Cellular Antioxidant Defense System Research Articles

Alteration of mitochondrial DNA content modulates antioxidant enzyme expressions and oxidative stress in myoblasts.

Mitochondrial dysfunction is closely associated with reactive oxygen species (ROS) generation and oxidative stress in cells. On the other hand, modulation of the cellular antioxidant defense system by changes in the mitochondrial DNA (mtDNA) content is largely unknown. To determine the relationship between the cellular mtDNA content and defense system against oxidative stress, this study examined a set of myoblasts containing a depleted or reverted mtDNA content. A change in the cellular mtDNA content modulated the expression of antioxidant enzymes in myoblasts. In particular, the expression and activity of glutathione peroxidase (GPx) and catalase were inversely correlated with the mtDNA content in myoblasts. The depletion of mtDNA decreased both the reduced glutathione (GSH) and oxidized glutathione (GSSG) slightly, whereas the cellular redox status, as assessed by the GSH/GSSG ratio, was similar to that of the control. Interestingly, the steady-state level of the intracellular ROS, which depends on the reciprocal actions between ROS generation and detoxification, was reduced significantly and the lethality induced by H2O2 was alleviated by mtDNA depletion in myoblasts. Therefore, these results suggest that the ROS homeostasis and antioxidant enzymes are modulated by the cellular mtDNA content and that the increased expression and activity of GPx and catalase through the depletion of mtDNA are closely associated with an alleviation of the oxidative stress in myoblasts.

Therapeutic Potential of Plants and Plant Derived Phytochemicals against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP), which is also known as paracetamol or N-acetyl-p-aminophenol is a safe and potent drug for fever, pain and inflammation when used at its normal therapeutic doses. It is available as over-the-counter drug and used by all the age groups. The overdose results in acute liver failure that often requires liver transplantation. Current clinical therapy for APAP-induced liver toxicity is the administration of N-acetyl-cysteine (NAC), a sulphydryl compound an approved drug which acts by replenishing cellular glutathione (GSH) stores in the liver. Over the past five decades, several studies indicate that the safety and efficacy of herbal extracts or plant derived compounds that are used either as monotherapy or as an adjunct therapy along with conventional medicines for hepatotoxicity have shown favorable responses. Phytochemicals mitigate necrotic cell death and protect against APAP-induced liver toxicityby restoring cellular antioxidant defense system, limiting oxidative stress and subsequently protecting mitochondrial dysfunction and inflammation. Recent experimental evidences indicat that these phytochemicals also regulate differential gene expression to modulate various cellular pathways that are implicated in cellular protection. Therefore, in this review, we highlight the role of the phytochemicals, which are shown to be efficacious in clinically relevant APAP-induced hepatotoxicity experimental models. In this review, we have made comprehensive attempt to delineate the molecular mechanism and the cellular targets that are modulated by the phytochemicals to mediate the cytoprotective effect against APAP-induced hepatotoxicity. In this review, we have also defined the challenges and scope of phytochemicals to be developed as drugs to target APAP-induced hepatotoxicity.

Inhibition of RAGE Attenuates Cigarette Smoke-Induced Lung Epithelial Cell Damage via RAGE-Mediated Nrf2/DAMP Signaling.

The oxidative stress and cellular apoptosis by environmental factor including cigarette smoke induces alveolar airway remodeling leading to chronic obstructive pulmonary disease (COPD). Recently, the receptor for advanced glycan end products (RAGE) which is highly expressed in alveolar epithelium is emerging as a biomarker for COPD susceptibility or progression. However, it still remains unknown how RAGE plays a role in cigarette smoke extract (CSE)-exposed human alveolar type II epithelial cell line. Therefore, we determined the efficacy of RAGE-specific antagonist FPS-ZM1 in response to CSE-induced lung epithelial cells. CSE induced the elevated generation of RONS and release of pro-inflammatory cytokines, and impaired the cellular antioxidant defense system. Further, CSE induced the alteration of RAGE distribution via the activation of redox-sensitive DAMP (Damage-associated molecular patterns) signaling through Nrf2 in cells. Although pre-treatment with SB202190 (p38 inhibitor) or SP600125 (JNK inhibitor) failed to recover the alteration of RAGE distribution, treatment of FPS-ZM1 significantly exhibited anti-inflammatory and anti-oxidative/nitrosative effects, also inhibited the activation of redox-sensitive DAMP signaling through Nrf2 (nuclear factor erythroid 2-related factor 2) migration in the presence of CSE. Taken together, our data demonstrate that RAGE and Nrf2 play a pivotal role in maintenance of alveolar epithelial integrity.

