Reactive Oxygen Species Generation In Response Research Articles

The sensitivity of acute myeloid leukemia cells to cytarabine is increased by suppressing the expression of Heme oxygenase-1 and hypoxia-inducible factor 1-alpha

BackgroundAcute myeloid leukemia (AML), a malignancy Often resistant to common chemotherapy regimens (Cytarabine (Ara-c) + Daunorubicin (DNR)), is accompanied by frequent relapses. Many factors are involved in causing chemoresistance. Heme Oxygenase-1 (HO-1) and Hypoxia-Inducible Factor 1-alpha (HIF-1α) are two of the most well-known genes, reported to be overexpressed in AML and promote resistance against chemotherapy according to several studies. The main chemotherapy agent used for AML treatment is Ara-c. We hypothesized that simultaneous targeting of HO-1 and HIF-1α could sensitize AML cells to Ara-c.MethodIn this study, we used our recently developed, Trans-Activator of Transcription (TAT) - Chitosan-Carboxymethyl Dextran (CCMD) - Poly Ethylene Glycol (PEG) - Nanoparticles (NPs), to deliver Ara-c along with siRNA molecules against the HO-1 and HIF-1α genes to AML primary cells (ex vivo) and cell lines including THP-1, KG-1, and HL-60 (in vitro). Subsequently, the effect of the single or combinational treatment on the growth, proliferation, apoptosis, and Reactive Oxygen Species (ROS) formation was evaluated.ResultsThe designed NPs had a high potential in transfecting cells with siRNAs and drug. The results demonstrated that treatment of cells with Ara-c elevated the generation of ROS in the cells while decreasing the proliferation potential. Following the silencing of HO-1, the rate of apoptosis and ROS generation in response to Ara-c increased significantly. While proliferation and growth inhibition were considerably evident in HIF-1α-siRNA-transfected-AML cells compared to cells treated with free Ara-c. We found that the co-inhibition of genes could further sensitize AML cells to Ara-c treatment.ConclusionsAs far as we are aware, this study is the first to simultaneously inhibit the HO-1 and HIF-1α genes in AML using NPs. It can be concluded that HO-1 causes chemoresistance by protecting cells from ROS damage. Whereas, HIF-1α mostly exerts prolific and direct anti-apoptotic effects. These findings imply that simultaneous inhibition of HO-1 and HIF-1α can overcome Ara-c resistance and help improve the prognosis of AML patients.

Reactive Oxygen Species Are Central Mediators of Vascular Dysfunction and Hypertension Induced by Ethanol Consumption.

Consumption of high amounts of ethanol is a risk factor for development of cardiovascular diseases such as arterial hypertension. The hypertensive state induced by ethanol is a complex multi-factorial event, and oxidative stress is a pathophysiological hallmark of vascular dysfunction associated with ethanol consumption. Increasing levels of reactive oxygen species (ROS) in the vasculature trigger important processes underlying vascular injury, including accumulation of intracellular Ca2+ ions, reduced bioavailability of nitric oxide (NO), activation of mitogen-activated protein kinases (MAPKs), endothelial dysfunction, and loss of the anticontractile effect of perivascular adipose tissue (PVAT). The enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a central role in vascular ROS generation in response to ethanol. Activation of the renin-angiotensin-aldosterone system (RAAS) is an upstream mechanism which contributes to NADPH oxidase stimulation, overproduction of ROS, and vascular dysfunction. This review discusses the mechanisms of vascular dysfunction induced by ethanol, detailing the contribution of ROS to these processes. Data examining the association between neuroendocrine changes and vascular oxidative stress induced by ethanol are also reviewed and discussed. These issues are of paramount interest to public health as ethanol contributes to blood pressure elevation in the general population, and it is linked to cardiovascular conditions and diseases.

ROS production in response to high-power microwave pulses induces p53 activation and DNA damage in brain cells: Radiosensitivity and biological dosimetry evaluation.

