Showing papers in "Redox biology in 2018"

Oxidative stress and the amyloid beta peptide in Alzheimer's disease.

Abstract: Oxidative stress is known to play an important role in the pathogenesis of a number of diseases. In particular, it is linked to the etiology of Alzheimer's disease (AD), an age-related neurodegenerative disease and the most common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Aβ) in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-β (Aβ). The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, …). This review highlights the existing link between oxidative stress and AD, and the consequences towards the Aβ peptide and surrounding molecules in terms of oxidative damage. In addition, the implication of metal ions in AD, their interaction with the Aβ peptide and redox properties leading to ROS production are discussed, along with both in vitro and in vivo oxidation of the Aβ peptide, at the molecular level.

13 reasons why the brain is susceptible to oxidative stress.

Abstract: The human brain consumes 20% of the total basal oxygen (O2) budget to support ATP intensive neuronal activity. Without sufficient O2 to support ATP demands, neuronal activity fails, such that, even transient ischemia is neurodegenerative. While the essentiality of O2 to brain function is clear, how oxidative stress causes neurodegeneration is ambiguous. Ambiguity exists because many of the reasons why the brain is susceptible to oxidative stress remain obscure. Many are erroneously understood as the deleterious result of adventitious O2 derived free radical and non-radical species generation. To understand how many reasons underpin oxidative stress, one must first re-cast free radical and non-radical species in a positive light because their deliberate generation enables the brain to achieve critical functions (e.g. synaptic plasticity) through redox signalling (i.e. positive functionality). Using free radicals and non-radical derivatives to signal sensitises the brain to oxidative stress when redox signalling goes awry (i.e. negative functionality). To advance mechanistic understanding, we rationalise 13 reasons why the brain is susceptible to oxidative stress. Key reasons include inter alia unsaturated lipid enrichment, mitochondria, calcium, glutamate, modest antioxidant defence, redox active transition metals and neurotransmitter auto-oxidation. We review RNA oxidation as an underappreciated cause of oxidative stress. The complex interplay between each reason dictates neuronal susceptibility to oxidative stress in a dynamic context and neural identity dependent manner. Our discourse sets the stage for investigators to interrogate the biochemical basis of oxidative stress in the brain in health and disease.

Empagliflozin rescues diabetic myocardial microvascular injury via AMPK-mediated inhibition of mitochondrial fission.

Abstract: Impaired cardiac microvascular function contributes to diabetic cardiovascular complications although effective therapy remains elusive. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor recently approved for treatment of type 2 diabetes, promotes glycosuria excretion and offers cardioprotective actions beyond its glucose-lowering effects. This study was designed to evaluate the effect of empagliflozin on cardiac microvascular injury in diabetes and the underlying mechanism involved with a focus on mitochondria. Our data revealed that empagliflozin improved diabetic myocardial structure and function, preserved cardiac microvascular barrier function and integrity, sustained eNOS phosphorylation and endothelium-dependent relaxation, as well as improved microvessel density and perfusion. Further study suggested that empagliflozin exerted its effects through inhibition of mitochondrial fission in an adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner. Empagliflozin restored AMP-to-ATP ratio to trigger AMPK activation, suppressed Drp1S616 phosphorylation, and increased Drp1S637 phosphorylation, ultimately leading to inhibition of mitochondrial fission. The empagliflozin-induced inhibition of mitochondrial fission preserved cardiac microvascular endothelial cell (CMEC) barrier function through suppressed mitochondrial reactive oxygen species (mtROS) production and subsequently oxidative stress to impede CMEC senescence. Empagliflozin-induced fission loss also favored angiogenesis by promoting CMEC migration through amelioration of F-actin depolymerization. Taken together, these results indicated the therapeutic promises of empagliflozin in the treatment of pathological microvascular changes in diabetes.

Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions.

Abstract: Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure in many developed countries. Mitochondrial oxidative stress is considered to be the predominant cellular event in APAP-induced liver injury. Accordingly, N-acetyl cysteine, a known scavenger of reactive oxygen species (ROS), is recommended as an effective clinical antidote against APAP-induced acute liver injury (AILI) when it is given at an early phase; however, the narrow therapeutic window limits its use. Hence, the development of novel therapeutic approaches that can offer broadly protective effects against AILI is clearly needed. To this end, it is necessary to better understand the mechanisms of APAP hepatotoxicity. Up to now, in addition to mitochondrial oxidative stress, many other cellular processes, including phase I/phase II metabolism, endoplasmic reticulum stress, autophagy, sterile inflammation, microcirculatory dysfunction, and liver regeneration, have been identified to be involved in the pathogenesis of AILI, providing new targets for developing more effective therapeutic interventions against APAP-induced liver injury. In this review, we summarize intracellular and extracellular events involved in APAP hepatotoxicity, along with emphatic discussions on the possible therapeutic approaches targeting these different cellular events.

