Protective effect of paeoniflorin against oxidative stress in human retinal pigment epithelium in vitro.
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This study was conducted to determine whether paeoniflorin (PF) could prevent H₂O₂-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection.Cultured ARPE-19 cells were subjected to oxidative stress with H₂O₂ in the presence and absence of PF. The preventive effective of PF on reactive oxygen species (ROS) production and retinal pigment epithelium (RPE) cell death induced by H₂O₂ was determined by 2',7'- dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescence and 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) assay. The ability of PF to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and 4', 6-diamidino-2-phenylindole (DAPI) staining. Furthermore, the protective effect of PF via the mitogen-activated protein kinase (MAPK) pathway was determined by western blot analysis.PF protected ARPE-19 cells from H₂O₂-induced cell death with low toxicity. H₂O₂-induced oxidative stress increased ROS production and caspase-3 activity, which was significantly inhibited by PF in a dose-dependent manner. Pretreatment with PF attenuated H₂O₂-induced p38MAPK and extracellular signal regulated kinase (ERK) phosphorylation in human RPE cells, which contributed to cell viability in ARPE-19 cells.This is the first report to show that PF can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. The results of this study open new avenues for the use of PF in treatment of ocular diseases, such as age-related macular degeneration (AMD), where oxidative stress plays a major role in disease pathogenesis.Keywords:
DAPI
Paeoniflorin
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<b><i>Aims:</i></b> Oxidative damage plays a vital role in the pathogenesis of age-related macular degeneration (AMD). Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, possesses several pharmacological functions, such as anti-inflammatory and antioxidative properties. However, the effects and mechanism of EX4 on oxidative stress in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) remain unclear. The present study aimed to investigate the protective mechanism of EX4 on human RPE cells subjected to oxidative stress. <b><i>Methods:</i></b> Human RPE ARPE-19 cells were treated with H<sub>2</sub>O<sub>2</sub> to induce oxidative damage. Cell viability was determined by Cell Counting Kit-8 and lactate dehydrogenase assay. Levels of intracellular reactive oxygen species (ROS), malonyldialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH) were measured using commercial kits. The expression of nuclear factor erythroid 2-related factor-2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase-1 (NQO-1) was measured using reverse transcription quantitative polymerase chain reaction assay and western blot, respectively. <b><i>Results:</i></b> H<sub>2</sub>O<sub>2</sub> significantly induced oxidative stress to reduce viability of RPE cells and increased intracellular ROS generation. EX4 significantly ameliorated H<sub>2</sub>O<sub>2</sub>-induced oxidative damage by reducing intracellular ROS generation, decreasing MDA concentration, and increasing antioxidant enzymes activities (SOD and GSH). In addition, EX4 markedly increased expression of <i>NRF2</i>, <i>HO-1</i>, and <i>NQO-1</i> and significantly improved protein expression of NRF2 and HO-1 in H<sub>2</sub>O<sub>2</sub>-treated ARPE-19 cells, caused by increased nuclear NRF2 protein expression. NRF2 knockdown by targeted siRNA alleviated EX4-mediated HO-1 expression and significantly nullified EX4-mediated RPE cell protection against H<sub>2</sub>O<sub>2</sub>. <b><i>Conclusions:</i></b> EX4 attenuated oxidative damage induced by H<sub>2</sub>O<sub>2</sub> in ARPE-19 cells through the activation of the NRF2 signaling pathway. The findings suggested that EX4 may be a potential therapeutic agent for the treatment of AMD.
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Oxidative stress damage to retinal pigment epithelial (RPE) cells is thought to play a critical role in the pathogenesis of age-related macular degeneration (AMD). This study was conducted to investigate the protective effect of canolol against oxidative stress-induced cell death in ARPE-19 cells and its underlying mechanism.ARPE-19 cells, a human retinal pigment epithelial cell line, were subjected to oxidative stress with 150 μM t-butyl hydroxide (t-BH) in the presence/absence of canolol in different concentrations. Cell viabilities were monitored by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) assay. The apoptosis was measured by flow cytometry using Annexin V-FITC and PI staining and intracellular reactive oxygen species (ROS) levels were measured by a fluorescence spectrophotometer. Gene expression of NF-E2-related factor (Nrf-2), heme oxygenase-1 (HO-1), catalase and glutathione S-transferase-pi (GST-pi) were measured by a reverse transcription polymerase chain reaction (RT-PCR) assay. Activation of the extracellular signal regulated kinase (ERK) protein was evaluated by western blot analysis.Canolol showed relatively high safety for ARPE-19 cells and recovered the cell death caused by t-BH dose-dependently at a concentration of 50-200 μM. Canolol also reduced t-BH-induced intracellular ROS generation and thus protected ARPE-19 cells from cell apoptosis. HO-1, catalase, GST-pi, and Nrf-2 were elevated in ARPE-19 cells after treatment with different concentrations of canolol for 24 h. Finally, canolol was found to activate extracellular signal regulated kinase (ERK) phosphorylation in ARPE-19 cells under the condition, with or without t-BH.Canolol protected ARPE-19 cells from t-BH-induced oxidative damage and the protective mechanism was associated, at least partly, with the upregulation (activation) of antioxidative enzymes, probably through an ERK mediated pathway. This suggests that canolol offers a remarkable protective effect against oxidative damage of RPE cells and may have a therapeutic effect on AMD and other oxidative stress-related retinal diseases.
