Oxidative Stress-induced Death Research Articles

Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo.

Oxidative stress has a critical role in the pathogenesis of vitiligo. However, the specific molecular mechanism involved in oxidative stress-induced melanocyte death is not well characterized. Given the powerful role of microRNAs (miRNAs) in the regulation of cell survival as well as the fact that the generation of miRNAs can be affected by oxidative stress, we hypothesized that miRNAs may participate in vitiligo pathogenesis by modulating the expression of vital genes in melanocytes. In the present study, we initially found that miR-25 was increased in both serum and lesion samples from vitiligo patients, and its serum level was correlated with the activity of vitiligo. Moreover, restoration of miR-25 promoted the H2O2-induced melanocyte destruction and led to the dysfunction of melanocytes. Further experiments proved that MITF, a master regulator in melanocyte survival and function, accounted for the miR-25-caused damaging impact on melanocytes. Notably, other than the direct role on melanocytes, we observed that miR-25 inhibited the production and secretion of SCF and bFGF from keratinocytes, thus impairing their paracrine protective effect on the survival of melanocytes under oxidative stress. At last, we verified that oxidative stress could induce the overexpression of miR-25 in both melanocytes and keratinocytes possibly by demethylating the promoter region of miR-25. Taken together, our study demonstrates that oxidative stress-induced overexpression of miR-25 in vitiligo has a crucial role in promoting the degeneration of melanocytes by not only suppressing MITF in melanocytes but also impairing the paracrine protective effect of keratinocytes. Therefore, it is worthy to investigate the possibility of miR-25 as a potential drug target for anti-oxidative therapy in vitiligo.

Neolignans from Aristolochia fordiana Prevent Oxidative Stress-Induced Neuronal Death through Maintaining the Nrf2/HO-1 Pathway in HT22 Cells.

Bioassay-guided fractionation of the ethanolic extract of the stems of Aristolochia fordiana led to the isolation of six new dihydrobenzofuran neolignans (1-3 and 7-9), three new 2-aryldihydrobenzofurans (4-6), a new 8-O-4' neolignan (10), and 14 known analogues (11-24). The structures of compounds 1-10 were established by spectroscopic methods, and their absolute configurations were determined by analyses of the specific rotation and electronic circular dichroism data. The neuroprotective effects of compounds 1-24 against glutamate-induced cell death were tested in hippocampal neuronal cell line HT22. Compounds 17 and 20-24 exhibited moderate neuroprotective activity by increasing the endogenous antioxidant defense system. In addition, the neolignans activated the Nrf2 (nuclear factor E2-related factor 2) pathway, resulting in the increase of the expression of endogenous antioxidant protein HO-1 (heme oxygenase-1). The active compounds also preserved the levels of antiapoptotic protein Bcl-2 (B cell lymphoma/leukemia-2), which was decreased by glutamate. Collectively, these results suggested that the active neolignans protect neurons against glutamate-induced cell death through maintaining the Nrf2/HO-1 signaling pathway as well as preserving the Bcl-2 protein and might be promising novel beneficial agents for oxidative stress-associated diseases.

One-step isolation of sappanol and brazilin from Caesalpinia sappan and their effects on oxidative stress-induced retinal death.

Caesalpinia sappan is a well-distributed plant that is cultivated in Southeast Asia, Africa, and the Americas. C. sappan has been used in Asian folk medicine and its extract has been shown to have pharmacological effects. Two homoisoflavonoids, sappanol and brazilin, were isolated from C. sappan by using centrifugal partition chromatography (CPC), and tested for protective effects against retinal cell death. The isolated homoisoflavonoids produced approximately 20-fold inhibition of N-retinylidene-N-retinyl-ethanolamine (A2E) photooxidation in a dose-dependent manner. Of the 2 compounds, brazilin showed better inhibition (197.93 ± 1.59 μM of IC50). Cell viability tests and PI/Hoechst 33342 double staining method indicated that compared to the negative control, sappanol significantly attenuated H2O2-induced retinal death. The compounds significantly blunted the up-regulation of intracellular reactive oxygen species (ROS), and sappanol inhibited lipid peroxidation in a concentration-dependent manner. Thus, both compounds represent potential antioxidant treatments for retinal diseases. [BMB Reports 2015; 48(5): 289-294]

HDAC2 selectively regulates FOXO3a-mediated gene transcription during oxidative stress-induced neuronal cell death.

