibited the phosphorylation of mitogen-activated protein kinases including p38, JNK, and ERK. The OC extracts restored IκBα degradation induced by H2O2, indicating that OC extracts suppressed the activation of nuclear factor-κB. Furthermore, the three OC extracts were shown to have antioxidant effects by up-regulating the intracellular expression of key antioxidant proteins such as SOD, NQO, and HO-1. Here we demonstrated the antioxidant and anti-apoptotic effects of the OC extracts on ARPE-19, indicating their potential role in improving eye health. These results suggest that three OC extracts plays a critical role in oxidative stress-induced cell death protects in ARPE-19 cells.

Therapeutic Application of Dendrobium fimbriatum Hook for Retinopathy Caused by Ultraviolet Radiation and Chemotherapy Using ARPE-19 Cells and Mouse Retina.

Retinopathy caused by ultraviolet radiation and cancer chemotherapy has increased dramatically in humans due to rapid environmental and social changes. Therefore, it is very important to develop therapeutic strategies to effectively alleviate retinopathy. In China, people often choose dendrobium to improve their eyesight. In this study, we explored how Dendrobium fimbriatum extract (DFE) protects ARPE-19 cells and mouse retinal tissue from damage of ultraviolet (UV) radiation and chemotherapy. We evaluated the antioxidant capacity of DFE using the 1,1-diphenyl-2-trinitophenylhydrazine (DPPH) assay. The protective effects of DEF from UV- and oxaliplatin (OXA)-induced damage were examined in ARPE-19 cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and immunofluorescence (IF) stains, and in mouse retinal tissue using immunohistochemistry (IHC) stains. Our results show that DFE has excellent antioxidant capacity. The ARPE-19 cell viability was decreased and the F-actin cytoskeleton structure was damaged by UV radiation and OXA chemotherapy, but both were alleviated after the DFE treatment. Furthermore, DFE treatment can alleviate OXA chemotherapy-induced reduced expressions of rhodopsin and SOD2 and increased expressions of TNF-α and caspase 3 in mouse retinal tissue. Thus, we suggest that DFE can act as suitable treatment for retinopathy through reducing oxidative stress, inflammation, and apoptosis.

SuperDopa (SD), SuperDopa amide (SDA) and Thioredoxin-mimetic peptides protect ARPE-19 cells from photic- and non-photic stress

Oxidative stress and inflammation in the retinal pigment epithelium (RPE) cells have been identified as significant risk factors in the development and progression of retinal associated diseases including age-related macular degeneration (AMD). In addition, AMD and myopia have been associated with impaired dopamine activity. Treatment of RPE cells with antioxidants or high concentrations of l-DOPA (levodopa), which down-regulates vascular endothelial growth factor (VEGF) via a G-protein-coupled receptor GPR143, slow AMD progression. To develop a targeted and effective treatment aimed at improving the viability of RPE cells we examined small molecular weight thiol-based and levodopa containing molecules. These include the N-acetylcysteine amide (AD4/NACA), SuperDopa-Amide (SDA), and members of the thioredoxin mimetic (TXM) family of peptides, TXM-CB13, TXM-CB30, and SuperDopa (SD). We show that these antioxidant/anti-inflammatory reagents protect ARPE-19 cells from photic stress mediated by rose Bengal (rB) and rhodopsin-rich POS, and from non-photic stress induced by oxidation with sodium iodate. Protection is correlated with a reduction in DPPH radical and singlet-oxygen quenching. Compared to GSH the bimolecular rate-constants of singlet oxygen quenching in aqueous solution by the levodopa derivatives SD and SDA were two-fold higher. Inhibition of auranofin-induced activation of the mitogen-activation-kinases (MAPK's) JNK1/2 and ERK1/2 confirmed the antioxidant/anti-inflammatory activity of the thiol-levodopa derivatives. The antioxidant and radical scavenging activities of TXM-CB13 and TXM-CB30, or SD and SDA, which combine redox activity with elevating cellular levodopa, might offer an efficient protection of RPE cells. These retino-protective peptides are potential drug candidates destined for slowing the onset and/or progression of RPE-related disorders.