Abstract 2421: SOD1 is required for tumor maintenance in a genetically engineered KRAS/TP53 non-small cell lung cancer mouse model

Abstract Lung cancer is the second most common cancer and the primary cause of cancer-related mortality globally. Over half a million people are estimated living with lung and bronchus cancer with a 5-year survival rate of 18%. Identifying and studying new drug targets are important for improving lung cancer patient survival. 80% to 85% of lung cancers are Non-small cell lung cancer (NSCLC). Knocking down and small molecule inhibition of Superoxide dismutase 1 (SOD1) has been shown to be cytotoxic to cultured NSCLC cells and mouse tumors, suggesting that SOD1 is important for NSCLC survival, thus presenting a promising therapeutic strategy. SOD1 rapidly converts superoxide anion to hydrogen peroxide. It is a critical component of the cellular antioxidant defense system. In addition, our laboratory has recently demonstrated that SOD1 has a novel function as a transcription factor that regulates oxidative stress response genes. Currently, there are no selective SOD1 inhibitors available to study physiological functions of SOD1. In our study, we show that genetically knockout SOD1 resulted in reduced tumor burden in lung cancer mouse model. Using mouse tumor-derived primary cancer cells, we further show that SOD1 is critical for NSCLC cell proliferation and survival. This study reveals novel functions of SOD1 that are of biological and translational significance, and provide further support for SOD1 as a promising therapeutic target for NSCLC. Citation Format: Xiaowen Wang, Justin Wong, Holly Van Remmen, Jessie Yanxiang Guo, Eileen White, X. F. Steven Zheng. SOD1 is required for tumor maintenance in a genetically engineered KRAS/TP53 non-small cell lung cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2421.

Multihierarchically Profiling the Biological Effects of Various Metal-Based Nanoparticles in Macrophages under Low Exposure Doses

Thus far, tremendous efforts have been made to understand the biosafety of metal-based nanoparticles (MNPs). Nevertheless, most previous studies focused on specific adverse outcomes of MNPs at unrealistically high concentrations with little relevance to the National Institute for Occupational Safety and Health (NIOSH) exposure thresholds, and failed to comprehensively evaluate their toxicity profiles. To address these challenges, we here endeavored to multihierarchically profile the hazard effects of various popularly used MNPs in macrophages under low exposure doses. At these doses, no remarkable cell viability drop and cell death were induced. However, a cellular antioxidant defense system was seen to be initiated in cells by all MNPs even at these low concentrations, albeit to a differential extent and through different pathways, as reflected by differential induction of the antioxidant enzymes and Nrf2 signaling. Regarding inflammation, rare earth oxide nanomaterials (REOs) except nCeO2 greatly increased IL-1β secretion in a NLRP3 inflammasome-dependent manner. By contrast, six REOs, AgNP-5nm, nFe2O3, nFe3O4, and nZnO were found to elevate TNF-α concentration through post-transcriptional regulation. Moreover, all MNPs except nCeO2 drastically altered cellular membrane/cytoskeleton meshwork, but leading to different outcomes, with condensed cellular size and reduced numbers of protrusions by REOs and elongated protrusions by other MNPs. Consequently, REOs (e.g., nDy2O3 and nSm2O3) impaired phagocytosis of macrophages, and other MNPs (such as AgNP-25nm and nZnO) reversely enhanced macrophagic phagocytosis. Alterations of membrane and cytoskeleton meshwork induced by these MNPs also caused disordered membrane potential and calcium ion flux. Collectively, our data profiled the biological effects of different MNPs in macrophages under low exposure doses, and deciphered a complex network that links multiparallel pathways and processes to differential adverse outcomes.

