Activation Of Antioxidant Response Element Research Articles

皂角刺 추출물의 Nrf2 활성화를 통한 간세포 보호 효과

Objectives : Oxidative stress is one of the most causes of hepatocyte injury. Gleditsia spina, the thorns ofGleditsia sinensisLam., has been known for its anti-cancer and anti-inflammatory effects in Korean medicine. The present study investigated hepatoprotective effect of Gleditsia spina water extract (GSE) against oxidative stress induced by arachidonic acid (AA) + iron in HepG2 cells.Methods : To investigate cytoprotective effect of GSE, cells were pretreated with GSE and then subsequently exposed to 10 μM AA for 12 h, followed by 5 μM iron. Cell viability was monitored by MTT assay, and expression of apoptosis-related proteins was examined by immunoblot analysis. To identify responsible molecular mechanisms, reactive oxygen species (ROS) production, GSH contents, and mitochondrial membrane potential were measured. In addition, effect of GSE on nuclear factor erythroid 2-related factor 2 (Nrf2) activation was determined by immunoblot and antioxidant response element (ARE)-driven reporter gene assays.Results : GSE pretreatment prevented AA + iron-mediated cytotoxicity in concentration dependent manner. In addition, ROS production, glutathione depletion, and mitochondrial impairment by AA + iron were significantly inhibited by GSE. Furthermore, GSE promoted translocation of Nrf2 to nucleus, which acts as essential transcription factor for induction of antioxidant genes. Increased nuclear Nrf2 that caused by GSE treatment promoted transcriptional activity of ARE. Finally, GSE up-regulated sestrin-2 which was widely recognized as target gene of Nrf2.Conclusions : This study demonstrates that GSE protects hepatocytes from oxidative stress via activation of Nrf2 signaling pathway.

Gleditsia Spina Extract Protects Hepatocytes from Oxidative Stress through Nrf2 Activation

Objectives : Oxidative stress is one of the most causes of hepatocyte injury. Gleditsia spina, the thorns of Gleditsia sinensis Lam., has been known for its anti-cancer and anti-inflammatory effects in Korean medicine. The present study investigated hepatoprotective effect of Gleditsia spina water extract (GSE) against oxidative stress induced by arachidonic acid (AA) + iron in HepG2 cells. Methods : To investigate cytoprotective effect of GSE, cells were pretreated with GSE and then subsequently exposed to 10 µM AA for 12 h, followed by 5 µM iron. Cell viability was monitored by MTT assay, and expression of apoptosis-related proteins was examined by immunoblot analysis. To identify responsible molecular mechanisms, reactive oxygen species (ROS) production, GSH contents, and mitochondrial membrane potential were measured. In addition, effect of GSE on nuclear factor erythroid 2-related factor 2 (Nrf2) activation was determined by immunoblot and antioxidant response element (ARE)–driven reporter gene assays. Results : GSE pretreatment prevented AA + iron-mediated cytotoxicity in concentration dependent manner. In addition, ROS production, glutathione depletion, and mitochondrial impairment by AA + iron were significantly inhibited by GSE. Furthermore, GSE promoted translocation of Nrf2 to nucleus, which acts as essential transcription factor for induction of antioxidant genes. Increased nuclear Nrf2 that caused by GSE treatment promoted transcriptional activity of ARE. Finally, GSE up-regul ated sestrin-2 which was widely recognized as target gene of Nrf2. Conclusions : This study demonstrates that GSE protects hepatocytes from oxidative stress via activation of Nrf2 signaling pathway.

Effects of Eicosapentaenoic Acid on the Cytoprotection Through Nrf2-Mediated Heme Oxygenase-1 in Human Endothelial Cells.