All neurodegenerative diseases are associated with oxidative stress-induced neuronal death. Forkhead box O3a (FOXO3a) is a key transcription factor involved in neuronal apoptosis. However, how FOXO3a forms complexes and functions in oxidative stress processing remains largely unknown. In the present study, we show that histone deacetylase 2 (HDAC2) forms a physical complex with FOXO3a, which plays an important role in FOXO3a-dependent gene transcription and oxidative stress-induced mouse cerebellar granule neuron (CGN) apoptosis. Interestingly, we also found that HDAC2 became selectively enriched in the promoter region of the p21 gene, but not those of other target genes, and inhibited FOXO3a-mediated p21 transcription. Furthermore, we found that oxidative stress reduced the interaction between FOXO3a and HDAC2, leading to an increased histone H4K16 acetylation level in the p21 promoter region and upregulated p21 expression in a manner independent of p53 or E2F1. Phosphorylation of HDAC2 at Ser 394 is important for the HDAC2-FOXO3a interaction, and we found that cerebral ischemia/reperfusion reduced phosphorylation of HDAC2 at Ser 394 and mitigated the HDAC2-FOXO3a interaction in mouse brain tissue. Our study reveals the novel regulation of FOXO3a-mediated selective gene transcription via epigenetic modification in the process of oxidative stress-induced cell death, which could be exploited therapeutically.

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Cytoprotective effects of 12-oxo phytodienoic acid, a plant-derived oxylipin jasmonate, on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity. MethodsThe cells were pretreated with individual jasmonates for 24h and exposed to hydrogen peroxide (H2O2) for 24h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA). ResultsAmong the jasmonates, only OPDA suppressed H2O2-induced cytotoxicity. OPDA pretreatment also inhibited the H2O2-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2. ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway. General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.

3,5,4′-Trihydroxy-6,7,3′-trimethoxyflavone protects astrocytes against oxidative stress via interference with cell signaling and by reducing the levels of intracellular reactive oxygen species

Oxidative stress is tightly involved in various neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and conditions such as ischemia. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support. Therefore, any damage to astrocytes will affect neuronal survival. In a previous study we have demonstrated that an extract prepared from the plant Achillea fragrantissima (Af) prevented the oxidative stress-induced death of astrocytes and attenuated the intracellular accumulation of ROS in astrocytes under oxidative stress. In the present study, using activity guided fractionation, we have purified from this plant the active compound, determined to be a flavonoid named 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF). The effects of TTF in any biological system have not been studied previously, and this is the first study to characterize the anti-oxidant and protective effects of this compound in the context of neurodegenerative diseases. Using primary cultures of astrocytes we have found that TTF prevented the hydrogen peroxide (H2O2)-induced death of astrocytes, and attenuated the intracellular accumulation of ROS following treatment of these cells with H2O2 or the peroxyl radicals generating molecule 2,2′-Azobis(amidinopropane) (ABAP). TTF also interfered with cell signaling events and inhibited the phosphorylation of the signaling proteins stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), extracellular signal regulated kinase (ERK 1/2) and mitogen activated protein kinase kinase (MEK1) and the phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB). The mechanism of the protective effect of TTF against H2O2-cytotoxicity could not be attributed to a direct H2O2 scavenging but rather to the scavenging of free radicals as was shown in cell free systems. Thus, TTF might be a therapeutic candidate for the prevention/treatment of neurodegenerative diseases where oxidative stress is part of the pathophysiology.

Potent effects of flavonoid-rich extract from Rosa laevigata Michx fruit against hydrogen peroxide-induced damage in PC12 cells via attenuation of oxidative stress, inflammation and apoptosis.

