Anti-Apoptotic Effect of Chrysophanol Isolated from Cassia tora Seed Extract on Blue-Light-Induced A2E-Loaded Human Retinal Pigment Epithelial Cells.

The seeds of Cassia tora (C. tora) species mainly contain anthraquinone, anthraquinone glycoside, and naphthalene derivatives. We investigated the anti-apoptotic effects of C. tora seed extract and its isolated compounds on blue-light-induced lipofuscin (A2E)-loaded human retinal pigment epithelial (RPE) cells. For analysis of the C. tora extract, high-performance liquid chromatography method was used. A2E-loaded human retinal pigment epithelial cells and blue light were used to create excessive photo-oxidation to induce cell death. Lactate dehydrogenase (LDH) assay was used to measure cell cytotoxicity, and the mRNA expression of genes involved in apoptosis was examined to evaluate the mechanism of cell death. C. tora extract, n-hexane fraction, and chrysophanol were found to inhibit apoptotic cell death. Additionally, C. tora extract, n-hexane fraction, and chrysophanol reduced the mRNA expression of genes involved in the apoptosis pathway. C. tora and chrysophanol were considered to inhibit apoptosis and oxidative stress response. The major component of C. tora has a protective effect against apoptosis. The ingredients of C. tora can be used as therapeutic substances or to prevent diseases caused by the excessive oxidation of A2E substances in the retina, such as in age-related macular degeneration.

Antioxidative and Mitochondrial Protection in Retinal Pigment Epithelium: New Light Source in Action.

Low-color-temperature light-emitting diodes (LEDs) (called 1900 K LEDs for short) have the potential to become a healthy light source due to their blue-free property. Our previous research demonstrated that these LEDs posed no harm to retinal cells and even protected the ocular surface. Treatment targeting the retinal pigment epithelium (RPE) is a promising direction for age-related macular degeneration (AMD). Nevertheless, no study has evaluated the protective effects of these LEDs on RPE. Therefore, we used the ARPE-19 cell line and zebrafish to explore the protective effects of 1900 K LEDs. Our results showed that the 1900 K LEDs could increase the cell vitality of ARPE-19 cells at different irradiances, with the most pronounced effect at 10 W/m2. Moreover, the protective effect increased with time. Pretreatment with 1900 K LEDs could protect the RPE from death after hydrogen peroxide (H2O2) damage by reducing reactive oxygen species (ROS) generation and mitochondrial damage caused by H2O2. In addition, we preliminarily demonstrated that irradiation with 1900 K LEDs in zebrafish did not cause retinal damage. To sum up, we provide evidence for the protective effects of 1900 K LEDs on the RPE, laying the foundation for future light therapy using these LEDs.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Qihuang Granule protects the retinal pigment epithelium from oxidative stress via regulation of the alternative complement pathway.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of vision loss in elderly people, and dry AMD is the most common type of AMD. Oxidative stress and alternative complement pathway activation may play essential roles in the pathogenesis of dry AMD. There are no available drugs for dry AMD. Qihuang Granule (QHG) is an herbal formula for the treatment of dry AMD, and it achieves a good clinical effect in our hospital. However, its potential mechanism is unclear. Our study investigated the effects of QHG on oxidative stress-associated retinal damage to reveal its underlying mechanism. METHODS: Oxidative stress models were established using H2O2 and NaIO3 in ARPE-19 cells and C57BL/6 mice. Cell apoptosis and viability were assessed using phase contrast microscopy and flow cytometry, respectively. Alterations in the mouse retinal structure were evaluated using Masson staining and transmission electron microscopy (TEM). The expression of complement factor H (CFH), complement component 3a (C3a) and complement component 5a (C5a) in retinal pigment epithelium (RPE) cells and mice was measured using RT‒PCR, Western blot analysis and ELISA. RESULTS: Pretreatment with QHG significantly prevented cell apoptosis and disorder of the RPE and inner segment/outer segment (IS/OS) in H2O2-treated RPE cells and NaIO3-injected mice. QHG alleviated mitochondrial damage in mouse RPE cells, as shown by TEM. QHG also promoted CFH expression and inhibited the expression of C3a and C5a. CONCLUSIONS: The results suggest that QHG protects the retinal pigment epithelium from oxidative stress, likely by regulating the alternative complement pathway.

