Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

Curcumin prevents high glucose damage in retinal pigment epithelial cells through ERK1/2-mediated activation of the Nrf2/HO-1 pathway

To study the effects of curcumin on human retinal pigment epithelial (RPE) cells exposed to high glucose (HG) insult, we performed in vitro studies on RPE cells cultured both in normal and HG conditions to assess the effects of curcumin on the cell viability, nuclear factor erythroid 2-related factor 2 (Nrf2) expression, HO-1 activity, and ERK1/2 expression. RPE cells exposed to HG insult were treated with curcumin. The cell viability, apoptosis, HO-1 activity, ERK, and Nrf2 expression were evaluated. The data indicated that treatment with curcumin caused a significant decrease in terms of apoptosis. Further, curcumin was able to induce HO-1 expression via Nrf2 activation and counteracts the damage elicited by HG. The present study demonstrated that curcumin provides protection against HG-induced damage in RPE cells through the activation of Nrf2/HO-1 signaling that involves the ERK pathway, suggesting that curcumin may have therapeutic value in the treatment of diabetic retinopathy.     

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19?cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation.     

Salidroside protects retinal endothelial cells against hydrogen peroxide-induced injury via modulating oxidative status and apoptosis

Oxidative stress can cause injury in retinal endothelial cells. Salidroside is a strong antioxidative and cytoprotective supplement in Chinese traditional medicine. In this study, we investigated the effects of salidroside on H₂O₂-induced primary retinal endothelial cells injury. Salidroside decreased H₂O₂-induced cell death, and efficiently suppressed cellular ROS production, malondialdehyde generation, and cell apoptosis induced by H₂O₂ treatment. Salidroside induced the intracellular mRNA expression, protein expression, and enzymatic activities of catalase and Mn-SOD and increased the ratio of Bcl2/Bax. Our results demonstrated that salidroside protected retinal endothelial cells against oxidative injury through increasing the Bcl2/Bax signaling pathway and activation of endogenous antioxidant enzymes. This finding presents salidroside as an attractive agent with potential to attenuate retinopathic diseases.     

Hyperbaric oxygen therapy suppresses hypoxia and reoxygenation injury to retinal pigment epithelial cells through activating peroxisome proliferator activator receptor-alpha signalling

Retinal ischemia followed by reperfusion (IR) is a common cause of many ocular disorders, such as age-related macular degeneration (AMD), which leads to blindness in the elderly population, and proper therapies remain unavailable. Retinal pigment epithelial (RPE) cell death is a hallmark of AMD. Hyperbaric oxygen (HBO) therapy can improve IR tissue survival by inducing ischemic preconditioning responses. We conducted an in vitro study to examine the effects of HBO preconditioning on oxygen–glucose deprivation (OGD)-induced IR-injured RPE cells. RPE cells were treated with HBO (100% O₂ at 3 atmospheres absolute for 90 min) once a day for three consecutive days before retinal IR onset. Compared with normal cells, the IR-injured RPE cells had lower cell viability, lower peroxisome proliferator activator receptor-alpha (PPAR-α) expression, more severe oxidation status, higher blood-retinal barrier disruption and more elevated apoptosis and autophagy rates. HBO preconditioning increased PPAR-α expression, improved cell viability, decreased oxidative stress, blood-retinal barrier disruption and cellular apoptosis and autophagy. A specific PPAR-α antagonist, GW6471, antagonized all the protective effects of HBO preconditioning in IR-injured RPE cells. Combining these observations, HBO therapy can reverse OGD-induced RPE cell injury by activating PPAR-α signalling.     

Ferulic acid (FA) protects human retinal pigment epithelial cells from H2O2‐induced oxidative injuries

The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H₂O₂)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H₂O₂‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H₂O₂‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H₂O₂‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.     

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

ABSTRACT

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.     

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.     

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.     

Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.     

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress

Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.     

Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling

Reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelial (RPE) cells, which is known as one major cause of age-related macular degeneration. Salvianolic acid A (Sal A) is the main effective aqueous extract of Salvia miltiorrhiza. The aim of this study was to test the potential role of Sal A against oxidative stress in cultured RPE cells and to investigate the underlying mechanistic signaling pathways. We observed that Sal A significantly inhibited hydrogen peroxide (H₂O₂)-induced primary and transformed RPE cell death and apoptosis. H₂O₂-stimulated mitogen-activated protein kinase activation, ROS production, and subsequent proapoptotic AMP-activated protein kinase activation were largely inhibited by Sal A. Further, Sal A stimulation resulted in a fast and dramatic activation of Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, followed by phosphorylation, accumulation, and nuclear translocation of the NF-E2-related factor 2 (Nrf2), along with increased expression of the antioxidant-response element-dependent gene heme oxygenase-1 (HO-1). Both Nrf2 and HO-1 were required for Sal A-mediated cytoprotective effect, as Nrf2/HO-1 inhibition abolished Sal A-induced beneficial effects against H₂O₂. Meanwhile, the PI3K/Akt/mTORC1 chemical inhibitors not only suppressed Sal A-induced Nrf2/HO-1 activation, but also eliminated its cytoprotective effect in RPE cells. These observations suggest that Sal A activates the Nrf2/HO-1 axis in RPE cells and protects against oxidative stress via activation of Akt/mTORC1 signaling.     

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.     

Rapamycin sensitive mTOR activation mediates nerve growth factor (NGF) induced cell migration and pro-survival effects against hydrogen peroxide in retinal pigment epithelial cells

Patients with age related macular degeneration (AMD) have a loss of vision in the center of the visual field. Oxidative stress plays an important role in this progress. Nerve growth factor (NGF) is important for the survival and maintenance of sympathetic and sensory neurons and NGF eye drops improve visual acuity and electro-functional activity in patients with AMD. However, the molecular mechanisms and signaling events involved in this have not been fully investigated. Using cultured human retinal pigment epithelial (RPE) cells, we demonstrate here that NGF protects RPE cells against hydrogen peroxide (H₂O₂)-induced cell apoptosis. NGF also induces RPE cell migration, the latter is important for retinal regeneration and the recovery from AMD. H₂O₂ decreases S6 phosphorylation and cell viability, which is restored by NGF. Rapamycin, the pharmacologic inhibitor of mammalian target of rapamycin (mTOR), diminished NGF-induced S6 phosphorylation, cell migration and protective effects against oxidative stress. Collectively, we conclude that activation of rapamycin sensitive mTOR signaling mediates NGF induced cell migration and pro-survival effects in H₂O₂ treated RPE cells.     

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2 (E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.