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Salidroside (SAL) is the major pharmacologically active constituent of Rhodiola rosea, which possesses a wide range of pharmacological functions, including anti‑aging, anti‑inflammatory, antioxidant, anticancer and neuroprotective activities. However, the effects and mechanisms of SAL on oxidative stress in retinal pigment epithelial (RPE) cells exposed to hydrogen peroxide (H2O2) remain unclear. The present study investigated the protective effects of SAL and the underlying mechanisms against H2O2‑induced oxidative stress in human RPE cells. ARPE‑19 cells were treated with various doses of SAL for 24 h and then exposed to 200 µM H2O2 for 24 h. Cell viability was analyzed by a MTT assay, and the intracellular levels of reactive oxygen species were measured using CellROX orange reagent. Cell apoptosis was analyzed by annexin V/propidium iodide double staining, followed by flow cytometry. The levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein, phospho (p)‑protein kinase B (Akt), Akt, p‑glycogen synthase kinase (GSK)‑3β and GSK‑3β were evaluated using western blotting. The results demonstrated that SAL markedly attenuated H2O2‑induced loss of cell viability. SAL also ameliorated H2O2‑induced oxidative stress and cell apoptosis in RPE cells. In addition, pretreatment with SAL significantly increased the phosphorylation levels of Akt and GSK‑3β in H2O2‑treated ARPE‑19 cells. In conclusion, the present study demonstrated that SAL protected RPE cells against H2O2‑induced cell injury through the activation of the Akt/GSK‑3β signaling pathway. This suggests that SAL may be a potential therapeutic strategy for the treatment of age‑related macular degeneration.
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Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population and oxidative stress-induced damage to retinal pigment epithelial (RPE) cells occurs as part of the pathogenesis of AMD. In the present study, we evaluated the protective effect of delphinidin (2-(3,4,5-trihydroxyphenyl) chromenylium-3,5,7-triol) against hydrogen peroxide (H2O2)-induced toxicity in human ARPE-19 cells and its molecular mechanism. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry demonstrated that pretreatment of ARPE-19 cells with delphinidin (25, 50, and 100 μg/ml) significantly increased cell viability and reduced the apoptosis from H2O2 (0.5 mM)-induced oxidative stress in a concentration-dependent manner, which was achieved by the inhibition of Bax, cytochrome c, and caspase-3 protein expression and enhancement of Bcl-2 protein. The same tendency was observed in ARPE-19 cells pre-treated with 15 mM of N-acetylcysteine (NAC) before the addition of H2O2 Furthermore, pre-incubation of ARPE-19 cells with delphinidin markedly inhibited the intracellular reactive oxygen species (ROS) generation and Nox1 protein expression induced by H2O2 Moreover, the decreased antioxidant enzymes activities of superoxide dismutase (SOD), catalase (CAT), and glutathione-peroxidase (GSH-PX) and elevated (MDA) level in H2O2-treated cells were reversed to the normal standard by the addition of delphinidin, which was regulated by increasing nuclear Nrf2 protein expression in ARPE-19 cells. Our results suggest that delphinidin effectively protects human ARPE-19 cells from H2O2-induced oxidative damage via anti-apoptotic and antioxidant effects.