The Mitochondrial Citrate Carrier (SLC25A1) Sustains Redox Homeostasis and Mitochondrial Metabolism Supporting Radioresistance of Cancer Cells With Tolerance to Cycling Severe Hypoxia.

Pronounced resistance of lung cancer cells to radiotherapy and chemotherapy is a major barrier to successful treatment. Herein, both tumor hypoxia and the upregulation of the cellular antioxidant defense systems observed during malignant progression can contribute to radioresistance. We recently found that exposure to chronic cycling severe hypoxia/reoxygenation stress results in glutamine-dependent upregulation of cellular glutathione (GSH) levels and associated radiation resistance opening novel routes for tumor cell-specific radiosensitization. Here, we explored the role of the mitochondrial citrate carrier (SLC25A1) for the improved antioxidant defense of cancer cells with tolerance to acute and chronic severe hypoxia/reoxygenation stress and the use of pharmacologic SLC25A1 inhibition for tumor cell radiosensitization. Exposure to acute or chronic cycling severe hypoxia/reoxygenation stress triggered upregulated expression of SLC25A1 in lung cancer, prostate cancer, and glioblastoma cells in vitro. Interestingly, exposure to ionizing radiation (IR) further promoted SLC25A1 expression. Inhibition of SLC25A1 by 1,2,3-benzene-tricarboxylic acid (BTA) disturbed cellular and mitochondrial redox homeostasis, lowered mitochondrial metabolism, and reduced metabolic flexibility of cancer cells. Even more important, combining IR with BTA was able to overcome increased radioresistance induced by adaptation to chronic cycling severe hypoxia/reoxygenation stress. This radiosensitizing effect of BTA-treated cells was linked to increased reactive oxygen species and reduced DNA repair capacity. Of note, key findings could be reproduced when using the SLC25A1-inhibitor 4-Chloro-3-[[(3-nitrophenyl)amino]sulfonyl]-benzoic acid (CNASB). Moreover, in silico analysis of publically available databases applying the Kaplan–Meier plotter tool (kmplot.com) revealed that overexpression of SLC25A1 was associated with reduced survival of lung cancer patients suggesting a potential link to aggressive cancers. We show that SLC25A1 can contribute to the increased antioxidant defense of cancer cells allowing them to escape the cytotoxic effects of IR. Since upregulation of SLC25A1 is induced by adverse conditions in the tumor environment, exposure to IR, or both pharmacologic inhibition of SLC25A1 might be an effective strategy for radiosensitization of cancer cells particularly in chronically hypoxic tumor fractions.

P-201 - Prevention of endothelial dysfunction via cross-talk between Nrf2/ARE and IKK/NF-κB pathways by wine pomace product

The hyperglycemia-induced endothelial cell dysfunction is a key event in the onset and progression of cardiovascular disease (CVD), protection of vascular endothelial cells represents an important strategy for the diagnosis of CVD. Previous studies suggest that the polyphenols of a wine pomace (WP) could be able to modulate endothelial dysfunction, increase the levels of nitric oxide and decrease the levels of ROS. The objective of this study was to elucidate some of the possible pathways involved in the potential protective effects against endothelial dysfunction and oxidative damage in the vasculature of WP product. For this, we evaluated the Nrf2/ARE and IKK/IκB/NF-κB pathway in high glucose-induced oxidative stress in the endothelial cell line HUVECs EA.hy926. Analysis revealed that WP product-mediated antioxidant effects were due to up-regulation of cellular anti-oxidant genes, such as HO-1 and NQO-1 via promotion of the transcriptional activity of Nrf2 and down-regulation of the NF-κB pathway. These results suggest that WP product may represent a promising intervention in diabetic-associated cardiovascular diseases by activating the Nrf2-dependent cellular anti-oxidant defense system.