Oxidative stress-induced neuronal death has an important role in the pathogenesis of neurodegenerative disorders. The effects and mechanisms of action of the total flavonoids (TFs) from Rosa laevigata Michx fruit against hydrogen peroxide (H2O2)-induced oxidative injury in PC12 cells were investigated in this study. The results demonstrated that the TFs protected against cell apoptosis, DNA and mitochondrial damage caused by H2O2 based on single cell gel electrophoresis, in situ terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM) assays. In addition, the TFs notably decreased cytochrome C release from mitochondria into the cytosol and intracellular Ca2+ levels, and diminished intracellular generation of reactive oxygen species (ROS). Furthermore, the TFs inhibited the phosphorylation levels of JNK, ERK and p38 MAPK as well as down-regulated the expressions of IL-1, IL-6, TNF-α, Fas, FasL, CYP2E1, Bak, caspase-3, caspase-9, p53, COX-2, NF-κB, AP-1, and up-regulated the expressions of Bcl-2 and Bcl-xl. In conclusion, these results suggest that the TFs from R. laevigata Michx fruit show good effects against H2O2-induced oxidative injury in PC12 cells by adjusting oxidative stress, and suppression of apoptosis and inflammation, and could be developed as a potential candidate to prevent oxidative stress in the future.

Inhibition of stress induced premature senescence in presenilin-1 mutated cells with water soluble Coenzyme Q10

A water-soluble formulation of CoQ10 (WS-CoQ10) was shown to stabilize mitochondria and prevent oxidative stress-induced neuronal death. Presenilin-1 (PS-1)-mutated Alzheimer's Disease (AD) fibroblasts (PSAF) were used for studying the effects of PS-1 mutation. PS-1 mutation correlated to increased reactive oxygen species (ROS) production and stress induced premature senescence (SIPS) in PSAF; WS-CoQ10 treatment decreased ROS generation, increased population doublings, and postponed SIPS. Treated PSAF had higher PCNA expression, and lower levels of MnSOD, p21, p16Ink4A, and Rb. WS-CoQ10 caused the resumption of autophagy in PSAF. Thus, WS-CoQ10 as inhibitor of SIPS and ameliorator of autophagy could be an effective prophylactic/therapeutic agent for AD.

Electromagnetic radiation (Wi-Fi) and epilepsy induce calcium entry and apoptosis through activation of TRPV1 channel in hippocampus and dorsal root ganglion of rats.

Incidence rates of epilepsy and use of Wi-Fi worldwide have been increasing. TRPV1 is a Ca(2+) permeable and non-selective channel, gated by noxious heat, oxidative stress and capsaicin (CAP). The hyperthermia and oxidant effects of Wi-Fi may induce apoptosis and Ca(2+) entry through activation of TRPV1 channel in epilepsy. Therefore, we tested the effects of Wi-Fi (2.45 GHz) exposure on Ca(2+) influx, oxidative stress and apoptosis through TRPV1 channel in the murine dorsal root ganglion (DRG) and hippocampus of pentylentetrazol (PTZ)-induced epileptic rats. Rats in the present study were divided into two groups as controls and PTZ. The PTZ groups were divided into two subgroups namely PTZ + Wi-Fi and PTZ + Wi-Fi + capsazepine (CPZ). The hippocampal and DRG neurons were freshly isolated from the rats. The DRG and hippocampus in PTZ + Wi-Fi and PTZ + Wi-Fi + CPZ groups were exposed to Wi-Fi for 1 hour before CAP stimulation. The cytosolic free Ca(2+), reactive oxygen species production, apoptosis, mitochondrial membrane depolarization, caspase-3 and -9 values in hippocampus were higher in the PTZ group than in the control although cell viability values decreased. The Wi-Fi exposure induced additional effects on the cytosolic Ca(2+) increase. However, pretreatment of the neurons with CPZ, results in a protection against epilepsy-induced Ca(2+) influx, apoptosis and oxidative damages. In results of whole cell patch-clamp experiments, treatment of DRG with Ca(2+) channel antagonists [thapsigargin, verapamil + diltiazem, 2-APB, MK-801] indicated that Wi-Fi exposure induced Ca(2+) influx via the TRPV1 channels. In conclusion, epilepsy and Wi-Fi in our experimental model is involved in Ca(2+) influx and oxidative stress-induced hippocampal and DRG death through activation of TRPV1 channels, and negative modulation of this channel activity by CPZ pretreatment may account for the neuroprotective activity against oxidative stress.

Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease

Dysfunction of myocyte enhancer factor 2D (MEF2D), a key survival protein and transcription factor, underlies the pathogenic loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Both genetic factors and neurotoxins associated with PD impair MEF2D function in vitro and in animal models of PD. We investigated whether distinct stress conditions target MEF2D via converging mechanisms. We showed that exposure of a DA neuronal cell line to 6-hyroxydopamine (6-OHDA), which causes PD in animals models, led to direct oxidative modifications of MEF2D. Oxidized MEF2D bound to heat-shock cognate protein 70 kDa, the key regulator for chaperone-mediated autophagy (CMA), at a higher affinity. Oxidative stress also increased the level of lysosomal-associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, and stimulated CMA activity. These changes resulted in accelerated degradation of MEF2D. Importantly, 6-OHDA induced MEF2D oxidation and increased LAMP2A in the substantia nigra pars compacta region of the mouse brain. Consistently, the levels of oxidized MEF2D were much higher in postmortem PD brains compared with the controls. Functionally, reducing the levels of either MEF2D or LAMP2A exacerbated 6-OHDA-induced death of the DA neuronal cell line. Expression of an MEF2D mutant that is resistant to oxidative modification protected cells from 6-OHDA-induced death. This study showed that oxidization of survival protein MEF2D is one of the pathogenic mechanisms involved in oxidative stress-induced DA neuronal death. Oxidation of survival factor MEF2D inhibits its function, underlies oxidative stress-induced neurotoxicity, and may be a part of the PD pathogenic process.

Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

Pre-Conditioning with CDP-Choline Attenuates Oxidative Stress-Induced Cardiac Myocyte Death in a Hypoxia/Reperfusion Model

Background. CDP-choline is a key intermediate in the biosynthesis of phosphatidylcholine, which is an essential component of cellular membranes, and a cell signalling mediator. CDP-choline has been used for the treatment of cerebral ischaemia, showing beneficial effects. However, its potential benefit for the treatment of myocardial ischaemia has not been explored yet. Aim. In the present work, we aimed to evaluate the potential use of CDP-choline as a cardioprotector in an in vitro model of ischaemia/reperfusion injury. Methods. Neonatal rat cardiac myocytes were isolated and subjected to hypoxia/reperfusion using the coverslip hypoxia model. To evaluate the effect of CDP-choline on oxidative stress-induced reperfusion injury, the cells were incubated with H2O2 during reperfusion. The effect of CDP-choline pre- and postconditioning was evaluated using the cell viability MTT assay, and the proportion of apoptotic and necrotic cells was analyzed using the Annexin V determination by flow cytometry. Results. Pre- and postconditioning with 50 mg/mL of CDP-choline induced a significant reduction of cells undergoing apoptosis after hypoxia/reperfusion. Preconditioning with CDP-choline attenuated postreperfusion cell death induced by oxidative stress. Conclusion. CDP-choline administration reduces cell apoptosis induced by oxidative stress after hypoxia/reperfusion of cardiac myocytes. Thus, it has a potential as cardioprotector in ischaemia/reperfusion-injured cardiomyocytes.

Protective effect of metformin on gentamicin-induced vestibulotoxicity in rat primary cell culture.

ObjectivesOne of the antidiabetic drugs, metformin, have shown that it prevented oxidative stress-induced death in several cell types through a mechanism involving the opening of the permeability transition pore and cytochrome c release. Thus, it is possible that the antioxidative effect of metformin can also serve as protection against gentamicin-induced cytotoxicity related to reactive oxygen species (ROS). The aim of this study was to examine the protective effect of metformin on gentamicin-induced vestibulotoxicity in primary cell culture derived from rat utricle.MethodsFor vestibular primary cell culture, rat utricles were dissected and incubated. Gentamicin-induced cytotoxicity was measured in both the auditory and vestibular cells. To examine the effects of metformin on gentamicin-induced cytotoxicity in the primary cell culture, the cells were pretreated with metformin at a concentration of 1 mM for 24 hours, and then exposed to 2.5 mM gentamicin for 48 hours. The intracellular ROS level was measured using a fluorescent dye, and also measured using a FACScan flow cytometer. Intracellular calcium levels in the vestibular cells were measured with calcium imaging using Fura-2 AM.ResultsVestibular cells were more sensitive to gentamicin-induced cytotoxicity than auditory hair cells. Metformin protects against gentamicin-induced cytotoxicity in vestibular cells. Metformin significantly reduced a gentamicin-induced increase in ROS, and also reduced an increase in intracellular calcium concentrations in gentamicin-induced cytotoxicity.ConclusionMetformin significantly reduced a gentamicin-induced increase in ROS, stabilized the intracellular calcium concentration, and inhibited gentamicin-induced apoptosis. Thus, Metformin showed protective effect on gentamicin-induced cytotoxicity in vestibular primary cell culture.