Background: Pulsed high-power microwave (HPM) has many applications and is constantly being researched to expand its uses in the future. As the number of applications grows, the biological effects and safety level of pulsed HPM become a serious issue, requiring further research. Objective: The brain is regarded as the most vulnerable organ to radiation, raising concerns about determining an acceptable level of exposure. The effect of nanosecond pulses and the mechanisms underlying HPM on the brain has not been studied. For the first time, we observed the effect of pulsed 3.5GHz HPM on brain normal astrocytes and cancer U87 MG cells, as well as the likely mechanisms involved. Methods: To generate 3.5GHz HPM, an axial virtual cathode oscillator was constructed on pulsed power generator "Chundoong". The cells were directly exposed to HPM (10, 25, 40, and 60) pulses (1mJ/pulse), with each pulse delivered after 1min of charging time to evaluate the dose dependent effects. Results: A strong electric field (∼23kV/cm) of HPM irradiation primarily causes the production of reactive oxygen species (ROS), altering cell viability, mitochondrial activity, and cell death rates in U87 and astrocytes at certain dosages. The ROS generation in response to HPM exposure was primarily responsible for DNA damage and p53 activation. The hazardous dosage of 60 pulses is acknowledged as having damaging effects on brain normal cells. Interestingly, the particular 25 pulses exhibited therapeutic effects on U87 cells via p53, Bax, and Caspase-3 activation. Conclusion: HPM pulses induced apoptosis-related events such as ROS burst and increased oxidative DNA damage at higher dosages in normal cells and specific 25 pulses in cancer U87. These findings are useful to understand the physiological mechanisms driving HPM-induced cell death, as well as the safety threshold range for HPM exposure on normal cells and therapeutic effects on cancer U87. As HPM technology advances, we believe this study is timely and will benefit humanity and future research.

Arsenic-Induced Injury of Mouse Hepatocytes through Lysosome and Mitochondria: An In Vitro Study.

Background and Aims The cellular mechanism of liver injury related to arsenic toxicity is ill defined. It is thought that oxidative stress and mitochondrial dysfunction may play some role in arsenic-induced liver damage. In this study, we evaluated subcellular events within the primary cultured mouse hepatocytes when exposed to inorganic arsenic. Methods Primary cultured mouse hepatocytes were treated with 10 μM arsenic for different time periods. Reactive oxygen species (ROS) formation, functional changes of the lysosome and mitochondria, and mode of hepatocytes death were studied by laser confocal microscopy, fluorescence spectroscopy, and flow cytometry. Expression of proapoptotic member of the BCL-2 family of genes BAX and antiapoptotic BCL-2 mRNA expression were studied by real-time PCR. Cytochrome c expression was studied by Western blotting. Results Fluorescence spectroscopy as well as flow cytometric analysis revealed that arsenic-induced formation of ROS was time dependent. Confocal microscopy showed initiation of ROS formation from periphery of the hepatocytes at 30 min of arsenic exposure that progressed to central part of the hepatocytes at 3 h of arsenic exposure. The ROS formation was found to be NADPH oxidase (NOX) dependent. This low level of intracellular ROS induced lysosomal membrane permeabilization (LMP) and subsequently released cathepsin B to the cytosol. The LMP further increased intracellular ROS which in turn triggered induction of mitochondrial permeability transition (MPT). Pretreatment of hepatocytes with LMP inhibitor bafilomycin A (BafA) significantly decreased, and LMP inducer chloroquine (ChQ) significantly increased the production of ROS suggesting that LMP preceded enhanced ROS generation in response to arsenic. MPT was accompanied with increase in BAX : BCL2 mRNA ratio resulting in upregulation of caspase 3 and increased hepatocyte apoptosis. Conclusion Although arsenic-related oxidative liver injury is well established, neither the site of origin of ROS nor the early sequence of events in arsenic toxicity due to ROS is known. We believe that our study provides evidences elucidating the early sequence of events that culminates in the death of the mouse hepatocytes during arsenic exposure.

Evodiamine inhibits stemness and metastasis by altering the SOX9-β-catenin axis in non-small-cell lung cancer.