Consumption of omega-3 polyunsaturated fatty acid, particularly eicosapentaenoic acid (EPA), is associated with a significant reduction in the risk of developing cardiovascular disease. The aim of this study was to investigate whether heme oxygenase-1 (HO-1) induction contributes to the cytoprotective effects of EPA in endothelial cells threatened with oxidative damage. In this study, we investigated the effect of EPA on the induction of HO-1 by NF-E2-related factor 2 (Nrf2) in human umbilical vein endothelial cells. In cells treated with low concentrations of EPA (10-25 μM), HO-1 expression increased in a time- and concentration-dependent manner. Additionally, EPA treatment increased Nrf2 nuclear translocation and antioxidant response element activity, leading to the upregulation of HO-1 expression. Furthermore, treatment with EPA reduced hydrogen peroxide (H(2)O(2))-induced cell death. The reduction in cell death was reversed by treatment with zinc protoporphyrin, an inhibitor of HO-1, indicating that HO-1 contributed to the protective effect of EPA. These data suggest that EPA protects against H(2)O(2)-induced oxidative stress in endothelial cells by activating Nrf2 and inducting HO-1 expression.

Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole‐cell‐stimulated Antioxidant Response

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE) (by Cravatt et al., Nature Methods, 2014, 11, 79-85)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains an untested hypothesis. Herein, using T-REX (targetable reactive electrophiles and oxidants)—an approach aimed at selectively flipping a single redox switch in cells at a precise time —we show that a modest level (~34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically-important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology. Research involving the development and applications of the new T-REX idea is supported by the NIH Director's New Innovator Award (1DP2GM114850), NSF CAREER Award (CHE-1351400), and the Beckman Young Investigator Award (to Y.A.).

The water extract of Angelica Sinensis Radix protects cultured PC12 cells against oxidative stress: suppression of reactive oxygen species and activation of anti-oxidant response elements

Objective: Angelica Sinensis Radix (ASR; Dang Gui; the root of Angelica sinensis) is a herbal supplement that has been used in invigorating blood circulation. Here, we provided different lines of evidence to support the beneficial role of ASR against oxidative stress in cultured PC12 cell, a rat pheochromocytoma cell line. Methods: The water extract of ASR inhibited the activity of xanthine oxidase in vitro. In cultures, the pre-treatment of ASR water extract reduced the cytotoxic effect of tert-butyl hydroperoxide (tBHP), an oxidative stress inducer. Results: The protecting mechanisms of ASR were shown to be mediated by: (i) suppression of tBHP-induced reactive oxygen species (ROS) formation; (ii) induction of caspase-3 and PARP activities; and (iii) stimulation of mRNAs encoding antioxidative genes, glutathione S-transferase A2 (GSTA2) and NAD(P)H dehydrogenase quinone oxidoreductase 1 (NQO1), via the transcriptional activation of antioxidant response element (ARE). The outcome was the prevention of tBHP-induced cell apoptosis. Conclusion: Interestingly, the protective effect of ASR extract in PC12 cells were insignificant when challenging by insults of -amlyoid (for Alzheimer’s disease) and MPP+ (for Parkinson’s disease). Taken together, we revealed a neurobeneficial role of ASR in protecting neuronal cells against oxidative damage, which might be useful in developing health food supplements for disease prevention in future.

Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

Abstract 20538: Nrf2 Signaling Ameliorates Ventilator-Induced Lung Inflammation

Ventilator-induced lung injury (VILI) is an acute lung injury that develops during mechanical ventilation with high tidal volume in patients in the intensive care unit (ICU). VILI is a common entity with an unacceptable mortality rate (~20%). As no current therapies exist for VILI, novel therapeutic targets for VILI and mechanical stress-induced tissue injury are needed. In this study, we defined Nrf2 signaling in VILI and mechanical stress-induced endothelial cell pathobiology. Nrf2 is a transcription factor that alters ROS balance and is a novel therapeutic target for VILI. First, we defined Nrf2 as a sensory marker for mechanical stress-induced endothelial cell injury. Antioxidant response element (ARE) luciferase plasmids were transfected to both endothelial cells and murine lung tissues. EC exposed to 18% cyclic stretch (18% CS) or mice exposed to excessive tidal volume ventilation (40 ml/kg) resulted in ARE activation, site of Nrf2 binding. We next confirmed Nrf2 signaling is essential for gene expression dysregulation by 18% CS via Nrf2 knockdown that significantly ameliorates 18% CS-induced gene expression pattern, including signaling pathways regulating endothelial barrier function including MYLK. Finally, we validated that Nrf2 is a novel and effective therapeutic target for VILI in pre-clinical models of VILI. Nrf2 activator sulforaphane (SF, 11.5 mg/kg) significantly attenuated VILI (40 ml/kg, 4 hrs)-induced lung inflammation, including BAL protein leakage and white blood cell infiltration. These novel findings characterized Nrf2 as a sensor for mechanical stress, a central mediator of mechanical stress induced gene expression dysregulation, and a novel therapeutic target for VILI.

