RESUMO
Bisphenol A (BPA) may adversely affect human health by inducing oxidative stress and irreversible damage to cells. Bioactive compounds found in some functional foods, individually or in combination, can attenuate the negative effects of BPA exposure; an example is the multi-supplement containing guarana (Gua), selenium (Se), and L-carnitine (LC) -GSC- which has already demonstrated antioxidant, genoprotective, and immunomodulatory activities. This study aimed to determine the effect of GSC and its constituents on oxidative and genotoxic alterations triggered by BPA exposure in the retinal epithelial cell line. The cells exposed to BPA (0.001, 0.01, 0.1, 1, 3, and 10 µM) to determine the lowest concentration required to induce cyto-genotoxicity. ARPE-19 cells were then concomitantly exposed to the selected BPA concentration, GSC, and its components (Gua, 1.07 mg/mL; Se, 0.178 µg/mL; and LC, 1.43 mg/mL). Flow cytometry, biochemical assays, qRT-PCR, genotoxicity, apoptosis, and cellular proliferation. Based on our results, 10 µM of BPA could induce cyto-genotoxic and oxidative alterations. BPA did not alter the Bcl-2/BAX expression ratio but induced Casp3 and Casp8 overexpression, suggesting that apoptosis was induced mainly via the extrinsic pathway. GSC partially reversed the alterations triggered by BPA in ARPE-19 cells. However, Se had unexpected negative effects on ARPE-19 cells. The multi-supplement GSC may attenuate changes in oxidative and genotoxic markers related to exposure of ARPE-19 cells to BPA. our results revealed that the antioxidant, anti-apoptotic, and genoprotective properties of GSC were not universally shared by its individual, once Se did not exhibit any positive impact.
Assuntos
Apoptose , Compostos Benzidrílicos , Carnitina , Estresse Oxidativo , Fenóis , Epitélio Pigmentado da Retina , Selênio , Fenóis/toxicidade , Compostos Benzidrílicos/toxicidade , Humanos , Selênio/farmacologia , Carnitina/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Paullinia/química , Dano ao DNA/efeitos dos fármacos , Antioxidantes/farmacologia , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo , Suplementos NutricionaisRESUMO
Resveratrol (RES) is a polyphenol with increasing interest for its inhibitory effects on a wide variety of viruses. Zika virus (ZIKV) is an arbovirus which causes a broad spectrum of ophthalmological manifestations in humans. Currently there is no certified therapy or vaccine to treat it, thus it has become a major global health threat. Retinal pigment epithelium (RPE) is highly permissive and susceptible to ZIKV. This work explored the protective effects of RES on ZIKV-infected human RPE cells. RES treatment resulted in a significant reduction of infectious viral particles in infected male ARPE-19 and female hTERT-RPE1 cells. This protection was positively influenced by the action of RES on mitochondrial dynamics. Also, docking studies predicted that RES has a high affinity for two enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis and viral polymerase. This evidence suggests that RES might be a potential antiviral agent to treat ZIKV-induced ocular abnormalities.
Assuntos
Antivirais/farmacologia , Resveratrol/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/virologia , Zika virus/efeitos dos fármacos , Antivirais/química , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Desenvolvimento de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Humanos , Ligantes , Dinâmica Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Resveratrol/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/patologia , Infecção por Zika virus/virologiaRESUMO
Purpose: This study investigated the safety and therapeutic efficacy of licarin A (LCA) in the treatment of intraocular inflammation. Methods:In vitro safety of LCA in retinal pigmented epithelial cells (ARPE-19) and human embryonic stem cell derived-retinal pigmented epithelial cells (hES-RPE) was evaluated using CellTiter-Blue® kit. The chorioallantoic membrane (CAM) assay was used to investigate LCA safety and antiangiogenic activity. In vivo safety of intravitreal LCA was accomplished by clinical examination (including assessment of intraocular pressure), electroretinography (ERG), and histopathology. Uveitis was induced in rats by subcutaneous and intravitreal injection of bacillus Calmette-Guérin (BCG) antigen of Mycobacterium bovis. Intraocular inflammation was graded by slit-lamp and fundus examination, ERG, and histopathology. Results: LCA was safe to cells and to the CAM at concentration below 12.0 µM. LCA significantly reduced the percentage of blood vessels in the CAM. Retinal safety and anti-inflammatory efficacy of intravitreal injection of LCA 6.0 µM were confirmed through clinical, functional, and histopathological evaluation. Significant reduction of inflammatory cytokines (tumor necrosis factor-α and interleukin-6) was also found, when compared to untreated animals. Conclusion: The results suggest that LCA is a potential new drug for the treatment of inflammatory eye disease.