Evodiamine (EVO), a natural dietary alkaloid extracted from the roots of the Evodia rutaecarpa, has shown anticancer activities. Here, we have investigated EVO's role in inhibiting cell proliferation and migration in A549 and NCI-H522 lung cancer cells. EVO decreased the cell viability in A549 and NCI-H522 cells in a dose- and time-dependent manner. It also induced apoptosis by downregulating the expression of antiapoptotic Bcl-2 and upregulating the expression of cleaved caspase-3 and PARP. In addition, the treatment of EVO elevated the level of reactive oxygen species (ROS) generation inside the cells to induce the cell death pathways. In contrast, the pretreatment of ROS scavenger, N-acetyl cysteine, reverses the effect of EVO and attenuates cell death. Moreover, excess ROS generation in response to EVO resulted in the depletion of mitochondrial membrane potential. Furthermore, it induced DNA damage and arrested the cell cycle at the G2/M phase in A549 and NCI-H522 cells. Our study also investigated that EVO significantly suppressed tumorigenicity by inhibiting colony formation and tumorsphere formation. However, the treatment of EVO downregulated the cancer stem cell markers CD44 and CD133 in non-small-cell lung cancer. The inhibitory effect of EVO on cell invasion was mediated by altering the expression of E-cadherin, ZO-1, N-cadherin, and Vimentin. Additionally, we have revealed that EVO treatment showed downregulation of SOX9, an upstream component of β-catenin. Lastly, we have demonstrated that EVO targets the SOX9-β-catenin axis by reducing SOX9 and β-catenin expression. These findings suggested that EVO could be a promising agent for treating human lung cancer.

Plasma obtained following murine hindlimb ischemic conditioning protects against oxidative stress in zebrafish models through activation of nrf2a and downregulation of duox.

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.

Variation in Neutrophil Function Among Healthy Subjects Influences Response to Bacteria

Introduction: We have recently shown that healthy subjects have variable neutrophil degranulation responses to antigen/antibody immune complexes (ICs; Duarte ME Blood 2019). With repeated testing over 2 years, we have shown that susceptibility to neutrophil activation represents a fixed phenotype: some healthy subjects have neutrophils which robustly degranulate in response to ICs, while others have neutrophils that are minimally reactive (>30-fold variation in release of MMP9, representative of tertiary granules). Whether this variation in degranulation responses is specific to ICs or is applicable to other stimulants is not known. Because neutrophils are involved in the first steps of host defense against bacterial pathogens, we aimed to determine if these phenotypes are associated with differences in neutrophil effector functions which may contribute to host defense. Methods/Results: To determine if observed neutrophil phenotypes are relevant to host defense, we compared neutrophil effector responses from "high" and "low" donors after being challenged with bacterial products including the bacterial tripeptide N-formyl-met-leu-phe (fMLP), LPS, and Staphylococcus aureus (USA300) supernatant. As a model IC, we chose to use KKO-platelet factor 4/heparin ICs, as neutrophil responses to this IC have been previously characterized by our lab. As a first step, we examined several healthy subjects and compared degranulation responses to ICs, fMLP, and LPS to determine if susceptibility to degranulation was preserved. As shown in Figure 1, the quantity of MMP9 released in response to ICs was highly correlated with MMP9 released in response to fMLP (Fig 1A) and to LPS (Fig 1B, r = 0.9 for both). We then determined if neutrophil phenotypes were preserved in response to bacteria. As shown in Figure 2, neutrophils from "high" and "low" donors differentially degranulated in response to Staphylococcus aureus supernatant, with continued preservation of the neutrophil phenotype. We next aimed to determine if the neutrophil phenotype was associated with differences in other neutrophil effector functions, besides degranulation, which are relevant to pathogen defense. First, we focused on neutrophil extracellular trap (NET) release, which has an important role in host defense. As shown in Figure 3, "high" and "low" donors differed in NET release in response to ICs, as quantified by visualization using immunofluorescent microscopy. This finding is supported by our previous report that neutrophil phenotypes are correlated with differences in MPO release, a NET component (Duarte ME, et al 2019). We also examined reactive oxygen species (ROS) generation, a primary mechanism by which neutrophils eliminate pathogens. As shown in Figure 4, using DCFH-DA, a fluorescent probe, we show that "high" and "low" donors differ in ROS generation in response to PMA and fMLP. Conclusions: Taken together, our studies demonstrate that neutrophil responses to a variety of stimuli (ICs, LPS, and fMLP) are highly concordant among healthy donors with "high" or "low" neutrophil phenotypes and that these differences in neutrophil effector functions are likely relevant to host defense. Further studies are needed to determine how differences in innate neutrophil function may affect clinical outcomes during bacterial infection. Disclosures Arepally: Apotex: Consultancy, Research Funding; Biokit: Consultancy, Patents & Royalties; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Annexon Biosciences: Consultancy, Other; Alexion: Other; Novartis: Consultancy.