Cyanidin-3-O-glucoside from blueberry anthocyanin extracts protects ARPE-19 cells against high glucose damage via REDD1/GSK3β pathway

The retinal pigment epithelium (RPE) serves dual roles: it acts as a selective barrier and also as a regulator for the photoreceptor layer situated above it. The RPE is considered a crucial target for halting the development and progression of diabetic retinopathy (DR). This study focuses on understanding the protective role of blueberry anthocyanin extracts (BAEs), particularly its anti-oxidant constituent, cyanidin-3-O-glucoside (C3G), against damage to RPE cells induced by high glucose (HG) levels. The findings revealed that the BAEs mitigated damage to HG-induced ARPE-19 cells by decreasing the intracellular reactive oxygen species (ROS) levels, which improved cell morphology and cell viability (p < 0.01). Importantly, these cytoprotective effects were linked to the C3G rich in BAEs. The underlying mechanism may involve C3G interrupting the ROS generation feedback loop by downregulating REDD1 expression, which in turn enhanced the retinal Nrf2 antioxidant response to HG through the promotion of the GSK3β phosphorylation at Ser-9. Moreover, C3G suppressed VEGFA expression by inhibiting REDD1 levels in HG-induced ARPE-19 cells, subsequently preventing an increase in vascular permeability. In conclusion, this study suggested that the C3G relieved HG-induced oxidative stress damage in ARPE-19 cells via REDD1/GSK3β pathway and inhibited an increase in vascular permeability by downregulating VEGFA expression. These insights could shed light on how this natural bioactive compound can help manage HG-associated eye disorders like DR.

Cis-urocanic acid improves cell viability and suppresses inflammasome activation in human retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a common eye disease among the elderly, which can result in impaired vision and irreversible loss of vision. The majority of patients suffer from the dry (also known as the atrophic) form of the disease, which is completely lacking an effective treatment. In the present study, we evaluated the potential of cis-urocanic acid (cis-UCA) to protect human ARPE-19 cells from cell damage and inflammasome activation induced by UVB light. Urocanic acid is a molecule normally present in human epidermis. Its cis-form has recently been found to alleviate UVB-induced inflammasome activation in human corneal epithelial cells. Here, we observed that cis-UCA is well-tolerated also by human retinal pigment epithelial (RPE) cells at a concentration of 100 μg/ml. Moreover, cis-UCA was cytoprotective and efficiently diminished the levels of mature IL-1β, IL-18, and cleaved caspase-1 in UVB-irradiated ARPE-19 cells. Interestingly, cis-UCA also reduced DNA damage, whereas its effect against ROS production was negligible. Collectively, cis-UCA protected ARPE-19 cells from UVB-induced phototoxicity and inflammasome activation. This study indicates that due to its beneficial properties of preserving cell viability and preventing inflammation, cis-UCA has potential in drug development of chronic ocular diseases, such as AMD.

Isolation and Identification of Dihydrophenanthrene Derivatives from Dendrobium virgineum with Protective Effects against Hydrogen-Peroxide-Induced Oxidative Stress of Human Retinal Pigment Epithelium ARPE-19 Cells

Oxidative stress is a significant factor in the development of age-related macular degeneration (AMD), which results from cell damage, dysfunction, and death in the retinal pigmented epithelium (RPE). The use of natural compounds with antioxidant properties to protect RPE cells from oxidative stress has been explored in Dendrobium, a genus of orchid plants belonging to the family Orchidaceae. Two new compounds and seven known compounds from the MeOH extract of the whole plant of Dendrobium virgineum were successfully isolated and structurally characterized. Out of all the compounds isolated, 2-methoxy-9,10-dihydrophenanthrene-4,5-diol (3) showed the highest protective effect against hydrogen peroxide (H2O2)-induced oxidative stress in human retinal pigment epithelial (ARPE-19) cells. Therefore, it was selected to evaluate its protective effect and mechanism on oxidative-stress-induced ARPE-19 cells. Cells were pre-treated with compound 3 at 25, 50, and 100 µg/mL for 24 h and then induced with 400 µM H2O2 for 1 h. The results demonstrated that compound 3 significantly (p < 0.05) increased cell viability by 10-35%, decreased ROS production by 10-30%, and reduced phosphorylation of p38, ERK1/2, and SAPK/JNK by 20-70% in a dose-dependent manner without toxicity. Furthermore, compound 3 significantly (p < 0.05) modulated the expression of apoptosis pathway proteins (cytochrome c, Bax and Bcl-2) by 20-80%, and enhanced SOD, CAT, and GPX activities, and GSH levels in a dose-dependent manner. These results suggest that compound 3 protects ARPE-19 cells against oxidative stress through MAPKs and apoptosis pathways, including the antioxidant system. Thus, compound 3 could be considered as an antioxidant agent for preventing AMD development by protecting RPE cells from oxidative stress and maintaining the retina. These findings open up new possibilities for the use of natural compounds in the treatment of AMD and other oxidative-stress-related conditions.

