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.     

Polyphenols suppress hydrogen peroxide‐induced oxidative stress in human bone‐marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell‐based tissue engineering and regeneration because of their self‐renewal and multi‐lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin‐3‐gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation‐modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H₂O₂ and S‐nitroso‐N‐acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H₂O₂‐induced loss of colony forming activity in hMSCs. In a dose‐dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H₂O₂ or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H₂O₂‐induced ROS in the absence of significant direct effect on H₂O₂ itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H₂O₂ exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application. J. Cell. Biochem. 114: 1163–1173, 2013. © 2012 Wiley Periodicals, Inc.     

Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide

Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinson’s, Alzheimer’s, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection.     

CREB‐mediated Bcl‐2 expression contributes to RCAN1 protection from hydrogen peroxide‐induced neuronal death

Regulator of calcineurin 1 (RCAN1) is located on the Down syndrome critical region (DSCR) locus in human chromosome 21. In this study, we investigated the functional role of RCAN1 in the reactive oxygen species (ROS)‐mediated neuronal death signaling. We found that RCAN1 was able to protect the cells from H₂O₂‐induced cytotoxicity. The expression of RCAN1 caused an inhibition of the H₂O₂‐induced activation of mitogen‐activated protein kinases (MAPKs) and AP‐1. In contrast, RCAN1 significantly enhanced the activity of cAMP response element‐binding protein (CREB). Furthermore, RCAN1 induced the expression of the CREB target gene, Bcl‐2. Consistently, knockdown of endogenous RCAN1 using shRNA down regulated the phosphorylation of CREB and the expression of Bcl‐2, which protects the cells from H₂O₂‐induced cytotoxicity. Our data provide a new mechanism for the cytoprotective function of RCAN1 in response to oxidant‐induced apoptosis. J. Cell. Biochem. 114: 1115–1123, 2013. © 2012 Wiley Periodicals, Inc.     

Release of cytokines by human nasal epithelial cells and peripheral blood mononuclear cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) infection is associated with asthma exacerbation in children. We hypothesized that Mp infection may cause airway inflammation by inducing the release of cytokines by respiratory epithelial cells. The levels of chemokines interleukin-8 (IL-8) and released upon activation, normal t cell expressed and secreted (RANTES) released by nasal epithelial cell (NEC) cultures established from asthmatic and nonasthmatic children were measured by ELISA at 4, 24, 48, and 72 hr after cells were inoculated with Mp, and were compared with baseline release of these factors. The presence of MP on apical membranes of NEC after infection was confirmed by transmission electron microscopy, and adherence was shown to be inhibited by erythromycin. Mp infection did not alter NEC release of IL-8 or RANTES at any time point. In contrast, tumor necrosis factor alpha (TNF-alpha) stimulated increased IL-8 at all time points, and respiratory syncytial virus (RSV) infection stimulated RANTES release at 48 and 72 hr by NEC. These results were not significantly different between NEC from asthmatic and nonasthmatic children. As a comparison, peripheral blood mononuclear cells from normal human volunteers were also incubated with Mp and had significantly increased release of IL-2, IL-6, and TNF-alpha. We conclude that Mp, unlike viral pathogens such as RSV, is unlikely to directly stimulate early airway surface cytokine responses via mechanisms involving epithelial cells. We speculate that the chronic presence of mononuclear cells at the airway surface of asthmatics provides a target for Mp-triggered cytokine production.     

MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca²⁺ concentration. Sodium selenite could ameliorate hydrogen peroxide (H₂O₂)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H₂O₂ or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca²⁺ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H₂O₂‐induced cell death in H9c2 cells.     

Benzyl isothiocyanate attenuates the hydrogen peroxide‐induced interleukin‐13 expression through glutathione S‐transferase P induction in T lymphocytic leukemia cells

We investigated the effect of benzyl isothiocyanate (BITC) on the hydrogen peroxide‐induced gene expression of a T‐helper‐2 cytokine, interleukin (IL)‐13, in T lymphocytic leukemia Jurkat cells. The 24‐h pretreatment of BITC significantly inhibited the IL‐13 expression enhanced by hydrogen peroxide. Although the BITC pretreatment did not change the enhanced level of the phosphorylated c‐Jun N‐terminal kinase (JNK), it significantly inhibited the nuclear translocation of c‐Jun induced by hydrogen peroxide. BITC also increased the protein expression of glutathione S‐transferase (GST) isozymes, GSTP1/2, as well as the total GST activity. A GSTP1/2‐specific inhibitor, 6‐(7‐nitro‐2,1,3‐benzoxadiazol‐4‐ylthio)hexanol (NBDHEX), significantly counteracted the inhibitory effect of BITC on the hydrogen peroxide‐enhanced IL‐13 upregulation as well as the c‐Jun nuclear translocation. Taken together, these results suggested that BITC inhibits the oxidative stress‐mediated IL‐13 mRNA expression, possibly through interference of the c‐Jun phosphorylation by GSTP.     