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Oxidative stress-induced damage to RPE cells has been suggested to be an important factor in the pathogenesis of age-related macular degeneration. Taxifolin, a flavonol, has been shown to exhibit significant antioxidant properties. The purpose of this study was to investigate the potential protective effects of taxifolin on RPE cells cultured under oxidative stress conditions and to elucidate the underlying mechanisms.Human RPE (ARPE-19) cells were treated with different concentrations of taxifolin and 0.4 mM of H2O2 for 24 h. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was quantitatively measured by annexin V/propidium iodide double staining, and the expression levels of poly (ADP-ribose) polymerase (PARP) were evaluated by western blotting. Reactive oxygen species (ROS) were measured using a commercially available ROS detection system. The expressions of phase II enzymes, including NAD(P)H quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase modifier (GCLM) and catalytic (GCLC) subunits, were examined using real-time PCR and western blotting. The nuclear localization of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) protein was detected by western blotting. Results: Taxifolin clearly inhibited the decrease in H2O2-induced cell viability, cell apoptosis, and intracellular ROS generation. In addition, taxifolin inhibited the H2O2-induced PARP cleavage. Moreover, treatment with taxifolin activated mRNA and the protein expression of NRF2 by inducing the translocation of NRF2 to the nucleus. Consequently, the mRNA and protein levels of the phase II enzymes NQO1, HO-1, GCLM, and GCLC increased. Conclusions: Taxifolin was shown to protect RPE cells against oxidative stress-induced apoptosis. The potential mechanism appears to involve the activation of NRF2 and the phase II antioxidant enzyme system.
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The production of reactive oxygen species (ROS) during hyperoxia contribute to alveolar epithelial apoptosis. In the present study, the molecular mechanisms of oxidative stress–induced alveolar epithelial cell apoptosis were investigated. The cytoprotective effects of N-acetylcysteine (NAC) were evaluated. Treatments using 500 μM H2O2 can induce primary alveolar type II epithelial cell apoptosis. During this procedure, c-Jun N-terminal kinase (JNK) was activated. SP600125, a specific inhibitor of JNK, can partially block H2O2-induced alveolar type II epithelial cells (ATII cells). SP600125 also attenuated Bax protein content and p53 nuclear accumulation induced by H2O2. NAC (5 mM) pretreatment decreased H2O2-induced ATII cell apoptosis. The high level of intracellular reactive oxygen species (ROS) induced by H2O2 was also attenuated by NAC pretreatment. Taken together, H2O2 can induce primary ATII cells apoptosis and increase JNK phosphorylation. NAC, a precursor of glutathione (GSH) synthesis, can protect ATII cells from H2O2-induced apoptosis through scavenging ROS.
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Because oxidative stress is assumed to be a key mechanism in the pathological process of age-related macular degeneration (AMD), increasing numbers of studies have focused on discovering new pathways and treatments for reducing oxidative damage. Our work investigates the potential role of the cannabinoid receptor 1 (CB1) in oxidative stress of primary human retinal pigment epithelial (RPE) cells, a cellular model of AMD.Primary human RPE cells were cultured and exposed to hydrogen peroxide for 24 h to induce oxidative damage. The expression of and changes in the CB1 receptor were determined with western blot assay and confocal imaging. The CB1 receptor in the RPE cells was inhibited with small interfering RNA (siRNA) or rimonabant (SR141716). Cell viability, apoptosis, and reactive oxygen species production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulforhodamine B assay, annexin V and propidium iodide staining, and the dichlorofluorescein fluorescence assay, respectively. Intracellular superoxide dismutase activity was assayed with a commercially available assay kit. Phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) protein expression and activation of signaling molecules were assessed with western blot analysis.We showed that human RPE cells express the CB1 receptor. In addition, oxidative stress upregulates the expression of the CB1 receptor. Deleting the CB1 receptor or treating with the CB1 receptor antagonist rimonabant (SR141716) rescued RPE cells from hydrogen peroxide-induced oxidative damage. Rimonabant pretreatment effectively reduced the apoptosis of RPE cells, inhibited the generation of intracellular reactive oxygen species and elevated the activity of superoxide dismutase. In addition, rimonabant significantly strengthened the oxidative stress-induced activation of the PI3K/Akt signaling pathway.The results demonstrate the expression and regulation of CB1 receptors in human RPE cells. Inhibiting the CB1 receptor may be an effective therapeutic strategy for AMD by downregulating oxidative stress signaling and facilitating PI3K/Akt activation.
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Background:We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells).Methods: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H 2 O 2 ).Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry.Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis.The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis.Results: ALS-L1023 clearly reduced H 2 O 2 -induced cell apoptosis and intracellular production of ROS.H 2 O 2 -induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023.Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells.Conclusions: ALS-L1023 protected human RPE cells against oxidative damage.This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration.
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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.
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Hydroquinone
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