Pinocembrin attenuates 6-OHDA-induced neuronal cell death through Nrf2/ARE pathway in SH-SY5Y cells.

Pinocembrin (PB), the most abundant flavonoid in propolis, has been known to display antioxidant activity. However, the mechanism as how PB can induce antioxidant activity remains elusive. The purpose of the present study was to investigate the potential neuroprotective role of PB and to delineate its mechanism of action against the Parkinson's disease-related neurotoxin 6-hydroxydopamine(6-OHDA)-induced cell death in neuroblastoma SH-SY5Y cells. Results indicate that pretreatment with PB for 4h significantly reduced the 6-OHDA-induced cell viability loss, apoptotic rate and decreased Bcl-2/Bax ratio. In addition, PB inhibited 6-OHDA-induced oxidative stress as measured by the formation of reactive oxygen species, the level of malondialdehyde, mitochondrial membrane potential, and superoxide dismutase. Moreover, we have revealed the PB treatment resulted in an increase in nuclear factor E2-related factor 2 (Nrf2) protein levels and subsequent activation of antioxidant response element (ARE) pathway genes of heme oxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS) in SH-SY5Y cells. Treatment of SH-SY5Y cells with Nrf2 small interference RNA abolished PB-induced HO-1 and γ-GCS expression and its protective effects. Taken together, these findings suggest that PB can protect the SH-SY5Y cells from 6-OHDA-induced oxidative cell death via Nrf2/ARE pathway. Thus, our study indicates that PB has a partial cytoprotective role in dopaminergic cell culture systems.

Iodoacetic acid activates Nrf2-mediated antioxidant response in vitro and in vivo.

Iodoacetic acid (IAA) is an unregulated drinking-water disinfection byproduct with potent cytotoxicity, genotoxicity, and tumorigenicity in animals. Oxidative stress is thought to be essential for IAA toxicity, but the exact mechanism remains unknown. Here we evaluated the toxicity of IAA by examining nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant response, luciferase antioxidant response element (ARE) activity, and intracellular glutathione (GSH) in HepG2 cells. IAA showed significant activation of ARE-luciferase reporter, mRNA, and protein expression of Nrf2 and its downstream genes (GCLC, NQO1, and HO-1). IAA also increased the intracellular GSH level in HepG2 cells in a time- and concentration-dependent manner. Moreover, we verified IAA induced Nrf2-mediated antioxidant response in rats. Subsequently, we confirmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells. Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity and led to an increase of IAA induced micronulei. We also examined the effects of antioxidant on Nrf2-mediated response in IAA treated cells. Pretreatment with curcumin markedly reduced cytotoxicity and genotoxicity (micronuclei formation) IAA in HepG2 cells. Our work here provides direct evidence that IAA activates Nrf2-mediated antioxidant response in vitro and in vivo and that oxidative stress plays a role in IAA toxicity.

Grain and bean lysates improve function of endothelial progenitor cells from human peripheral blood: involvement of the endogenous antioxidant defenses.

Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective effect of lysates on EPCs exposed to oxidative stress through the involvement of antioxidant systems. Lisosan G seems to activate the Nrf-2/ARE pathways.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation.

Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells

The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3β). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3β pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3β inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3β pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3β and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer cells and suggest that patients׳ hormonal status through this activity may play a significant role in some therapeutic outcomes.