Sphingosine-1-phosphate and ceramide-1-phosphate promote migration, pro-inflammatory and pro-fibrotic responses in retinal pigment epithelium cells.

Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 µM S1P or 10 µM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.

Lycium barbarum L. and Salvia miltiorrhiza Bunge protect retinal pigment epithelial cells through endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum L. and Salvia miltiorrhiza Bunge (Gouqi and Danshen, LS) are traditional herbs for the treatment of retinal degeneration in China. LS have been integrated into pharmacopoeia and health care system of many countries around the world. However, the mechanisms by which LS protect retina are not fully clarified. AIM OF THE STUDY: We aimed at exploration of the effect of LS on retinal pigment epithelium (RPE) cells apoptosis as well as the endoplasmic reticulum (ER) stress mechanisms. MATERIAL AND METHODS: ARPE-19 cells were exposed to tunicamycin to induce ER stress, followed by LS treatment for 24 h. The cell morphology was photographed using the Incucyte S3 instrument, and the potential cytotoxic effect and viability were evaluated by CCK-8 assays. The Annexin V-FITC/PI staining and TUNEL assay were conducted to detect cells apoptotic. Western blot and digital PCR were used to detected related protein and gene expression. RESULTS: The ARPE-19 cells are increased in number and aligned after treating with LS. 1 mg/ml is the LS high dose group dose and treatment with LS increased cell vitality. LS significantly inhibit ARPE-19 cells apoptosis. Moreover, LS were markedly decreased the expression levels of ER stress-related factors in the ARPE-19 cells. CONCLUSIONS: This study reveals that LS relieve ARPE-19 cells apoptosis by inhibiting ER stress, and here we can speculate that LS have a certain protective effect on retina.

Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium.

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula.

Purpose: With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods: Eyes (N = 139 donors, 61 women and 78 men, aged 40-80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results: A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions: Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

Riboflavin deficiency leads to irreversible cellular changes in the RPE and disrupts retinal function through alterations in cellular metabolic homeostasis.

Ariboflavinosis is a pathological condition occurring as a result of riboflavin deficiency. This condition is treatable if detected early enough, but it lacks timely diagnosis. Critical symptoms of ariboflavinosis include neurological and visual manifestations, yet the effects of flavin deficiency on the retina are not well investigated. Here, using a diet induced mouse model of riboflavin deficiency, we provide the first evidence of how retinal function and metabolism are closely intertwined with riboflavin homeostasis. We find that diet induced riboflavin deficiency causes severe decreases in retinal function accompanied by structural changes in the neural retina and retinal pigment epithelium (RPE). This is preceded by increased signs of cellular oxidative stress and metabolic disorder, in particular dysregulation in lipid metabolism, which is essential for both photoreceptors and the RPE. Though many of these deleterious phenotypes can be ameliorated by riboflavin supplementation, our data suggests that some patients may continue to suffer from multiple pathologies at later ages. These studies provide an essential cellular and mechanistic foundation linking defects in cellular flavin levels with the manifestation of functional deficiencies in the visual system and paves the way for a more in-depth understanding of the cellular consequences of ariboflavinosis.

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells.

BACKGROUND: Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics. METHODS: We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins. RESULTS: We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells. CONCLUSIONS: Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

Nutraceuticals/Drugs Promoting Mitophagy and Mitochondrial Biogenesis May Combat the Mitochondrial Dysfunction Driving Progression of Dry Age-Related Macular Degeneration.