DUSP1 alleviates cardiac ischemia/reperfusion injury by suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK pathways.

Abstract: Mitochondrial fission and selective mitochondrial autophagy (mitophagy) form an essential axis of mitochondrial quality control that plays a critical role in the development of cardiac ischemia-reperfusion (IR) injury. However, the precise upstream molecular mechanism of fission/mitophagy remains unclear. Dual-specificity protein phosphatase1 (DUSP1) regulates cardiac metabolism, but its physiological contribution in the reperfused heart, particularly its influence on mitochondrial homeostasis, is unknown. Here, we demonstrated that cardiac DUSP1 was downregulated following acute cardiac IR injury. In vivo, compared to wild-type mice, DUSP1 transgenic mice (DUSP1TG mice) demonstrated a smaller infarcted area and the improved myocardial function. In vitro, the IR-induced DUSP1 deficiency promoted the activation of JNK which upregulated the expression of the mitochondrial fission factor (Mff). A higher expression level of Mff was associated with elevated mitochondrial fission and mitochondrial apoptosis. Additionally, the loss of DUSP1 also amplified the Bnip3 phosphorylated activation via JNK, leading to the activation of mitophagy. Increased mitophagy overtly consumed mitochondrial mass resulting into the mitochondrial metabolism disorder. However, the reintroduction of DUSP1 blunted Mff/Bnip3 activation and therefore alleviated the fatal mitochondrial fission/mitophagy by inactivating the JNK pathway, providing a survival advantage to myocardial tissue following IR stress. The results of our study suggest that DUSP1 and its downstream JNK pathway are therapeutic targets for conferring protection against IR injury by repressing Mff-mediated mitochondrial fission and Bnip3-required mitophagy.

Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration.

Abstract: In this review we provide an analysis of the biochemistry of peroxynitrite and tyrosine nitration. Peroxynitrite is the product of the diffusion-controlled reaction between superoxide (O2•-) and nitric oxide (•NO). This process is in competition with the enzymatic dismutation of O2•- and the diffusion of •NO across cells and tissues and its reaction with molecular targets (e.g. guanylate cyclase). Understanding the kinetics and compartmentalization of the O2•- / •NO interplay is critical to rationalize the shift of •NO from a physiological mediator to a cytotoxic intermediate. Once formed, peroxynitrite (ONOO- and ONOOH; pKa = 6,8) behaves as a strong one and two-electron oxidant towards a series of biomolecules including transition metal centers and thiols. In addition, peroxynitrite anion can secondarily evolve to secondary radicals either via its fast reaction with CO2 or through proton-catalyzed homolysis. Thus, peroxynitrite can participate in direct (bimolecular) and indirect (through secondary radical intermediates) oxidation reactions; through these processes peroxynitrite can participate as cytotoxic effector molecule against invading pathogens and/or as an endogenous pathogenic mediator. Peroxynitrite can cause protein tyrosine nitration in vitro and in vivo. Indeed, tyrosine nitration is a hallmark of the reactions of •NO-derived oxidants in cells and tissues and serves as a biomarker of oxidative damage. Protein tyrosine nitration can mediate changes in protein structure and function that affect cell homeostasis. Tyrosine nitration in biological systems is a free radical process that can be promoted either by peroxynitrite-derived radicals or by other related •NO-dependent oxidative processes. Recently, mechanisms responsible of tyrosine nitration in hydrophobic biostructures such as membranes and lipoproteins have been assessed and involve the parallel occurrence and connection with lipid peroxidation. Experimental strategies to reveal the proximal oxidizing mechanism during tyrosine nitration in given pathophysiologically-relevant conditions include mapping and identification of the tyrosine nitration sites in specific proteins.

Antioxidant response elements: Discovery, classes, regulation and potential applications.

Abstract: Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress.

Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: A mechanism involving calcium overload/XO/ROS/mPTP pathway.

Abstract: Receptor-interacting protein 3 (Ripk3)-mediated necroptosis contributes to cardiac ischaemia-reperfusion (IR) injury through poorly defined mechanisms. Our results demonstrated that Ripk3 was strongly upregulated in murine hearts subjected to IR injury and cardiomyocytes treated with LPS and H2O2. The higher level of Ripk3 was positively correlated to the infarction area expansion, cardiac dysfunction and augmented cardiomyocytes necroptosis. Function study further illustrated that upregulated Ripk3 evoked the endoplasmic reticulum (ER) stress, which was accompanied with an increase in intracellular Ca2+ level ([Ca2+]c) and xanthine oxidase (XO) expression. Activated XO raised cellular reactive oxygen species (ROS) that mediated the mitochondrial permeability transition pore (mPTP) opening and cardiomyocytes necroptosis. By comparison, genetic ablation of Ripk3 abrogated the ER stress and thus blocked the [Ca2+]c overload-XO-ROS-mPTP pathways, favouring a pro-survival state that ultimately resulted in the inhibition of cardiomyocytes necroptosis in the setting of cardiac IR injury. In summary, the present study helps to elucidate how necroptosis is mediated by ER stress, via the calcium overload /XO/ROS/mPTP opening axis.