Cavitation-Inducible Mesoporous Silica-Titania Nanoparticles for Cancer Sonotheranostics.

Sonodynamic therapy has received increasing attention for cancer treatments as an alternative to photodynamic therapy. However, its clinical applications have been limited by the lack of a sonosensitizer that is capable of producing sufficient amounts of reactive oxygen species (ROS) in response to ultrasound (US) exposure. Herein, PEGylated mesoporous silica-titania nanoparticles (P-MSTNs) are prepared and used as US-responsive nanocarriers for cancer sonotheranostics. Perfluorohexane (PFH), which is chosen as the gas precursor, is physically encapsulated into P-MSTNs using the oil-in-water emulsion method. Owing to the vaporization of the gas precursor, PFH@P-MSTNs (137 nm in diameter) exhibit a strong photoacoustic signal in vivo for at least 6 h. Compared to P-MSTNs, PFH@P-MSTNs generate significantly higher amounts of ROS due to the nanobubble-induced cavitation in the presence of US. When systemically administered to tumor-bearing mice, PFH@P-MSTNs effectively accumulate in the tumor site due to the passive targeting mechanism. Consequently, PFH@P-MSTNs show much higher antitumor efficacy than P-MSTNs due to the enhanced cavitation-mediated ROS generation in response to US exposure. It is considered that PFH@P-MSTNs may hold significant potential for cancer sonotheranostics.

N-Acetyl cysteine abrogates silver-induced reactive oxygen species in human cells without altering silver-based antimicrobial activity

Silver-based antimicrobials are widely used topically to treat infections associated with multi-drug resistant (MDR) pathogens. Expanding this topical use to aerosols to treat lung infections requires understanding and preventing silver toxicity in the respiratory tract. A key mechanism resulting in silver-induced toxicity is the production of reactive oxygen species (ROS). In this study, we have verified ROS generation in silver-treated bronchial epithelial cells prompting evaluation of three antioxidants, N-acetyl cysteine (NAC), ascorbic acid, and melatonin, to identify potential prophylactic agents. Among them, NAC was the only candidate that abrogated the ROS generation in response to silver acetate exposure resulting in the rescue of these cells from silver-associated toxicity. Further, this protective effect directly translated to preservation of metabolic activity, as demonstrated by the normal levels of citric acid cycle metabolites in NAC-pretreated silver acetate-exposed cells. Because the citric acid cycle remained functional, silver-exposed cells pre-incubated with NAC demonstrated significantly higher levels of adenosine triphosphate levels compared with NAC-free controls. Moreover, we found that this prodigious capacity of NAC to rescue silver acetate-exposed cells was due not only to its antioxidant activity, but also to its ability to directly bind silver. Despite binding to silver, NAC did not alter the antimicrobial activity of silver acetate.

Ehrlichia chaffeensis Uses an Invasin To Suppress Reactive Oxygen Species Generation by Macrophages via CD147-Dependent Inhibition of Vav1 To Block Rac1 Activation.