Study on anti-oxidative effect of extracts from Cichorium endivia on HepG2 cells and its mechanism

To investigate the protective effect of extracts from Cichorium endivia (CEE) in H2O2-induced HepG2 cell oxidative stress injury, and explore the antioxidant mechanism of CEE in HepG2 cells. The viability of H2O2-induced HepG2 cells and the intracellular ROS level were measured by MTT assay and DCFH-DA fluorescence staining assay. The antioxidant-response element (ARE)-Luciferase activity was tested in HepG2 cells stably transected by ARE reporter gene. The fluorescence quantitative RT-PCR was adopted to determine the mRNA expressions of genes containing ARE sequence in HepG2 cells. The cell viability reduced, while the ROS level increased after HepG2 cells were treated by H2O2. Different concentrations of CEE could be added to significantly improve the above results. After HepG2 cells transected by ARE reporter gene were treated with different concentrations of CEE, the intracellular ARE activity could increase in a concentration-dependent manner. In addition, the mRNA expressions of regulatory genesGCLC, GCLM and HMOX-1 containing ARE sequence in HepG2 cells were up-regulated in a concentration-dependent manner by CEE. CEE inhibited the H2O2-injured HepG2 cells by reducing the ROS level. CEE's antioxidant mechanism for HepG2 cells may be closely related to the antioxidant defense system associated with its effect of activating Nrf2-ARE pathway in HepG2 cells.

Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death

In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB(25)R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB(25)R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.

Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts

IntroductionOxidative stress can impair fracture healing. To protect against oxidative damage, a system of detoxifying and antioxidative enzymes works to reduce the cellular stress. The transcription of these enzymes is regulated by antioxidant response element (ARE). The nuclear factor (erythroid-derived 2)-like2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes. Recently it has been shown that vascular endothelial growth factor (VEGF) prevents oxidative damage via activation of the Nrf2 pathway in vitro. Platelet-released growth factor (PRGF) is a mixture of autologous proteins and growth factors, prepared from a determined volume of platelet-rich plasma (PRP). It has already used to enhance fracture healing in vitro. The aim of the present study was to elucidate if platelets can lead to upregulation of VEGF and if platelets can regulate the activity of Nrf2–ARE system in primary human osteoblast (hOB) and in osteoblast-like cell line (SAOS-2). MethodsPlatelets and PRGF were obtained from healthy human donors.HOB and SAOS-2 osteosarcoma cell line were used. The ARE activity was analysed using a dual luciferase reporter assay system. We used Western blot to detect the nuclear accumulation of Nrf2 and the amount of cytosolic antioxidant Thioredoxin Reductase-1 (TXNRD-1), Heme Oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO1). Gene expression analysis was performed by real-time RT PCR. ELISA was used for the quantification of growth factors. ResultsThe activity of ARE was increased in the presence of PRGF up to 50%. Western blotting demonstrated enhanced nuclear accumulation of Nrf2. This was followed by an increase in the protein expression of the aforementioned downstream targets of Nrf2. Real-time RT PCR data showed an upregulation in the gene expression of the VEGF after PRGF treatment. This was confirmed by ELISA, where the treatment with PRGF induced the protein level of VEGF in both cells. ConclusionsThese results provide a new insight into PRGF's mode of action in osteoblasts. PRGF not only leads to increase the endogenous VEGF, but also it may be involved in preventing oxidative damage through the Nrf2–ARE signalling. Nrf2 activation via PRGF may have great potential as an effective therapeutic drug target in fracture healing.