In patients with age-related macular degeneration (AMD), the crucial retinal pigment epithelial (RPE) cells are characterized by mitochondria that are structurally and functionally defective. Moreover, deficient expression of the mRNA-editing enzyme Dicer is noted specifically in these cells. This Dicer deficit up-regulates expression of Alu RNA, which in turn damages mitochondria-inducing the loss of membrane potential, boosting oxidant generation, and causing mitochondrial DNA to translocate to the cytoplasmic region. The cytoplasmic mtDNA, in conjunction with induced oxidative stress, triggers a non-canonical pathway of NLRP3 inflammasome activation, leading to the production of interleukin-18 that acts in an autocrine manner to induce apoptotic death of RPE cells, thereby driving progression of dry AMD. It is proposed that measures which jointly up-regulate mitophagy and mitochondrial biogenesis (MB), by replacing damaged mitochondria with "healthy" new ones, may lessen the adverse impact of Alu RNA on RPE cells, enabling the prevention or control of dry AMD. An analysis of the molecular biology underlying mitophagy/MB and inflammasome activation suggests that nutraceuticals or drugs that can activate Sirt1, AMPK, Nrf2, and PPARα may be useful in this regard. These include ferulic acid, melatonin urolithin A and glucosamine (Sirt1), metformin and berberine (AMPK), lipoic acid and broccoli sprout extract (Nrf2), and fibrate drugs and astaxanthin (PPARα). Hence, nutraceutical regimens providing physiologically meaningful doses of several or all of the: ferulic acid, melatonin, glucosamine, berberine, lipoic acid, and astaxanthin, may have potential for control of dry AMD.

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 (H2 O2 ) 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 H2 O2 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.

Ilex paraguariensis Extracts Prevent Oxidative Damage in a Mouse Model of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), a chronic disease of the retina, leads to severe visual loss. AMD affects the retinal pigment epithelium (RPE) and the visual cells (photoreceptors). RPE failure, the first step of this disease, is associated with oxidative stress. Since antioxidants can slow down AMD progression, the intake of foods and drinks rich in antioxidant compounds may reduce retinal damage. Ilex paraguariensis (yerba mate, YM) extracts reduce oxidative damage of RPE cells in vitro as shown in previous study. Here, the effects of YM drinking on RPE and photoreceptor survival after oxidative damage with sodium iodate (NaIO3; SI) in a murine AMD model are described. Funduscopy and histology show that YM treatment prevents RPE and photoreceptor damage. YM also increases the expression of NRF2, the master antioxidant gene, and its effectors HO-1 and SOD2. In mice receiving YM and SI, the antioxidant response is larger than in mice receiving YM or SI alone. The YM drink also increases expression of RPE65, a gene that is involved in the functionality and survival of photoreceptors and RPE cells. The results suggest YM can play an important role in the prevention of retinal damage associated with oxidative stress, such as AMD.

HDL is the primary transporter for carotenoids from liver to retinal pigment epithelium in transgenic ApoA-I-/-/Bco2-/- mice.

Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â‰« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.

Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model.

Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.

A Potential New Role for Zinc in Age-Related Macular Degeneration through Regulation of Endothelial Fenestration.

Age-related macular degeneration (AMD) is a common blinding disease in the western world that is linked to the loss of fenestration in the choriocapillaris that sustains the retinal pigment epithelium and photoreceptors in the back of the eye. Changes in ocular and systemic zinc concentrations have been associated with AMD; therefore, we hypothesized that these changes might be directly involved in fenestrae formation. To test this hypothesis, an endothelial cell (bEND.5) model for fenestrae formation was treated with different concentrations of zinc sulfate (ZnSO4) solution for up to 20 h. Fenestrae were visualized by staining for Plasmalemmal Vesicle Associated Protein-1 (PV-1), the protein that forms the diaphragms of the fenestrated endothelium. Size and distribution were monitored by transmission electron microscopy (TEM). We found that zinc induced the redistribution of PV-1 into areas called sieve plates containing ~70-nm uniform size and typical morphology fenestrae. As AMD is associated with reduced zinc concentrations in the serum and in ocular tissues, and dietary zinc supplementation is recommended to slow disease progression, we propose here that the elevation of zinc concentration may restore choriocapillaris fenestration resulting in improved nutrient flow and clearance of waste material in the retina.

Assessing the protective effects of cryptotanshinone on CoCl2­induced hypoxia in RPE cells.