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1.

Abstract: Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

Diverse roles of mitochondria in ischemic stroke.

Abstract: Stroke is the leading cause of adult disability and mortality in most developing and developed countries. The current best practices for patients with acute ischemic stroke include intravenous tissue plasminogen activator and endovascular thrombectomy for large-vessel occlusion to improve clinical outcomes. However, only a limited portion of patients receive thrombolytic therapy or endovascular treatment because the therapeutic time window after ischemic stroke is narrow. To address the current shortage of stroke management approaches, it is critical to identify new potential therapeutic targets. The mitochondrion is an often overlooked target for the clinical treatment of stroke. Early studies of mitochondria focused on their bioenergetic role; however, these organelles are now known to be important in a wide range of cellular functions and signaling events. This review aims to summarize the current knowledge on the mitochondrial molecular mechanisms underlying cerebral ischemia and involved in reactive oxygen species generation and scavenging, electron transport chain dysfunction, apoptosis, mitochondrial dynamics and biogenesis, and inflammation. A better understanding of the roles of mitochondria in ischemia-related neuronal death and protection may provide a rationale for the development of innovative therapeutic regimens for ischemic stroke and other stroke syndromes.

Reactive oxygen species promote tubular injury in diabetic nephropathy: The role of the mitochondrial ros-txnip-nlrp3 biological axis

Abstract: NLRP3/IL-1β activation via thioredoxin (TRX)/thioredoxin-interacting protein (TXNIP) following mitochondria ROS (mtROS) overproduction plays a key role in inflammation. However, the involvement of this process in tubular damage in the kidneys of patients with diabetic nephropathy (DN) is unclear. Here, we demonstrated that mtROS overproduction is accompanied by decreases in TRX expression and TXNIP up-regulation. In addition, we discovered that mtROS overproduction is also associated with increases in NLRP3/IL-1β and TGF-β expression in the kidneys of patients with DN and db/db mice. We reversed these changes in db/db mice by administering a peritoneal injection of MitoQ, an antioxidant targeting mtROS. Similar results were observed in human tubular HK-2 cells subjected to high-glucose (HG) conditions and treated with MitoQ. Treating HK-2 cells with MitoQ suppressed the dissociation of TRX from TXNIP and subsequently blocked the interaction between TXNIP and NLRP3, leading to the inhibition of NLRP3 inflammasome activation and IL-1β maturation. The effects of MitoQ were enhanced by pretreatment with TXNIP siRNA and abolished by pretreatment with monosodium urate (MSU) and TRX siRNA in vitro. These results suggest that mitochondrial ROS-TXNIP/NLRP3/IL-1β axis activation is responsible for tubular oxidative injury, which can be ameliorated by MitoQ via the inhibition of mtROS overproduction.

Iron accumulation in senescent cells is coupled with impaired ferritinophagy and inhibition of ferroptosis.

Abstract: Cellular senescence is characterised by the irreversible arrest of proliferation, a pro-inflammatory secretory phenotype and evasion of programmed cell death mechanisms. We report that senescence alters cellular iron acquisition and storage and also impedes iron-mediated cell death pathways. Senescent cells, regardless of stimuli (irradiation, replicative or oncogenic), accumulate vast amounts of intracellular iron (up to 30-fold) with concomitant changes in the levels of iron homeostasis proteins. For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity. Cellular senescence preceded iron accumulation and was not perturbed by sustained iron chelation (deferiprone). Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis. Lysosomal dysfunction in senescent cells was confirmed through several markers, including the build-up of microtubule-associated protein light chain 3 (LC3-II) in autophagosomes. Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency. Accordingly, senescent cells were highly resistant to ferroptosis. Promoting ferritin degradation by using the autophagy activator rapamycin averted the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron, but failed to re-sensitize these cells to ferroptosis. Finally, the enrichment of senescent cells in mouse ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin and mirrored our observations using cultured senescent cells.

Therapeutic effect of Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: The role of the ERK-CREB pathway and Bnip3-mediated mitophagy.