Chitooligosaccharides Derivatives Protect ARPE-19 Cells against Acrolein-Induced Oxidative Injury.

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly. The progression of AMD is closely related to oxidative stress in the retinal pigment epithelium (RPE). Here, a series of chitosan oligosaccharides (COSs) and N-acetylated derivatives (NACOSs) were prepared, and their protective effects on an acrolein-induced oxidative stress model of ARPE-19 were explored using the MTT assay. The results showed that COSs and NACOs alleviated APRE-19 cell damage induced by acrolein in a concentration-dependent manner. Among these, chitopentaose (COS-5) and its N-acetylated derivative (N-5) showed the best protective activity. Pretreatment with COS-5 or N-5 could reduce intracellular and mitochondrial reactive oxygen species (ROS) production induced by acrolein, increase mitochondrial membrane potential, GSH level, and the enzymatic activity of SOD and GSH-Px. Further study indicated that N-5 increased the level of nuclear Nrf2 and the expression of downstream antioxidant enzymes. This study revealed that COSs and NACOSs reduced the degeneration and apoptosis of retinal pigment epithelial cells by enhancing antioxidant capacity, suggesting that they have the potential to be developed into novel protective agents for AMD treatment and prevention.

Circ_0006667 contributes to high glucose-induced retinal pigment epithelial cell dysfunction by mediating miR-7-5p/TGFA axis in diabetic retinopathy.

Diabetic retinopathy (DR) is a common complication of diabetes mellitus and it can lead to visual impairment and blindness. The loss of retinal pigment epithelial (RPE) cells is associated with the etiology of DR. Moreover, dysregulated circular RNAs (circRNAs) are implicated in DR progression. Therefore, this project aims to explore the role and potential mechanism of circ_0006667 in DR. RPE cells (ARPE-19) were stimulated with high glucose (33mM; HG group) for 24h to establish the DR cell model. Circ_0006667, microRNA-7-5p (miR-7-5p), and transforming growth factor alpha (TGFA) expression was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, and apoptosis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry. CyclinD1, Cleaved-caspase-3, and TGFA protein levels were detected using western blot. Using Circinteractome and starBase analysis, the binding miR-7-5p and circ_0006667 or TGFA was predicted, and then validated using dual-luciferase reporter and RNA Immunoprecipitation (RIP). Circ_0006667 expression was up-regulated in DR patients and HG-induced ARPE-19 cells. HG stimulation suppressed ARPE-19 cell proliferation and promoted cell apoptosis and inflammation, which were alleviated via circ_0006667 silence. Circ_0006667 acted as a molecular sponge for miR-7-5p, and circ_0006667 absence-mediated protective effects in HG-induced ARPE-19 cells were largely overturned by the interference of miR-7-5p. miR-7-5p directly targeted TGFA, and miR-7-5p overexpression protected ARPE-19 cells from HG-induced dysfunction largely by down-regulating TGFA. Circ_0006667 can up-regulate the expression of TGFA by sponging miR-7-5p in ARPE-19 cells. Circ_0006667 silencing protected ARPE-19 cells from HG-induced dysfunction by mediating miR-7-5p/TGFA axis.