MDM2 controls NRF2 antioxidant activity in prevention of diabetic kidney disease

Oxidative stress and P53 contribute to the pathogenesis of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a master regulator of cellular antioxidant defense system, is negatively regulated by P53 and prevents DKD. Recent findings revealed an important role of mouse double minute 2 (MDM2) in protection against DKD. However, the mechanism remained unclear. We hypothesized that MDM2 enhances NRF2 antioxidant signaling in DKD given that MDM2 is a key negative regulator of P53. The MDM2 inhibitor nutlin3a elevated renal P53, inhibited NRF2 signaling and induced oxidative stress, inflammation, fibrosis, DKD-like renal pathology and albuminuria in the wild-type (WT) non-diabetic mice. These effects exhibited more prominently in nutlin3a-treated WT diabetic mice. Interestingly, nutlin3a failed to induce greater renal injuries in the Nrf2 knockout (KO) mice under both the diabetic and non-diabetic conditions, indicating that NRF2 predominantly mediates MDM2's action. On the contrary, P53 inhibition by pifithrin-α activated renal NRF2 signaling and the expression of Mdm2, and attenuated DKD in the WT diabetic mice, but not in the Nrf2 KO diabetic mice. In high glucose-treated mouse mesangial cells, P53 gene silencing completely abolished nutlin3a's inhibitory effect on NRF2 signaling. The present study demonstrates for the first time that MDM2 controls renal NRF2 antioxidant activity in DKD via inhibition of P53, providing MDM2 activation and P53 inhibition as novel strategies in the management of DKD.

Evaluation of attenuative effect of tert-butylhydroquinone against diazinon-induced oxidative stress on hematological indices in male Wistar rats.

Diazinon is an organophosphate toxicant that has been identified to induce oxidative stress within biological systems through altering biochemical and hematological indices, by generating free radicals while deteriorating the cellular antioxidant defense system. The present study was designed to evaluate the effectiveness of tert-butylhydroquinone (TBHQ), a synthetic food additive, in serving as an antioxidant against sub-lethal diazinon-induced oxidative stress in male Wistar rats. Animals were randomly divided into 4 groups: Control (treated with corn oil as a vehicle), diazinon (10 mg/kg/day), TBHQ (0.028 g/kg of diet) and a combination group of TBHQ (0.028 g/kg of diet) plus diazinon (10 mg/kg/day). All animals were treated orally once a day by gastric tube and treatments were continued for 7 weeks. Hematological indices, including red blood cell (RBC) indices and white blood cell (WBC) and platelet counts, were measured at the end of the treatment schedule. On comparison of the treatment groups with the untreated control group, RBC count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were significantly decreased in the diazinon and TBHQ+diazinon groups, while MCHC in the TBHQ group and MCV in the diazinon and TBHQ+diazinon groups were significantly increased. When the diazinon and TBHQ+diazinon groups were compared with the TBHQ group, RBC, Hb, Hct, and MCHC were significantly decreased whereas MCV was increased in both groups. Additionally, when comparing the TBHQ+diazinon group with the diazinon group, MCHC was determined to be significantly decreased. The results of the current experiments suggested that TBHQ could not efficiently protect blood cells against diazinon toxicity.

Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells

Nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) is a transcription factor that binds to the antioxidant response element (ARE) in the upstream promoter region of various antioxidant-responsive genes. Hence, at least in nonruminants, the NFE2L2-ARE signaling pathway plays an important role in the cellular antioxidant defense system. Whether oxidative stress in bovine mammary epithelial cells alters NFE2L2 or the NFE2L2-ARE pathway is unclear. Therefore, the objective of this study was to examine the response in NFE2L2- and NFE2L2-ARE-related components in bovine mammary epithelial cells (BMEC) under oxidative stress. An in silico analysis to identify potential phosphorylation sites on NFE2L2 and the protein kinases was performed with Netphos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite (http://scansite.mit.edu) software. Isolated BMEC were exposed to H2O2 (600 μM) for 6 h to induce oxidative stress. In silico analysis revealed ataxia telangiectasia-mutated (ATM) serine/threonine kinase as a key kinase responsible for the phosphorylation of NFE2L2. Thus, after the 6 h incubation with H2O2, BMEC were transiently transfected with ATM-small interfering RNA (siRNA) 1, 2, or 3. Compared with the control, transfection with ATM-siRNA3 resulted in proliferation rates that were 60.7 and 36.2% lower with or without H2O2. In addition, production of reactive oxygen species and malondialdehyde increased markedly, but activities of superoxide dismutase, glutathione peroxidase, catalase, and glutathione-S-transferase decreased markedly in transfected cells without or with H2O2 compared with the control. Transfected cells had markedly lower protein and mRNA expression of NFE2L2 without or with H2O2 compared with the control. In addition, fluorescent activity of the ARE in transfected BMEC indicated that NFE2L2-driven transcriptional activation decreased under oxidative stress. Overall, results indicate that ATM is a physiologically relevant NFE2L2 kinase. Furthermore, inhibition of ATM activity can cause marked alterations in oxidative stress leading to cell death as a result of diminished capacity of BMEC to cope with H2O2-induced cytotoxicity. The relevance of this kinase in vivo merits further study.

Rosmarinic acid attenuates β-amyloid-induced oxidative stress via Akt/GSK-3β/Fyn-mediated Nrf2 activation in PC12 cells.

Oxidative stress is an important pathogenic factor in Alzheimer's disease (AD). Recently, nuclear factor E2-related factor 2 (Nrf2) has emerged as a master regulator for the endogenous antioxidant response, and thus represents an attractive therapeutic target against AD. The aim of this study is to test the hypothesis that rosmarinic acid (RosA) attenuates amyloid-β (Aβ)-evoked oxidative stress through activating Nrf2-inducible cellular antioxidant defense system. Here, we reported that RosA attenuated Aβ-induced cellular reactive oxygen species (ROS) generation and lipid hydroperoxides (LPO). Interestingly, knockdown of Nrf2 by plasmid-based short hairpin RNA (shRNA) abrogated, at least in part, RosA-mediated neuroprotection in Aβ-challenged PC12 cells. Mechanistically, RosA enhanced the nuclear translocation of Nrf2 and binding to antioxidant response element (ARE) core element but did not induced Nrf2 transcription. Simultaneously, RosA induced a set of Nrf2 downstream target genes encoding phase-II antioxidant enzymes. Furthermore, RosA enhanced protein kinase B (Akt) phosphorylation, glycogen synthase kinase-3β (GSK-3β) phosphorylation at Ser9, and Fyn phosphorylation. Noteworthy, pharmacological inhibition or gene knockdown studies demonstrated that Akt locate upstream of GSK-3β and regulate Nrf2 through Fyn in the context of PC12 cells pre-incubated with RosA following exposed to Aβ. Conversely, the antioxidant effects of RosA could be blocked by Akt inhibitors LY294002, GSK-3β inhibitor LiCl, Nrf2 shRNA, or Fyn shRNA in Aβ-challenged PC12 cells. Consequently, the antioxidant effects of RosA are mediated predominantly by Akt/GSK-3β/Fyn pathway through increased activity of Nrf2. These results suggest, although do not prove, that RosA can be a promising candidate for neuroprotective treatment of AD.

Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies.

The ketogenic diet (KD) is a high-fat, low-carbohydrate diet based on the induction of the synthesis of ketone bodies (KB). Despite its widespread use, the systemic impact of KD is not completely understood. The purpose of this study was to evaluate the effects of physiological levels of KB on HMEC-1 endothelial cells. To this aim, DNA oxidative damage and the activation of Nrf2, a known transcriptional factor involved in cell responses to oxidative stress, were assessed. The exposure of cells to KB exerted a moderate genotoxic effect, measured by a significant increase in DNA oxidative damage. However, cells pre-treated with KB for 48 h and subjected to a secondary oxidative insult (H2O2), significantly decreased DNA damage compared to control oxidized cells. This protection occurred by the activation of Nrf2 pathway. In KB-treated cells, we found increased levels of Nrf2 in nuclear extracts and higher gene expression of HO-1, a target gene of Nrf2, compared to control cells. These results suggest that KB, by inducing moderate oxidative stress, activate the transcription factor Nrf2, which induces the transcription of target genes involved in the cellular antioxidant defense system.