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.IMPORTANCEEhrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

Dyslipidemia-associated atherogenic oxidized lipids induce platelet hyperactivity through phospholipase Cγ2-dependent reactive oxygen species generation

Oxidized low-density lipoprotein (oxLDL) and associated oxidized phosphocholine-headgroup phospholipids (oxPCs) activate blood platelets through ligation of the scavenger receptor CD36. Previously, we found that oxLDL stimulated phosphorylation of phospholipase Cγ2 (PLCγ2). However, the functional relevance of PLCγ2 phosphorylation in oxLDL-mediated platelet hyperactivity remained elusive. Here, we set out to explore the functional importance of PLCγ2 in oxLDL-mediated platelet activation using human and genetically modified murine platelets. The CD36-specific oxidized phospholipid (oxPCCD36) triggered the generation of reactive oxygen species (ROS) in platelets under static and arterial flow conditions. The ROS generation in response to oxPCCD36 was sustained for up to 3 h but ablated in CD36- and PLCγ2-deficient platelets. The functional importance of ROS generation in response to atherogenic lipid stress was examined through measurement of P-selectin expression. OxPCCD36 induced P-selectin expression, but required up to 60 min incubation, consistent with the timeline for ROS generation. P-selectin expression was not observed in CD36- and PLCγ2-deficient mice. The ability of oxPCCD36 and oxLDL to stimulate P-selectin expression was prevented by incubation of platelets with the ROS scavenger N-acetyl-cysteine (NAC) and the NOX-2 inhibitor gp91ds-tat, but not with the NOX-1 inhibitor ML171. In summary, we provide evidence that prolonged exposure to oxLDL-associated oxidized phospholipids induces platelet activation via NOX-2-mediated ROS production in a CD36- and PLCγ2-dependent manner.

Activation of NLRX1-mediated autophagy accelerates the ototoxic potential of cisplatin in auditory cells

To date, the mechanism (s) underlying the cisplatin-elicited ototoxicity has not been elucidated fully. Nucleotide-binding domain and leucine-rich-repeat-containing family member ×1 (NLRX1), a cytoplasmic pattern recognition receptor, is tightly related to mitochondrial function, reactive oxygen species (ROS) production, and autophagy. In this work, autophagy alteration, NLRX1 expression, ROS generation and cell injury were investigated correspondingly by immunofluorescence staining, western-blot, TEM, flow cytometry and MTT in HEI-OC1 cells of both NLRX1 overexpression and silencing in response to cisplatin stimulus. We found that NLRX1 expression was increased concurrent with the increase of autophagy activation in HEI-OC1 cells under the cisplatin insult. NLRX1 overexpression led to the amount of accumulation of autophagsomes in HEI-OC1 cells in normal condition and a higher activation of autophagy concurrent with cell injury in HEI-OC1 cells treated with cisplatin, whereas, NLRX1 silencing decreased the activation level of autophagy concurrent with increased cell viability in HEI-OC1 cells treated with cisplatin. Mechanistic studies showed that NLRX1 potentiated mitochondrial-derived ROS generation in response to cisplatin exposure. Inhibition of ROS generation significantly prevented autophagy activation and apoptosis both in HEI-OC1cells and cochlear explants treated with cisplatin. The findings from this work reveal that NLRX1 sensitizes auditory cells in vitro to cisplatin-induced ototoxity via autophagic cell death pathway, providing another strategy against cisplatin-induced ototoxity.

Differential ROS Generation in Response to Stress in Symbiodinium spp.

Oxidative stress inside cells occurs when the production of reactive oxygen species (ROS) is no longer efficiently counterbalanced by the generation of antioxidants. In this study, we measured the intracellular production of ROS, including hydrogen peroxide (H2O2), superoxide (O2-), and singlet oxygen (1O2), in cultured dinoflagellates of the genus Symbiodinium under thermal and oxidative stress. ROS tagged with fluorescent probes were measured by flow cytometry. Dissimilar Symbiodinium internal transcribed spacer 2 (ITS2) clades or phylotypes (A1, B2, E, F1) produced ROS in different quantities in response to stress. For example, when comparing the control (26 °C) to the high-temperature treatment (35 °C), Symbiodinium E showed no change in the intracellular concentrations of any of the ROS; but phylotype A1 displayed a 10-fold increase in the overall ROS concentration and a 4-fold increase in O2-. Under oxidative stress, when 8 mmol l-1 H2O2 was added to the cells, these same two Symbiodinium phylotypes increased their overall concentrations of ROS, but only Symbiodinium E showed an increase in the concentrations of O2- (2×) and 1O2 (3×). Therefore, not only were the stress responses of the various Symbiodinium phylotypes different but also the responses of individual phylotypes to thermal and oxidative stress were different in terms of ROS production. Variation in the quality and quantity of ROS generation and its implications for subsequent antioxidant production suggest that different stress mechanisms are at play. While our experiments were done under laboratory conditions that did not necessarily mirror ecological ones, these results provide new insight into processes inside Symbiodinium cells during stress events and add new explanations for a phylotype's susceptibility to stress.