Anti-Inflammatory Activity and Mechanism of Sweet Corn Extract on Il-1β-Induced Inflammation in a Human Retinal Pigment Epithelial Cell Line (ARPE-19).

Age-related macular degeneration (AMD) is an eye disease associated with aging. Development of AMD is related to degeneration and dysfunction of the retinal pigment epithelium (RPE) caused by low-grade chronic inflammation in aged RPE cells leading to visual loss and blindness. Sweet corn is a good source of lutein and zeaxanthin, which were reported to exert various biological activities, including anti-inflammatory activity. The present study aims to investigate the anti-inflammatory activity and mechanisms of SCE to inhibit the production of inflammatory biomarkers related to AMD development. Cells were pretreated with SCE for 1 h followed by stimulation with IL-1β for another 24 h. The results demonstrated that SCE attenuated IL-1β-induced production of IL-6, IL-8, and MCP-1 and the expression of ICAM-1 and iNOS in a dose-dependent manner. In addition, SCE suppressed the phosphorylation of ERK1/2, SAPK/JNK, p38, and NF-κB (p65) in IL-1β-stimulated ARPE-19 cells. These results proved that SCE protected ARPE-19 cells from IL-1β-induced inflammation by inhibiting inflammatory markers partly via suppressing the activation of MAPK and NF-κB signaling pathways. Overall, SCE is a potential agent for the prevention of AMD development, which should be further evaluated in animals.

Phloroglucinol Attenuates DNA Damage and Apoptosis Induced by Oxidative Stress in Human Retinal Pigment Epithelium ARPE-19 Cells by Blocking the Production of Mitochondrial ROS.

Phloroglucinol, a phenolic compound, is known to possess a potent antioxidant ability. However, its role in retinal cells susceptible to oxidative stress has not been well elucidated yet. Thus, the objective of this study was to evaluate whether phloroglucinol could protect against oxidative damage in cultured human retinal pigment epithelium ARPE-19 cells. For this purpose, ARPE-19 cells were stimula ted with hydrogen peroxide (H2O2) to mimic oxidative stress. Cell viability, cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial function, DNA damage, and autophagy were then assessed. Our results revealed that phloroglucinol ameliorated cell viability, cytotoxicity, and DNA damage in H2O2-exposued ARPE-19 cells and blocked production of ROS. Phloroglucinol also counteracted H2O2-induced apoptosis by reducing Bax/Bcl-2 ratio, blocking activation of caspase-3, and inhibiting degradation of poly (ADP-ribose) polymerase. H2O2 caused mitochondrial impairment and increased expression levels of mitophagy markers such as PINK1and PARKIN known to be associated with mitochondrial ROS (mtROS) generation and cytosolic release of cytochrome c. However, these changes were significantly attenuated by phloroglucinol. Mito-TEMPO, a selective mitochondrial antioxidant, further enhanced the protective effect of phloroglucinol against dysfunctional mitochondria. Furthermore, H2O2 induced autophagy, but not when ARPE-19 cells were pretreated with phloroglucinol, meaning that autophagy by H2O2 contributed to the pro-survival mechanism and that phloroglucinol protected ARPE-19 cells from apoptosis by blocking autophagy. Taken together, these results suggest that phloroglucinol can inhibit oxidative stress-induced ARPE-19 cell damage and dysfunction by protecting DNA damage, autophagy, and subsequent apoptosis through mitigation of mtROS generation. Thus, phloroglucinol might have therapeutic potential to prevent oxidative stress-mediated damage in RPE cells.

Diosgenin protects retinal pigment epithelial cells from inflammatory damage and oxidative stress induced by high glucose by activating AMPK/Nrf2/HO-1 pathway.