Protective effect of mitochondrial-targeted antioxidant MitoQ against iron ion 56Fe radiation induced brain injury in mice

Exposure to iron ion 56Fe radiation (IR) during space missions poses a significant risk to the central nervous system and radiation exposure is intimately linked to the production of reactive oxygen species (ROS). MitoQ is a mitochondria-targeted antioxidant that has been shown to decrease oxidative damage and lower mitochondrial ROS in a number of animal models. Therefore, the present study aimed to investigate role of the mitochondrial targeted antioxidant MitoQ against 56Fe particle irradiation-induced oxidative damage and mitochondria dysfunction in the mouse brains. Increased ROS levels were observed in mouse brains after IR compared with the control group. Enhanced ROS production leads to disruption of cellular antioxidant defense systems, mitochondrial respiration dysfunction, altered mitochondria dynamics and increased release of cytochrome c (cyto c) from mitochondria into cytosol resulting in apoptotic cell death. MitoQ reduced IR-induced oxidative stress (decreased ROS production and increased SOD, CAT activities) with decreased lipid peroxidation as well as reduced protein and DNA oxidation. MitoQ also protected mitochondrial respiration after IR. In addition, MitoQ increased the expression of mitofusin2 (Mfn2) and optic atrophy gene1 (OPA1), and decreased the expression of dynamic-like protein (Drp1). MitoQ also suppressed mitochondrial DNA damage, cyto c release, and caspase-3 activity in IR-treated mice compared to the control group. These results demonstrate that MitoQ may protect against IR-induced brain injury.

Inhibitory effect of catecholic colonic metabolites of rutin on fatty acid hydroperoxide and hemoglobin dependent lipid peroxidation in Caco-2 cells.

To determine the preventive effect of dietary rutin on oxidative damages occurring in the digestive tract, 13-hydroperoxyoctadecadienoic acid and hemoglobin were exposed to Caco-2 intestinal cells after the pretreatment with colonic rutin metabolites. Among four catechol-type metabolites, quercetin and 3,4-dihydroxytoluene exerted significant protection on 13-hydroperoxyoctadecadienoic and hemoglobin-dependent lipid peroxidation of this epithelial cell. Compared with quercetin, a much lower concentration allowed 3,4-dihydroxytoluene to maximize the protective effect, though it needed a longer pre-incubation period. Neither quercetin nor 3,4-dihydroxytoluene affected the expression of peroxiredoxin-6 protein, which comprises the cellular antioxidant defense system. It is concluded that 3,4-dihydroxytoluene is a plausible rutin colonic metabolite that can suppress oxidative damages of intestinal epithelial cells by directly inhibiting lipid peroxidation. This result may illuminate the preventive role of dietary rutin against colorectal cancer incidence in relation to the consumption of red and processed meat.

SP600125 suppresses Keap1 expression and results in NRF2-mediated prevention of diabetic nephropathy.

c-Jun N-terminal kinase (JNK) contributes to the pathogenesis of diabetic nephropathy (DN). The JNK inhibitor SP600125 was reported to ameliorate DN. However, the mechanism remained unclear. We previously reported that SP600125 activated nuclear factor erythroid 2-related factor 2 (NRF2), a governor of the cellular antioxidant defense system, in the aortas of the diabetic mice. Given the critical role of NRF2 in preventing DN, the present study aimed to test whether or not NRF2 is required for SP600125's protection against DN. To test the role of NRF2 in SP600125's effect, streptozotocin-induced C57BL/6 wild-type (WT) and Nrf2-knockout (KO) diabetic mice were treated in the presence or absence of SP600125, for 24 weeks. To explore the mechanism by which SP600125 activates NRF2, mouse mesangial cells (MMCs) were treated with high glucose (HG), in the presence or absence of either SP600125 or JNK siRNA. SP600125 significantly attenuated the diabetes-induced renal oxidative stress, inflammation, fibrosis, pathological change and dysfunction in the WT, but not the Nrf2 KO mice. SP600125 inactivated JNK, inhibited kelch-like ECH-associated protein 1 expression, preserved NRF2 protein and facilitated its nuclear translocation in the kidneys of the WT mice, the effects of which were similarly produced by either SP600125 or JNK siRNA in HG-treated MMCs. Further, both SP600125 and JNK siRNA alleviated HG-induced mesangial oxidative stress and expression of inflammatory and fibrotic genes. The present study demonstrates that NRF2 is required for SP600125's protection against DN. SP600125 activates NRF2 possibly via inhibition of JNK-induced Keap1 expression.