Hyaluronic acid optimises therapeutic effects of hydrogen peroxide‐induced oxidative stress on breast cancer

Distinguishing the multiple effects of reactive oxygen species (ROS) on cancer cells is important to understand their role in tumour biology. On one side, ROS can be oncogenic by promoting hypoxic conditions, genomic instability and tumorigenesis. Conversely, elevated levels of ROS‐induced oxidative stress can induce cancer cell death. This is evidenced by the conflicting results of research using antioxidant therapy, which in some cases promoted tumour growth and metastasis. However, some antioxidative or ROS‐mediated oxidative therapies have also yielded beneficial effects. To better define the effects of oxidative stress, in vitro experiments were conducted on 4T1 and splenic mononuclear cells (MNCs) under hypoxic and normoxic conditions. Furthermore, hydrogen peroxide (H₂O₂; 10–1,000 μM) was used as an ROS source alone or in combination with hyaluronic acid (HA), which is frequently used as drug delivery vehicle. Our result indicated that the treatment of cancer cells with H₂O₂ + HA was significantly more effective than H₂O₂ alone. In addition, treatment with H₂O₂ + HA led to increased apoptosis, decreased proliferation, and multiphase cell cycle arrest in 4T1 cells in a dose‐dependent manner under normoxic or hypoxic conditions. As a result, migratory tendency and the messenger RNA levels of vascular endothelial growth factor, matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 were significantly decreased in 4T1 cells. Of note, HA treatment combined with 100–1,000 μM H₂O₂ caused more damage to MNCs as compared to treatment with lower concentrations (10–50 μM). Based on these results, we propose to administer high‐dose H₂O₂ + HA (100–1000 μM) for intratumoural injection and low doses for systemic administration. Intratumoural route could have toxic and inhibitory effects not only on the tumour but also on residential myeloid cells defending it, whereas systemic treatment could stimulate peripheral immune responses against the tumour. More in vivo research is required to confirm this hypothesis.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

A novel pH‐controlled hydrogen sulfide donor protects gastric mucosa from aspirin‐induced injury

Hydrogen sulphide (H₂S) serves as a vital gastric mucosal defence under acid condition. Non‐steroidal anti‐inflammatory drugs (NSAIDs) are among widely prescribed medications with effects of antipyresis, analgesia and anti‐inflammation. However, their inappropriate use causes gastric lesions and endogenous H₂S deficiency. In this work, we reported the roles of a novel pH‐controlled H₂S donor (JK‐1) in NSAID‐related gastric lesions. We found that JK‐1 could release H₂S under mild acidic pH and increase solution pH value. Intragastrical administration of aspirin (ASP), one of NSAIDs, to mice elicited significant gastric lesions, evidenced by mucosal festering and bleeding. It also led to infiltration of inflammatory cells and resultant releases of IL‐6 and TNF‐α, as well as oxidative injury including myeloperoxidase (MPO) induction and GSH depletion. In addition, the ASP administration statistically inhibited H₂S generation in gastric mucosa, while up‐regulated cyclooxygenase (COX)‐2 and cystathionine gamma lyase (CSE) expression. Importantly, these adverse effects of ASP were prevented by the intragastrical pre‐administration of JK‐1. However, JK‐1 alone did not markedly alter the property of mouse stomachs. Furthermore, in vitro cellular experiments showed the exposure of gastric mucosal epithelial (GES‐1) cells to HClO, imitating MPO‐driven oxidative injury, decreased cell viability, increased apoptotic rate and damaged mitochondrial membrane potential, which were reversed by pre‐treatment with JK‐1. In conclusion, JK‐1 was proved to be an acid‐sensitive H₂S donor and could attenuate ASP‐related gastric lesions through reconstruction of endogenous gastric defence. This work indicates the possible treatment of adverse effects of NSAIDs with pH‐controlled H₂S donors in the future.     

Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) cells may contribute to the progression of age-related macular degeneration. Thymoquinone (TQ), an active component derived from Nigella sativa, possesses antioxidative effect. However, the role of TQ in RPE cells under oxidative stress condition remains unclear. The present study aimed to examine the protective effect of TQ against hydrogen peroxide (H₂O₂)-induced oxidative stress in human RPE cells. Our results showed that TQ improved the cell viability and apoptosis in H₂O₂-induced ARPE cells. We also found that the levels of reactive oxygen species and malondialdehyde induced by H₂O₂ were reduced after the pretreatment of TQ. In addition, the inhibitory effect of H₂O₂ on the glutathione (GSH) level and superoxide dismutase activity was markedly attenuated by TQ pretreatment. Moreover, TQ enhanced the activation of Nrf2/heme oxygenase 1 (HO-1) signaling pathway in H₂O₂-induced ARPE cells. Knockdown of Nrf2 abolished the protective effect of TQ on H₂O₂-induced oxidative damage. These results suggested that TQ protected ARPE cells from H₂O₂-induced oxidative stress and apoptosis via the Nrf2/HO-1 signaling pathway.     