Diosgenin is a natural steroidal compound with reported antidiabetic and many other protective properties. This study aimed to explore the protective effect of diosgenin on high-glucose (HG)-induced retinal pigment epithelial cells. HG-induced ARPE-19 cells were considered as a cell model of diabetic retinopathy (DR). The viability and apoptosis of ARPE-19 cells induced by HG treated with either diosgenin or Compound C (CC; dorsomorphin) were detected by Cell Counting Kit-8 assay and flow cytometric analysis. The expression of apoptosis-related proteins, inflammation-related proteins, and AMPK/Nrf2/HO-1 pathway-related proteins was detected by western blotting. The levels of inflammatory cytokines and detection of oxidative stress indexes were performed using the appropriate assay kits. The messenger RNA expression of inflammatory cytokines was detected by real-time quantitative polymerase chain reaction. There was no obvious effect of diosgenin on the viability of ARPE-19 cells and the viability of ARPE-19 cells was significantly reduced after HG induction. However, diosgenin increased the viability, inhibited the apoptosis, and reduced the inflammatory response and oxidative stress of ARPE-19 cells induced by HG. In addition, diosgenin could activate the AMPK/Nrf2/HO-1 pathway. CC, an AMPK inhibitor, could reverse the above changes caused by diosgenin treatment in ARPE-19 cells induced by HG. Diosgenin could protect ARPE-19 cells from inflammatory damage and oxidative stress induced by HG, by activating the AMPK/Nrf2/HO-1 pathway.

Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage.

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

Sulforaphane alleviates LPS-induced inflammatory injury in ARPE-19 cells by repressing the PWRN2/NF-kB pathway

Background Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population and its pathogenesis has been associated with inflammatory damage to retinal pigment epithelial (RPE) cells. Here, we explored the ability of sulforaphane to protect ARPE-19 cells from lipopolysaccharide (LPS)-induced inflammatory injury and elucidated the underlying molecular mechanism. Methods Cell viability, apoptosis, inflammation, PWRN2 expression, nuclear transcription factor-kappa B (NF-kB) activity, and the interaction between PWRN2 and the IkBa protein were assessed in RPE cells under- or over-expressing PWRN2 that had been treated with LPS and sulforaphane. Results Overexpression of PWRN2 in LPS-treated cells promoted NF-kB activation by interacting with IkBa, thus reducing cell viability. In contrast, PWRN2 downregulation repressed LPS-induced NF-kB activation and apoptosis in RPE cells. Similarly, sulforaphane downregulated PWRN2 and inhibited NF-kB activation in a concentration-dependent manner. Conversely, PWRN2 overexpression or NF-kB upregulation weakened the anti-inflammatory effects of sulforaphane. Conclusion Our results suggest that sulforaphane protects RPE cells from LPS-induced inflammatory injury by suppressing the PWRN2/NF-kB pathway.

Circ_NNT suppresses the apoptosis and inflammation in glucose-induced human retinal pigment epithelium by regulating miR-320b/TIMP3 axis in diabetic retinopathy

BackgroundDiabetic retinopathy (DR) is a frequent complication of diabetes. Recent reports have showed that circular RNAs (circRNAs) play important roles in DR progression. Herein, the aim of this study was to explore the role and molecular mechanism of circ_NNT in DR process. MethodsHuman retinal pigment epithelial cells ARPE-19 were treated with high glucose (HG) in experimental group. The expression of circ_NNT, miR-320b, and TIMP3 (TIMP Metallopeptidase Inhibitor 3) were determined using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by 5-ethynyl-2′-deoxyuridine (EdU) assay, MTT assay, flow cytometry, Western blot, and ELISA. The binding interaction was confirmed using dual-luciferase reporter and pull-down assays. ResultsHG stimulation led to a decrease of circ_NNT and TIMP3 expression, and an increase of miR-320b expression in ARPE-19 cells. Functionally, circ_NNT up-regulation reversed HG-evoked apoptosis and inflammation in ARPE-19 cells. Mechanistically, circ_NNT acted as a sponge for miR-320b to elevate TIMP3 expression. Further rescue experiments showed that miR-320b elevation attenuated the protective effects of circ_NNT on HG-induced ARPE-19 cells. Moreover, inhibition of miR-320b protected ARPE-19 cells against HG-evoked apoptosis and inflammation, which were abolished by TIMP3 knockdown. ConclusionCirc_NNT protected ARPE-19 cells against HG-evoked apoptosis and inflammation via elevating TIMP3 through sequestering miR-320b, indicating that up-regulation of circ_NNT might contribute to the inhibition of DR process.