PPARα activation by MHY908 attenuates age-related renal inflammation through modulation of the ROS/Akt/FoxO1 pathway

2-[4-(5-Chlorobenzothiazothiazol-2-yl)phenoxy]-2-methyl-propionic acid (MHY908) has been shown to prevent insulin resistance-induced hyperinsulinemia in aged rats. However, the mechanism underlying MHY908-mediated amelioration of renal inflammation with insulin resistance during aging remains unknown. This study investigated the effects of MHY908 on age-related changes in the IRS/Akt/forkhead box (FoxO) 1 signaling pathway in the kidneys of aged rats and HEK293T cells. Experiments were performed in young, old, and MHY908-fed old rats (1mg or 3mg/kg/day MHY908 for 4 weeks). We found that MHY908-fed old rats suppressed phosphorylation of IRS/Akt and induced FoxO1 activation, leading to increased expression of MnSOD and catalase. In addition, in insulin-treated cells, MHY908 prevented the FoxO1 inactivation and increased the expression of MnSOD and catalase by inactivating IRS and Akt. In contrast, NF-κB signaling pathway decreased with MHY908 treatment in insulin-treated cells. Furthermore, MHY908 exclusively activated peroxisome proliferator-activated receptor (PPAR) α in the kidneys, leading to the inhibition of insulin-induced NADPH oxidase subunit 4 (NOX4)-derived reactive oxygen species (ROS) generation and FoxO1 inactivation. In conclusion, MHY908 improved the hyperinsulinemia-induced pro-inflammatory response through NF-κB inactivation and FoxO1 activation in aged rat kidneys. These phenomena suggest that PPARα activation by MHY908 attenuates NOX4-derived ROS generation in response to insulin.

PRDX2 protects hepatocellular carcinoma SMMC-7721 cells from oxidative stress.

Peroxiredoxin2 (PRDX2) is a member of the peroxiredoxin family of antioxidant enzymes. A number of previous studies have indicated that PRDX2 may serve a cell type-dependent role in tumorigenesis. Recently, PRDX2 has been identified to be the new target of miR-122a, which has been demonstrated to be frequently downregulated in hepatocellular carcinoma (HCC). Thus, PRDX2 may have a pro-tumorigenic role in HCC. Because the role of PRDX2 in HCC has not yet been reported, it is of interest to explore how PRDX2 may affect reactive oxygen species (ROS)-mediated cell death in HCC cells. The present study analyzed the effects of PRDX2 knockdown or overexpression on hydrogen peroxide (H2O2)-induced cell death in HCC SMMC-7721 cells. Tumor necrosis factor-α (TNF-α)-induced cell death upon PRDX2 knockdown or overexpression was also examined in SMMC-7721 cells. It was found that PRDX2 knockdown augmented H2O2-induced cell death in SMMC-7721 cells, whereas PRDX2 overexpression exhibited opposite effects. By contrast, PRDX2 knockdown enhanced TNF-α-induced apoptosis, whereas PRDX2 overexpression reduced it, even though both treatments showed little effects on TNF-α-induced necrosis in SMMC-7721 cells. Further exploration confirmed PRDX2 knockdown led to enhanced ROS generation in response to H2O2. Taken together, the present study supports that PRDX2 serves a pro-tumorigenic role in HCC through, at least partially, limiting ROS-mediated apoptosis under oxidative stress.