Brain‐derived neurotrophic factor protects cementoblasts from serum starvation‐induced cell death

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNF's actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009. © 2009 Wiley‐Liss, Inc.     

L-Carnitine protects mammalian cells from chromosome aberrations but not from inhibition of cell proliferation induced by hydrogen peroxide

L-carnitine is a small essential molecule indispensable in fatty acid metabolism and required in several biological pathways regulating cellular homeostasis. Despite considerable progress in understanding of L-carnitine biosynthesis and metabolism, very few data are reported concerning the protective role of L-carnitine from oxidative stress-induced DNA damage that is known to be a factor in cell transformation and tumourigenesis. In order to detect the capability of L-carnitine to protect mammalian cells from oxidative stress-induced chromosomal effects, we analysed chromosome aberrations in mitotic CHO cells, which represent an appropriate cytogenetic model to study compounds that enhance cell protection against externally induced DNA damage. We chose H2O2 as an inducer of oxidative stress. Our results demonstrate for the first time a marked and reproducible reduction of H2O2-induced chromosome damage involving an L-carnitine-mediated capacity to buffer intracellular formation of reactive oxygen species (ROS). Furthermore, by studying the mitotic index and cell cycle progression, we also demonstrated that this protective effect is highly specific, since L-carnitine itself was not able to prevent the inhibition of cell growth caused by H2O2.     

Resistin protects against 6‐hydroxydopamine‐induced cell death in dopaminergic‐like MES23.5 cells

Resistin is originally reported as an adipose tissue‐specific hormone and is thought to represent a link between obesity and insulin‐resistant diabetes. Adipokines exert energy‐regulation and has been reported to have neuroprotective effect like leptin, adiponectin, and ghrelin. However, the role of resistin in neuroprotective effect has not been explored. 6‐hydroxydopamine (6‐OHDA), one of the most investigated Parkinson's disease neurotoxins, is widely used to study mechanisms of cell death in dopaminergic neurons. In the present study, our results show that treatment of resistin protects 6‐OHDA‐induced cell death in dopaminergic‐like MES23.5 cells. Resistin also antagonizes 6‐OHDA‐induced apoptotic cell death measured by fluorescence‐activated cell sorter (FACS) analysis and Hochest 33342 staining. Furthermore, treatment of resistin also dramatically reduces 6‐OHDA‐mediated ROS production and mitochondria transmembrane potential dissipation. Moreover, expression of 6‐OHDA‐induced apoptotic markers, such as Bcl‐2 degradation, Bax expression, PARP degradation and caspase 3 activity increase, are all attenuated by resistin treatment. Our results also show that resistin induces up‐regulation of heat shock protein (Hsp) 32 (heme oxygenase‐1, HO‐1) and Hsc (heat shock cognate) 70. The protective effect of resistin on 6‐OHDA‐induced cell death is abolished by HO‐1 inhibitor zinc protoporphyrin IX and HSP inhibitor KNK437. These results suggest the neuroprotective effects of resistin against 6‐OHDA‐induced cell death with the underlying mechanisms of inhibiting oxidative stress and apoptosis. Therefore, we suggest that resistin may provide a useful therapeutic strategy for neurodegenerative diseases such as Parkinson's disease. J. Cell. Physiol. 228: 563–571, 2013. © 2012 Wiley Periodicals, Inc.     

Lentinan protects pancreatic β cells from STZ‐induced damage

Pancreatic β‐cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of Lentinus edodes, on pancreatic β‐cell apoptosis and dysfunction caused by streptozotocin (STZ) and the possible mechanisms implicated. The rat insulinoma cell line INS‐1 were pre‐treated with the indicated concentration of LNT for 30 min. and then incubated for 24 hrs with or without 0.5 mM STZ. We found that STZ treatment causes apoptosis of INS‐1 cells by enhancement of intracellular reactive oxygen species (ROS) accumulation, inducible nitric oxide synthase (iNOS) expression and nitric oxide release and activation of the c‐jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways. However, LNT significantly increased cell viability and effectively attenuated STZ‐induced ROS production, iNOS expression and nitric oxide release and the activation of JNK and p38 MAPK in a dose‐dependent manner in vitro. Moreover, LNT dose‐dependently prevented STZ‐induced inhibition of insulin synthesis by blocking the activation of nuclear factor kappa beta and increasing the level of Pdx‐1 in INS‐1 cells. Together these findings suggest that LNT could protect against pancreatic β‐cell apoptosis and dysfunction caused by STZ and therefore may be a potential pharmacological agent for preventing pancreatic β‐cell damage caused by oxidative stress associated with diabetes.