Protective Effect of Ribes nigrum Extract against Blue Light-Induced Retinal Degeneration In Vitro and In Vivo.

Although blackcurrant has several health benefits, such as antioxidant and anti-inflammatory properties, its effects on the retina remain unclear. In this study, we investigated the efficacy of black currant extract (BCE) in an in vitro and in vivo model of dry age-related macular degeneration (AMD) induced by blue light. Dry macular degeneration is characterized by the abnormal accumulation of lipofuscin (e.g., N-retinylidene-N-retinylethanolamine, A2E) in the retina. Blue light (BL) significantly decreased the viability of A2E-laden human retinal pigment epithelial cells (ARPE-19). However, BCE treatment protected ARPE-19 cells from A2E and BL. A2E, which is oxidized by blue light, generates reactive oxygen species in RPE cells. Treatment with BCE significantly decreased (80.8%) reactive oxygen species levels induced by A2E and BL in a concentration-dependent manner. BCE inhibited A2E accumulation in ARPE-19 cells and significantly downregulated the expression of genes increased by A2E and BL in ARPE-19 cells. In vivo, oral administration of BCE (25–100 mg/kg) ameliorated ocular lesions of BL-induced retinal damage in a mouse model and rescued the thickness of the whole retina, photoreceptor segment layer, outer nuclear layer, and inner nuclear layer. The decrease in the number of nuclei in the outer nuclear layer induced by BL was also rescued by BCE. Additionally, BCE administration rescued (40.0%) the BL-induced reduction in the expression level of superoxide dismutase 1. Taken together, our results suggest that BCE may have preventive and therapeutic effects on dry AMD through its antioxidant activity and inhibition of lipofuscin accumulation in the retina.

Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages.

Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age‐related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo‐excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age‐related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L‐Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE‐19) cells were exposed to hydrogen peroxide (H2O2) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE‐19 cells against H2O2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL‐8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress.

Deficiency of thyroid hormone receptor protects retinal pigment epithelium and photoreceptors from cell death in a mouse model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Progressive dystrophy of the retinal pigment epithelium (RPE) and photoreceptors is the characteristic of dry AMD, and oxidative stress/damage plays a central role in the pathogenic lesion of the disease. Thyroid hormone (TH) regulates cell growth, differentiation, and metabolism, and regulates development/function of photoreceptors and RPE in the retina. Population-/patient-based studies suggest an association of high free-serum TH levels with increased risk of AMD. We recently showed that suppressing TH signaling by antithyroid treatment reduces cell damage/death of the RPE and photoreceptors in an oxidative-stress/sodium iodate (NaIO3)-induced mouse model of AMD. This work investigated the effects of TH receptor (THR) deficiency on cell damage/death of the RPE and photoreceptors and the contribution of the receptor subtypes. Treatment with NaIO3 induced RPE and photoreceptor cell death/necroptosis, destruction, and oxidative damage. The phenotypes were significantly diminished in Thrα1−/−, Thrb−/−, and Thrb2−/− mice, compared with that in the wild-type (C57BL/6 J) mice. The involvement of the receptor subtypes varies in the RPE and retina. Deletion of Thrα1 or Thrb protected RPE, rods, and cones, whereas deletion of Thrb2 protected RPE and cones but not rods. Gene-expression analysis showed that deletion of Thrα1 or Thrb abolished/suppressed the NaIO3-induced upregulation of the genes involved in cellular oxidative-stress responses, necroptosis/apoptosis signaling, and inflammatory responses. In addition, THR antagonist effectively protected ARPE-19 cells and hRPE cells from NaIO3-induced cell death. This work demonstrates the involvement of THR signaling in cell damage/death of the RPE and photoreceptors after oxidative-stress challenge and the receptor-subtype contribution. Findings from this work support a role of THR signaling in the pathogenesis of AMD and the strategy of suppressing THR signaling locally in the retina for protection of the RPE/retina in dry AMD.

Luteolin Alleviates Epithelial-Mesenchymal Transformation Induced by Oxidative Injury in ARPE-19 Cell via Nrf2 and AKT/GSK-3β Pathway.