Assuntos
Inibidores da Angiogênese/farmacologia , Inflamação/tratamento farmacológico , Lignanas/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Membrana Corioalantoide/metabolismo , Modelos Animais de Doenças , Descoberta de Drogas , Eletrorretinografia/métodos , Oftalmopatias/patologia , Inflamação/diagnóstico , Pressão Intraocular/efeitos dos fármacos , Injeções Intravítreas , Lignanas/administração & dosagem , Lignanas/uso terapêutico , Masculino , Ratos , Ratos Wistar , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Segurança , Resultado do Tratamento , Uveíte/induzido quimicamente , Uveíte/patologiaRESUMO
Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33â¯mM) or to normal glucose concentration (NG, 5.5â¯mM) for 4, 24 or 72â¯h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72â¯h of incubation. In addition, short term HG exposure (4â¯h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24â¯h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.
Assuntos
Retinopatia Diabética/metabolismo , Glucose/farmacologia , Fosfolipase D/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Interleucina-6/genética , Interleucina-8/genética , Microscopia Confocal , Microscopia de Fluorescência , Estresse Oxidativo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/metabolismo , Quinase Induzida por NF-kappaBRESUMO
PURPOSE: Investigate the potential application of corosolic acid (CA) in the treatment of diseases causing retinal neovascularization. METHODS: CA cytotoxicity effect was evaluated in ARPE-19 cells by sulforhodamine B colorimetric method, and antiangiogenic activity was studied using chorioallantoic membrane (CAM) assay. An amount of 0.01 mL of CA formulations at 5, 10 and 25 µM was injected in the right eyes of Wistar rats, and the contralateral eyes received the vehicle to verify the safety of ophthalmic use. Electroretinography (ERG) was performed before, 7 and 15 days after CA administration. Animals were killed on the 15th day, and the histological analysis of retina was carried out under light microscopy. RESULTS: CA did not present cytotoxicity at concentrations below 35.5 µM after 48 h of treatment. The antiangiogenic activity was confirmed by CAM assay, since CA (range from 5 to 25 µM) induced a significant reduction in vascularity without any signs of toxicity. ERG recordings and histological evaluation did not show any signs of retinal toxicity. CONCLUSIONS: CA was effective in reducing vascularity in a CAM model and was found to be safe for potential ophthalmic use.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Retina/efeitos dos fármacos , Triterpenos/administração & dosagem , Inibidores da Angiogênese/toxicidade , Animais , Linhagem Celular , Membrana Corioalantoide/irrigação sanguínea , Eletrorretinografia/efeitos dos fármacos , Injeções Intravítreas , Masculino , Neovascularização Patológica/tratamento farmacológico , Ratos , Ratos Wistar , Epitélio Pigmentado da Retina/efeitos dos fármacos , Triterpenos/toxicidade , Corpo Vítreo/efeitos dos fármacosRESUMO
PURPOSE: To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. METHODS: PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. RESULTS: After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. CONCLUSIONS: Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.
Assuntos
Imunossupressores/farmacocinética , Imunossupressores/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sirolimo/farmacocinética , Sirolimo/toxicidade , Corpo Vítreo/metabolismo , Implantes Absorvíveis , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular , Sistemas de Liberação de Medicamentos , Implantes de Medicamento , Eletrorretinografia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Injeções Intravítreas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Retina/efeitos dos fármacosRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Assuntos
Autofagia/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular , Citometria de Fluxo , Humanos , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.
Assuntos
Humanos , Proteínas Quinases/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Glucose/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Quinases/metabolismo , Autofagia/fisiologia , Transdução de Sinais/fisiologia , Linhagem Celular , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Eletrônica de Transmissão , Epitélio Pigmentado da Retina/citologia , Citometria de Fluxo , Proteínas de Membrana/metabolismoRESUMO
Breakdown of the blood-retinal barrier (BRB), as occurs in diabetic retinopathy and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing vision loss. Vasoinhibins are N-terminal fragments of prolactin that prevent BRB breakdown during diabetes. They modulate the expression of some transient receptor potential (TRP) family members, yet their role in regulating the TRP vanilloid subtype 4 (TRPV4) remains unknown. TRPV4 is a calcium-permeable channel involved in barrier permeability, which blockade has been shown to prevent and resolve pulmonary edema. We found TRPV4 expression in the endothelium and retinal pigment epithelium (RPE) components of the BRB, and that TRPV4-selective antagonists (RN-1734 and GSK2193874) resolve BRB breakdown in diabetic rats. Using human RPE (ARPE-19) cell monolayers and endothelial cell systems, we further observed that (i) GSK2193874 does not seem to contribute to the regulation of BRB and RPE permeability by vasoinhibins under diabetic or hyperglycemic-mimicking conditions, but that (ii) vasoinhibins can block TRPV4 to maintain BRB and endothelial permeability. Our results provide important insights into the pathogenesis of diabetic retinopathy that will further guide us toward rationally-guided new therapies: synergistic combination of selective TRPV4 blockers and vasoinhibins can be proposed to mitigate diabetes-evoked BRB breakdown.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Humanos , Masculino , Piperidinas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Wistar , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2-26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2-26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2-26 peptide, in a concentration of 1.7µM and 33.8µM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2-26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2-26 peptide at 33.8µM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2-26 peptide as an innovative therapy for the treatment of ocular inflammation.