Sichuan pepper attenuates H2O2-induced apoptosis via antioxidant activity and up-regulating heme oxygenase-1 gene expression in primary rat hepatocytes

Sichuan pepper, a well-known traditional Chinese spice, is popularly used in Chinese cuisine. In this study, the antioxidant activities of Sichuan pepper water extract (SP) were evaluated both chemically [in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay] and biologically [in 2,2′-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay]. The protective effect of SP against H2O2-induced cytotoxicity in primary Sprague Dawley rat hepatocytes was investigated by using cell viability assay, nuclear staining, intracellular reactive oxygen species (ROS) measurement and flow cytometry. It was found to possess high total phenolic content as assessed by the Folin–Ciocalteu method. The antiapoptotic effect of SP may be mediated, in part, by significantly inhibiting intracellular ROS generation and depletion of the mitochondrial membrane potential. SP was also found to enhance the cellular antioxidant defense system through up-regulating heme oxygenase-1 expression. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases. Practical applications The present study demonstrated that Sichuan pepper water extract (SP) could enhance the cellular antioxidant ability in hepatocytes. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases.

Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells.

In the present study, we investigated the effects of antrodin C (ADC), a maleimide derivative isolated from mycelia of Antrodia cinnamomea, on high glucose (HG, 30 mM)-accelerated endothelial dysfunction in vitro. HG-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) was significantly ameliorated by ADC. In addition, treatment with ADC significantly prevented HG-induced senescence, growth arrest at the G1-S transition phase and apoptosis in HUVECs. Moreover, the increased level of intracellular reactive oxygen species (ROS) under HG condition was significantly ameliorated by ADC. Further analysis revealed that ADC-mediated anti-oxidant effects were due to up-regulation of cellular anti-oxidant genes, such as HO-1 and NQO-1 via promotion of the transcriptional activity of Nrf2, which was further confirmed by the failure of ADC to protect HUVECs from HG-induced dysfunction under HO-1 inhibition or Nrf2 silencing. Furthermore, hyperosmotic glucose (HOG, 60 mM)-induced uncontrolled production of ROS, rapid apoptotic cell death and HUVEC injury were significantly prevented by ADC, whereas these preventive effects were barely observed in HO-1 inhibited or Nrf2 silenced cells. Taken together, these results suggest that ADC may represent a promising intervention in diabetic-associated cardiovascular diseases by activating the Nrf2-dependent cellular anti-oxidant defense system.

Dietary Phytochemicals Promote Health by Enhancing Antioxidant Defence in a Pig Model.

Phytochemical-rich diets are protective against chronic diseases and mediate their protective effect by regulation of oxidative stress (OS). However, it is proposed that under some circumstances, phytochemicals can promote production of reactive oxygen species (ROS) in vitro, which might drive OS-mediated signalling. Here, we investigated the effects of administering single doses of extracts of red cabbage and grape skin to pigs. Blood samples taken at baseline and 30 min intervals for 4 hours following intake were analyzed by measures of antioxidant status in plasma, including Trolox equivalent antioxidant capacity (TEAC) and glutathione peroxidase (GPx) activity. In addition, dose-dependent production of hydrogen peroxide (H2O2) by the same extracts was measured in untreated commercial pig plasma in vitro. Plasma from treated pigs showed extract dose-dependent increases in non-enzymatic (plasma TEAC) and enzymatic (GPx) antioxidant capacities. Similarly, extract dose-dependent increases in H2O2 were observed in commercial pig plasma in vitro. The antioxidant responses to extracts by treated pigs were highly correlated with their respective yields of H2O2 production in vitro. These results support that dietary phytochemicals regulate OS via direct and indirect antioxidant mechanisms. The latter may be attributed to the ability to produce H2O2 and to thereby stimulate cellular antioxidant defence systems.