Oxidative stress plays a critical role in age-related macular degeneration (AMD), and epithelial-mesenchymal transition (EMT) is involved in this process. The aim of this study was to investigate the protective effects of luteolin, a natural flavonoid with strong antioxidant activity, on H2O2-induced EMT in ARPE-19 cells. ARPE-19 cells were incubated with H2O2 at 200 μΜ to induce oxidative stress-associated injury. Cell viability assay showed that luteolin at 20 and 40 μM significantly promoted cell survival in H2O2-treated ARPE-19 cells. Luteolin also markedly protected ARPE-19 cells from H2O2-induced apoptosis. Cell migration assay presented that luteolin significantly reduced H2O2-induced migration in APRE-19 cells. EMT in ARPE-19 cells was detected by western blotting and immunofluorescence. The results showed that H2O2 significantly upregulated the expression of α-SMA and vimentin and downregulated the expression of ZO-1 and E-cadherin, while cells pretreated with luteolin showed a reversal. Meanwhile, the assessment of effects of luteolin on the Nrf2 pathway indicated that luteolin promoted Nrf2 nuclear translocation and upregulated the expressions of HO-1 and NQO-1. In addition, luteolin significantly increased the activities of SOD and GSH-PX and decreased intracellular levels of ROS and MDA in H2O2-treated ARPE-19 cells. Meanwhile, we observed that the expression of TGF-β2, p-AKT, and p-GSK-3β was upregulated in H2O2-treated ARPE-19 cells and downregulated in luteolin-treated cells, revealing that luteolin inhibited the activation of the AKT/GSK-3β pathway. However, these effects of luteolin were all annulled by transfecting ARPE-19 cells with Nrf2 siRNA. Our current data collectively indicated that inhibition of luteolin on EMT was induced by oxidative injury in ARPE-19 cell through the Nrf2 and AKT/GSK-3β pathway, suggesting that luteolin could be a potential drug for the treatment of dry AMD.

A polysaccharide from Lycium barbarum L.: Structure and protective effects against oxidative stress and high-glucose-induced apoptosis in ARPE-19 cells

Lycium barbarum polysaccharides (LBPs) are beneficial for vision; however, relevant research has mainly focused on entire crude polysaccharides, with the basis and exact structure of the polysaccharide rarely explored. In this study, LICP009-3F-2a, a novel polysaccharide from Lycium barbarum L., was separated and then purified using anion-exchange and size-exclusion chromatography. Structural characteristics were investigated using chemical and spectroscopic methods, which revealed that LICP009-3F-2a has an Mw of 13720 Da and is an acidic heteropolysaccharide composed of rhamnose (39.1%), arabinose (7.4%), galactose (22.5%), glucose (8.3%), galacturonic acid (13.7%), and glucuronic acid (4.0%). Linkage and NMR data revealed that LICP009-3F-2a has the following backbone: →2)-α-L-Rha-(1 → 2,4)-α-L-Rha-(1 → 4)-α-D-GalAp-(1 → 3,6)-β-D-Galp-(1 → 3,6)-β-D-Galp-(1 → 6)-β-D-Galp-(1→, with three main branches, including: α-L-Araf-(1 → 5)-α-L-Araf-(1 → 6)-β-D-Glcp-(1 → 2,4)-α-L-Rha-(1→, β-D-Glcp-(1 → 4)-β-D-Glcp-(1 → 3,6)-β-D-Galp-(1→, and β-D-Galp-(1 → 3)-β-D-Galp-(1 → 3,6)-β-D-Galp-(1 → . Differential scanning colorimetry and thermogravimetric analysis showed that LICP009-3F-2a is thermally stable, while X-ray diffractometry showed that LICP009-3F-2a has a semi-crystalline structure. In addition, LICP009-3F-2a protects ARPE-19 cells from H2O2-induced oxidative damage by regulating the expression of antioxidant SOD1 and CAT enzymes and down-regulating MMP2 expression. Moreover, LICP009-3F-2a promotes the proliferation of ARPE-19 cells in a concentration-dependent manner, and protects ARPE-19 cells from hyperglycemia by inhibiting apoptosis.