The volatile oil of Nardostachyos Radix et Rhizoma inhibits the oxidative stress-induced cell injury via reactive oxygen species scavenging and Akt activation in H9c2 cardiomyocyte

Ethnopharmacological relevanceNardostachyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a well-known medicinal herb widely used in Chinese, Uyghur and Ayurvedic medicines for the treatment of cardiovascular disorders. The oxidative stress-induced cardiomyocyte loss is the major pathogenesis of heart disorders. Here, the total volatile oil of NRR was isolated, and its function in preventing the cell death of cardiomyocyte was demonstrated. Materials and methodsThe cyto-protective effect of volatile oil of NRR against tBHP-induced H9c2 cardiomyocyte injury was measured by MTT assay. A promoter-report construct (pARE-Luc) containing four repeats of antioxidant response element (ARE) was applied to study the transcriptional activation of ARE. The amounts of phase ΙΙ antioxidant enzymes were analyzed by quantitative real-time polymer chain reaction (qPCR) upon the volatile oil treatment at 30μg/mL for 24h. The activation of Akt pathway was analyzed by western blot. ResultsIn cultured H9c2 cardiomyocytes, application of NRR volatile oil exhibited strong potency in preventing tBHP-induced cell death and accumulation of intracellular reactive oxygen species (ROS) in a concentration-dependent manner. In addition, the application of NRR volatile oil in cultures stimulated the gene expressions of self-defense antioxidant enzymes, which was mediated by the transcriptional activation of antioxidant response element (ARE). The induced genes were glutathione S-transferase, NAD(P)H quinone oxidoreductase, glutamate-cysteine ligase catalytic and modulatory subunits. In addition, the volatile oil of NRR activated the phosphorylation of Akt in cultured H9c2 cells. The treatment of LY294002, an Akt inhibitor, significantly inhibited the volatile oil-mediated ARE transcriptional activity, as well as the cell protective effect of NRR oil. ConclusionThese results demonstrated that NRR volatile oil prevented the oxidative stress-induced cell death in H9c2 cells by (i) reducing intracellular ROS production, (ii) inducing antioxidant enzymes and (iii) activating Akt phosphorylation.

Application of Complementary and Alternative Medicine on Neurodegenerative Disorders 2013

Application of Complementary and Alternative Medicine on Neurodegenerative Disorders 2013

Activation of the Nuclear Factor E2-Related Factor 2 Pathway by Novel Natural Products Halomadurones A–D and a Synthetic Analogue

Two novel chlorinated pyrones, halomadurones A and B, and two novel brominated analogues, halomadurones C and D, were isolated from a marine Actinomadura sp. cultivated from the ascidian Ecteinascidia turbinata. Additionally, a non-halogenated analogue, 2-methyl-6-((E)-3-methyl-1,3-hexadiene)-γ-pyrone, was synthesized to understand the role of the halogens for activity. Halomadurones C and D demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases.

Naringenin protects against 6-OHDA-induced neurotoxicity via activation of the Nrf2/ARE signaling pathway

There is increasing evidence that oxidative stress is critically involved in the pathogenesis of Parkinson's disease (PD), suggesting that pharmacological targeting of the antioxidant machinery may have therapeutic value. Naringenin, a natural flavonoid compound, has been reported to possess neuroprotective effect against PD related pathology; however the mechanisms underlying its beneficial effects are poorly defined. Thus, the purpose of the present study was to investigate the potential neuroprotective role of naringenin and to delineate its mechanism of action against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in models of PD both in vitro and in vivo. Naringenin treatment resulted in an increase in nuclear factor E2-related factor 2 (Nrf2) protein levels and subsequent activation of antioxidant response element (ARE) pathway genes in SH-SY5Y cells and in mice. Exposure of SH-SY5Y cells to naringenin provided protection against 6-OHDA-induced oxidative insults that was dependent on Nrf2, since treatment with Nrf2 siRNA failed to block against 6-OHDA neurotoxicity or induce Nrf2-dependent cytoprotective genes in SH-SY5Y cells. In mice, oral administration of naringenin resulted in significant protection against 6-OHDA-induced nigrostriatal dopaminergic neurodegeneration and oxidative damage. Our results indicate that activation of Nrf2/ARE signaling by naringenin is strongly associated with its neuroprotective effects against 6-OHDA neurotoxicity and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in PD.

The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors

The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib.We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib.In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies' culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies' mortality rate, healthspan and overall longevity.Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures:No relevant conflicts of interest to declare.