A Comparative Study of In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N‐heteroaromatic Schiff Base (E)‐2‐[N′‐(1‐pyridin‐2‐yl‐ethylidene)hydrazino]acetate

In search for novel biologically active metal based compounds, an evaluation of in vitro cytotoxic, antioxidant, and antimicrobial activity of new Pt(II) complex and its Zn(II), Cu(II), and Co(III) analogues, with NNO tridentately coordinated N-heteroaromatic Schiff base ligand (E)-2-[N'-(1-pyridin-2-yl-ethylidene)hydrazino]acetate, was performed. Investigation of antioxidative properties showed that all of the compounds have strong radical scavenging potencies. The Zn(II) complex showed potent inhibition of DNA cleavage by hydroxyl radical. A cytotoxic action of investigated compounds was evaluated on cultures of human promyelocitic leukaemia (HL-60), human glioma (U251), rat glioma (C6), and mouse melanoma (B16) cell lines. It was shown that binuclear pentacoordinated Zn(II) complex possesses a strong dose-dependent cytotoxic activity, of the same order of magnitude as cisplatin on B16, C6, and U251 cells. Furthermore, Zn(II) complex causes oxidative stress-induced apoptotic death of HL-60 leukemic cells, associated with caspase activation, phosphatidylserine externalization, and DNA fragmentation.

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Antioxidant action of 7,8-dihydroxyflavone protects PC12 cells against 6-hydroxydopamine-induced cytotoxicity

Oxidative stress-induced neuronal death plays a pivotal role in pathogenesis of neurodegenerative disorders. Recently, 7,8-dihydroxyflavone (7,8-DHF) has been shown to exert neuroprotective effects by acting as a selective tyrosine kinase receptor B (TrkB) agonist. In addition, the antioxidant action of 7,8-DHF may protect neuronal cells against oxidative injury. In the present study, we used PC12 cells, a cell line generally thought to lack TrkB, to investigate the effect of 7,8-DHF on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and the underlying mechanism. We found that 7,8-DHF effectively prevented cell death, apoptosis and mitochondrial dysfunction induced by 6-OHDA. In a cell free system, 7,8-DHF did not slow down extracellular auto-oxidation of 6-OHDA which may generate H2O2. However, We found that 7,8-DHF dramatically reduced cellular malondialdehyde content and phospho-histone H2A.X protein level. 7,8-DHF also elevated total superoxide dismutase activity in 6-OHDA-treated cells. These results indicate that 7,8-DHF might protect PC12 cells against 6-OHDA-induced cytotoxicity through its powerful antioxidant activity. By acting as a potent TrkB agonist and an antioxidant together with its easiness to pass across blood–brain barrier, 7,8-DHF may be developed into a promising candidate in treatment of neurodegenerative diseases.

Carvedilol, a third-generation β-blocker prevents oxidative stress-induced neuronal death and activates Nrf2/ARE pathway in HT22 cells

Carvedilol, a nonselective β-adrenoreceptor blocker with pleiotropic activities has been shown to exert neuroprotective effect due to its antioxidant property. However, the neuroprotective mechanism of carvedilol is still not fully uncovered. Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. Here we investigated the effect of carvedilol on oxidative stress-induced cell death (glutamate 2mM and H2O2 600μM) and the activity of Nrf2/ARE pathway in HT22 hippocampal cells. Carvedilol significantly increased cell viability and decreased ROS in HT22 cells exposed to glutamate or H2O2. Furthermore, carvedilol activated the Nrf2/ARE pathway in a concentration-dependent manner, and increased the protein levels of heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1(NQO-1), two downstream factors of the Nrf2/ARE pathway. Collectively, our results indicate that carvedilol protects neuronal cell against glutamate- and H2O2-induced neurotoxicity possibly through activating the Nrf2/ARE signaling pathway.

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H2O2) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H2O2 treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.

Inflammatory Cytokines Protect Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Death

PurposeTo investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19.MethodsConfluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting.ResultsViability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment.ConclusionOxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.