Evaluating a Second Generation Phytochemical Nrf2 Activator on Proteostasis and Cytoprotective Gene Expression in Vivo

Aging is associated with increases in oxidative stress. Redox imbalance occurs when production of reactive oxygen species (ROS) exceeds the capacity of antioxidant enzymes to eliminate ROS. Increased levels of intracellular ROS can compromise proteostasis by causing irreversible damage to proteins. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) mediates the cellular endogenous antioxidant defense system by regulating antioxidant enzymes that are cytoprotective against ROS. Nrf2 can be activated phytochemically through the supplement Protandim. Previous work from our lab has demonstrated that a phytochemical based Nrf2 activator improves proteostasis in skeletal muscle in vivo. Recently, we have begun to characterize a second generation Nrf2 activator (PB125) that has increased anti-inflammatory action in addition to anti-oxidant properties. Since inflammation can blunt protein synthetic responses, we speculated that PB125 might provide additional benefits on proteostatic processes. PURPOSE: The purpose of the present study was to examine in vivo the effects of three different doses (10, 100, and 300 ppm) of PB125 supplementation on Nrf2 activation and proteostasis. METHODS: 60 male CB6F1 mice aged 10–11 months were assigned to diets containing low, medium, or high doses of product PB125 in a 5 week feeding study. Mice were isotopically labeled with 8% deuterium oxide (D2O) to simultaneously measure protein and DNA fractional synthesis rates (FSR) in liver, heart, and skeletal muscle. Nrf2 activation was assessed through analysis of gene expression profiles via Affymetrix GeneChip microarray. RESULTS: Proteostatic mechanisms were increased in the liver mitochondrial fraction in the 10 ppm treatment group (18.9 vs 16.3 FSR%/day, p<0.05). However, there were no differences in proteostatic mechanisms in heart or skeletal muscle. At 100 ppm, there was up-regulation of Nrf2-dependent cytoprotective genes (Akr1d1, Gpx2, Gclm, Fthl17b, 3.82, 1.84, 1.42, 1.64-fold increase). CONCLUSION: From our data we were able to conclude that all three doses were safe, and that 100 ppm was effective at activating Nrf2. In addition, there was an indication of increased proteostatic processes in the liver, but not heart or skeletal muscle, perhaps due to the healthy status of the mice.

Prochlorperazine interaction with melanin and melanocytes.

Prochlorperazine is a phenothiazine-class antipsychotic drug usually used to treat nausea, vomiting and schizophrenia. Phenothiazine derivatives have been known to cause serious side effects, like extrapyramidal symptoms, but also skin disorders which mechanism has not been fully established. The aim of this study was to examine the interaction between prochlorperazine and melanin as well as to estimate the effect of prochlorperazine on cell viability, melanogenesis and antioxidant defense system in normal human melanocytes. We have demonstrated that prochlorperazine forms stable complexes with melanin, characterized by two classes of independent binding sites with the association constants K1∼106 M-1 and K2∼102 M-1. It has been shown that prochlorperazine induces concentration-dependent loss in cell viability. The value of EC50 was calculated to be 18.49 μM. Prochlorperazine in a concentration of 0.001 μM stimulated melanogenesis, while in concentrations 1.0 and 10.0 μM melanization process was inhibited. Furthermore, the drug in concentrations of 0.1, 1.0 and 10.0 μM caused changes in cellular antioxidant defense system, what indicated the induction of oxidative stress. The observed changes in cell viability, melanization and antioxidant defense system in normal human melanocytes after prochlorperazine treatment may explain a potential role of melanin, oxidative stress and melanocytes in mechanisms of undesirable side effects after accumulation of this drug in pigmented tissues.