The development of several retinal diseases is closely related to hypoxia. As a component of the Traditional Chinese medicine Salvia miltiorrhiza, the effects of cryptotanshinone (CT) on retinal cells under hypoxic conditions are not well understood. The aim of the present study was to explore how CT exerted its protective effects on retinal pigment epithelium (RPE) cells under hypoxic conditions induced by cobalt chloride (CoCl2). The effects of CT were investigated using a Cell Counting Kit­8 assay, Annexin V­FITC/PI staining, reverse transcription­quantitative PCR and western blotting in ARPE­19 cells. CT (10 and 20 µM) reduced the CoCl2­induced increase in vascular endothelial growth factor expression and hypoxia­inducible transcription factor­1α expression in ARPE­19 cells. Additionally, CT alleviated hypoxia­induced apoptosis by regulating Bcl­2 and Bax protein expression. CT treatment also reduced the increase in the mRNA levels of IL­6, IL­1ß and TNF­α induced by CoCl2. In summary, CT may protect RPE cells against apoptosis and inflammation in CoCl2­induced hypoxia, and these results warrant further in vivo study into its value as a drug for treating hypoxic eye diseases.

MiR-195 inhibits the ubiquitination and degradation of YY1 by Smurf2, and induces EMT and cell permeability of retinal pigment epithelial cells.

The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin-eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.

Lutein and zeaxanthin reduce A2E and iso-A2E levels and improve visual performance in Abca4-/-/Bco2-/- double knockout mice.

Accumulation of bisretinoids such as A2E and its isomer iso-A2E is thought to mediate blue light-induced oxidative damage associated with age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1). We hypothesize that increasing dietary intake of the macular carotenoids lutein and zeaxanthin in individuals at risk of AMD and STGD1 can inhibit the formation of bisretinoids A2E and iso-A2E, which can potentially ameliorate macular degenerative diseases. To study the beneficial effect of macular carotenoids in a retinal degenerative diseases model, we used ATP-binding cassette, sub-family A member 4 (Abca4-/-)/ß,ß-carotene-9',10'-oxygenase 2 (Bco2-/-) double knockout (KO) mice that accumulate elevated levels of A2E and iso-A2E in the retinal pigment epithelium (RPE) and macular carotenoids in the retina. Abca4-/-/Bco2-/- and Abca4-/- mice were fed a lutein-supplemented chow, zeaxanthin-supplemented chow or placebo chow (~2.6 mg of carotenoid/mouse/day) for three months. Visual function and electroretinography (ERG) were measured after one month and three months of carotenoid supplementation. The lutein and zeaxanthin supplemented Abca4-/-/Bco2-/- mice had significantly lower levels of RPE/choroid A2E and iso-A2E compared to control mice fed with placebo chow and improved visual performance. Carotenoid supplementation in Abca4-/- mice minimally raised retinal carotenoid levels and did not show much difference in bisretinoid levels or visual function compared to the control diet group. There was a statistically significant inverse correlation between carotenoid levels in the retina and A2E and iso-A2E levels in the RPE/choroid. Supplementation with retinal carotenoids, especially zeaxanthin, effectively inhibits bisretinoid formation in a mouse model of STGD1 genetically enhanced to accumulate carotenoids in the retina. These results provide further impetus to pursue oral carotenoids as therapeutic interventions for STGD1 and AMD.

Procyanidin B2 and rutin in Ginkgo biloba extracts protect human retinal pigment epithelial (RPE) cells from oxidative stress by modulating Nrf2 and Erk1/2 signalling.

Oxidative stress plays an important role in the pathogenesis of human retinal diseases. Ginkgo biloba products are widely consumed herbal supplements that contain ingredients with anti-oxidant potentials. However, the active agents in ginkgo biloba extracts (GBE) are unclear. This study assessed the anti-oxidant effects of 19 natural compounds isolated from GBE to provide a rational basis for their use in preventing retinal diseases. The compounds were tested in retinal pigment epithelial (RPE) cells subjected to tert-butyl hydroperoxide (t-BHP)-induced oxidative stress. Cell viability and intracellular reactive oxygen species (ROS) were assessed and flow cytometry was used to delineate the cell death profile. The expression of nuclear factor erythroid 2-related factor-2 (Nrf2) was activated in RPE cells by t-BHP accompanied with an activation of Erk1/2 signaling. GBE-derived rutin and procyanidin B2 ameliorated t-BHP-induced cell death and promoted cell viability by suppressing intracellular ROS generation. These agents also enhanced Nrf2 expression with activating Erk1/2 signaling in RPE cells. In contrast, the other compounds tested were minimally active and did not prevent the loss of cell viability elicited by t-BHP. The present findings suggest that rutin and procyanidin B2 may have potential therapeutic values in the prevention of retinal diseases induced by oxidative damage.