Phytoextract of Indian mustard seeds acts by suppressing the generation of ROS against acetaminophen-induced hepatotoxicity in HepG2 cells

Context: Indian mustard [Brassica juncea (L.) Czern. & Coss. (Brassicaceae)] is reported to possess diverse pharmacological properties. However, limited information is available concerning its hepatoprotective activity and mechanism of action.Objective: To study the protective mechanism of mustard seed extract against acetaminophen (APAP) toxicity in a hepatocellular carcinoma (HepG2) cell line.Materials and methods: Hepatotoxicity models were established using APAP (2.5–22.5 mM) based on the cytotoxicity profile. An antioxidant-rich fraction from mustard seeds was extracted and evaluated for its hepatoprotective potential. The mechanism of action was elucidated using various in vitro antioxidant assays, the detection of intracellular generation of reactive oxygen species (ROS), and cell cycle analysis. The phytoconstituents isolated via HPLC-DAD were also evaluated for hepatoprotective activity.Results: Hydromethanolic seed extract exhibited hepatoprotective activity in post- and pre-treatment models of 20 mM APAP toxicity and restored the elevated levels of liver indices to normal values (p < 0.05). Post-treatment suppressed the generation of ROS by 58.37% and pre-treatment effectively prevented the generation of ROS by 90.5%. The mechanism of ROS suppression was further supported by antioxidant activity (IC50) data from DPPH (103.37 ± 4.2 µg AAE/mg), FRAP (83.26 ± 1.1 µg AAE/mg), ORAC (1115 µM GAE/ml), ABTS (83.05 µg GAE/ml), and superoxide (345.22 ± 5.15 µg AAE/mg) scavenging assays and by the restoration of cell cycle alterations. HPLC-DAD analysis revealed the presence quercetin, vitamin E, and catechin, which exhibited hepatoprotective activity.Discussion and conclusions: A phytoextract of mustard seeds acts by suppressing the generation of ROS in response to APAP toxicity.

RhoA and Rac1 Gtpases Differentially Regulate Agonist-Receptor Mediated ROS Generation in Platelets

Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and hence the risk of thrombosis. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Previous studies have shown that small GTPases Rac1 and RhoA are involved in NOX activation. Rac GTPase activates NOX by directly binding to NOX as well as by interacting with p67phox to promote its binding to NOX (Physiol Rev 87: 245–313, 2007), whereas RhoA triggers ROS generation via the ROCK/p38MAPK cascade mediated phosphorylation of p47phox, a critical component of the NOX complex, (Exp Mol Med 37:575-87, 2005). To date, however, the roles of Rac1 and RhoA in platelet ROS production remain unclear. This study was conducted to define the contributions of Rac1- and RhoA- signaling to ROS generation and platelet function. ROS generation was quantified by flow cytometry in dcf-da (10 µM) loaded washed platelets. Thrombin has been shown to generate ROS in human platelets (Blood 106: 2757-2760, 2005). In this study we confirmed that platelets stimulated with thrombin generate ROS in a time- and a concentration- dependent manner. Addition of thrombin to human platelets pre-treated with NSC23766, a Rac-specific inhibitor, or murine platelets with Rac1 gene deletion, produced significantly less ROS than the matching control samples. Further, Phox-I, a pharmacologic inhibitor of Rac-p67phox interaction (Chem Biol 19: 228-24, 2012), potently suppressed thrombin induced ROS production, indicating that a Rac1-p67phox signaling axis is involved in thrombin mediated ROS production. Separately, treatment of washed human platelets with a RhoA specific inhibitor, Rhosin (Chem Biol 19:699-710, 2012) resulted in: (a) inhibition of the U46619, a stable analog of TXA2, induced activation of RhoA, but not that of Rac1or Cdc42; (b) U46619 induced phosphorylation p38MAPK and p47phox; and (c) U46619 or thrombin induced ROS generation. We further investigated the role of RhoA/ROCK/p38MAPK in ROS production by using platelets from RhoA-/- mice, Y27632 (a ROCK inhibitor) and SB203580 (a p38MAPK inhibitor). RhoA-/- platelets or human platelets treated with Y27632 or SB203580 exhibited significantly diminished ROS generation in response to thrombin. Next, we investigated the physiological effects of Rhosin on platelet activation. A pre-incubation of washed human platelets with Rhosin inhibited U46619 or thrombin induced platelet shape change, release of P-selectin, secretion of ATP and aggregation. The anti-platelet effects of Rhosin were reversible as washing of platelets after incubation with Rhosin abolished the inhibitory effect of Rhosin on platelet aggregation. These results suggest that (a) RhoA signaling, through ROCK/MAPK/p47phox activation, leads to ROS generation and platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC, and (b) Rac1 and RhoA differentially regulate platelet ROS generation by directly binding to NOX, promoting binding of p67phox to NOX and by phosphorylation of p47phox, respectively. DisclosuresNo relevant conflicts of interest to declare.

Redox signaling mediates symbiosis between the gut microbiota and the intestine

The microbiota that populates the intestinal tract affects many physiological processes, such as cell proliferation, epithelial barrier function, and immune responses. However, the molecular mechanisms by which the microbiota influences these events remain unknown. It was recently reported by our research group that specific taxa of intestinal bacteria induce the rapid and transient enzymatic production of reactive oxygen species (ROS) within enterocytes. Whereas NADPH oxidase 2 (Nox2) catalyzed ROS generation in response to microbial perception by bone marrow-derived phagocytes is well-studied, the function of ROS generated by Nox1 in enterocytes in response to microbial signals is not fully understood. It is established that ROS can act as signaling molecules in diverse transduction pathways by the rapid and transient oxidation of oxidant-sensitive thiol groups harbored within sensor regulatory proteins. Because commensal-bacterial-stimulated ROS generation in enterocytes has been shown to induce a wide range of physiological processes, in our recent manuscript, we proposed a paradigm wherein the influence of the microbiota on intestinal physiology is mediated in part by redox-dependant signaling.

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca2+ and Zn2+ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca2+ or Zn2+ loading. Induction of rapid cytosolic accumulation of either Ca2+ (via NMDA exposure) or Zn2+ (via Zn2+/Pyrithione exposure in 0 Ca2+) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca2+-induced ROS production with little effect on the Zn2+- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca2+ or Zn2+ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn2+-triggered ROS, while partially attenuating the Ca2+-triggered ROS. Furthermore, block of the mitochondrial Ca2+ uniporter (MCU), through which Zn2+ as well as Ca2+ can enter the mitochondrial matrix, substantially diminished Zn2+ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn2+ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine, which impairs Zn2+ binding to cytosolic metalloproteins, far lower Zn2+ exposures were able to induce mitochondrial Zn2+ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn2+ and Ca2+ each can trigger injurious ROS generation, Zn2+ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn2+ buffering is impaired due to acidosis and oxidative stress.

Vitamin D receptor activation and downregulation of renin-angiotensin system attenuate morphine-induced T cell apoptosis

Opiates have been reported to induce T cell loss. We evaluated the role of vitamin D receptor (VDR) and the activation of the renin-angiotensin system (RAS) in morphine-induced T cell loss. Morphine-treated human T cells displayed downregulation of VDR and the activation of the RAS. On the other hand, a VDR agonist (EB1089) enhanced T cell VDR expression both under basal and morphine-stimulated states. Since T cells with silenced VDR displayed the activation of the RAS, whereas activation of the VDR was associated with downregulation of the RAS, it appears that morphine-induced T cell RAS activation was dependent on the VDR status. Morphine enhanced reactive oxygen species (ROS) generation in a dose-dependent manner. Naltrexone (an opiate receptor antagonist) inhibited morphine-induced ROS generation and thus, suggested the role of opiate receptors in T cell ROS generation. The activation of VDR as well as blockade of ANG II (by losartan, an AT(1) receptor blocker) also inhibited morphine-induced T cell ROS generation. Morphine not only induced double-strand breaks (DSBs) in T cells but also attenuated DNA repair response, whereas activation of VDR not only inhibited morphine-induced DSBs but also enhanced DNA repair. Morphine promoted T cell apoptosis; however, this effect of morphine was inhibited by blockade of opiate receptors, activation of the VDR, and blockade of the RAS. These findings indicate that morphine-induced T cell apoptosis is mediated through ROS generation in response to morphine-induced downregulation of VDR and associated activation of the RAS.