Abstract: Increased mitochondrial damage is related to the progression of a diet-induced nonalcoholic fatty liver disease. The aim of our study is to investigate the role of Sirtuin 3 (Sirt3) in treating nonalcoholic fatty liver disease with a focus on mitophagy and the ERK-CREB pathway. Our data indicated that Sirt3 was downregulated in liver tissue in response to chronic HFD treatment. Interestingly, re-introduction of Sirt3 protected hepatic function, attenuated liver fibrosis, alleviated the inflammatory response, and prevented hepatocyte apoptosis. Molecular investigations demonstrated that lipotoxicity was associated with an increase in mitochondrial apoptosis as evidenced by reduced mitochondrial potential, augmented ROS production, increased cyt-c leakage into the nucleus, and activated caspase-9 apoptotic signalling. Additionally, Sirt3 overexpression protected hepatocytes against mitochondrial apoptosis via promoting Bnip3-required mitophagy. Functional studies showed that Sirt3 reversed Bnip3 expression and mitophagy activity via the ERK-CREB signalling pathway. Blockade of the ERK-CREB axis repressed the promotive effects of Sirt3 on Bnip3 activation and mitophagy augmentation, finally negating the anti-apoptotic influences of Sirt3 on hepatocytes in the setting of high-fat-stress. Collectively, our data show that high-fat-mediated liver damage is associated with Sirt3 downregulation, which is followed by ERK-CREB pathway inactivation and Bnip3-mediated inhibition of mitophagy, causing hepatocytes to undergo mitochondria-dependent cell death. Based on this, strategies for enhancing Sirt3 activity and activating the ERK-CREB-Bnip3-mitophagy pathways could be used to treat nonalcoholic fatty liver disease.

α-Lipoic acid improves abnormal behavior by mitigation of oxidative stress, inflammation, ferroptosis, and tauopathy in P301S Tau transgenic mice.

Abstract: Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by neurofibrillary tangles (NFTs) composed of Tau protein. α-Lipoic acid (LA) has been found to stabilize the cognitive function of AD patients, and animal study findings have confirmed its anti-amyloidogenic properties. However, the underlying mechanisms remain unclear, especially with respect to the ability of LA to control Tau pathology and neuronal damage. Here, we found that LA supplementation effectively inhibited the hyperphosphorylation of Tau at several AD-related sites, accompanied by reduced cognitive decline in P301S Tau transgenic mice. Furthermore, we found that LA not only inhibited the activity of calpain1, which has been associated with tauopathy development and neurodegeneration via modulating the activity of several kinases, but also significantly decreased the calcium content of brain tissue in LA-treated mice. Next, we screened for various modes of neural cell death in the brain tissue of LA-treated mice. We found that caspase-dependent apoptosis was potently inhibited, whereas autophagy did not show significant changes after LA supplementation. Interestingly, Tau-induced iron overload, lipid peroxidation, and inflammation, which are involved in ferroptosis, were significantly blocked by LA administration. These results provide compelling evidence that LA plays a role in inhibiting Tau hyperphosphorylation and neuronal loss, including ferroptosis, through several pathways, suggesting that LA may be a potential therapy for tauopathies.

Yap promotes hepatocellular carcinoma metastasis and mobilization via governing cofilin/F-actin/lamellipodium axis by regulation of JNK/Bnip3/SERCA/CaMKII pathways.

Abstract: Despite the increasingly important role of Hippo-Yap in hepatocellular carcinoma (HCC) development and progression, little insight is available at the time regarding the specifics interaction of Yap and cancer cells migration. Here, we identified the mechanism by which tumor-intrinsic Yap deletion resulted in HCC migratory inhibition. Yap was greatly upregulated in HCC and its expression promoted the cells migration. Functional studies found that knockdown of Yap induced JNK phosphorylation which closely bound to the Bnip3 promoter and contributed to Bnip3 expression. Higher Bnip3 employed excessive mitophagy leading to mitochondrial dysfunction and ATP shortage. The insufficient ATP inactivated SERCA and consequently triggered intracellular calcium overload. As the consequence of calcium oscillation, Ca/calmodulin-dependent protein kinases II (CaMKII) was signaled and subsequently inhibited cofilin activity via phosphorylated modification. The phosphorylated cofilin failed to manipulate F-actin polymerization and lamellipodium formation, resulting into the impairment of lamellipodium-based migration. Collectively, our results identified Hippo-Yap as the tumor promoter in hepatocellular carcinoma that mediated via activation of cofilin/F-actin/lamellipodium axis by limiting JNK-Bnip3-SERCA-CaMKII pathways, with potential application to HCC therapy involving cancer metastasis.

MicroRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2.

Abstract: Clinical application of doxorubicin (DOX), an anthracycline antibiotic with potent anti- tumor effects, is limited because of its cardiotoxicity. However, its pathogenesis is still not entirely understood. The aim of this paper was to explore the mechanisms and new drug targets to treat DOX-induced cardiotoxicity. The in vitro model on H9C2 cells and the in vivo models on rats and mice were developed. The results showed that DOX markedly decreased H9C2 cell viability, increased the levels of CK, LDH, caused histopathological and ECG changes in rats and mice, and triggered myocardial oxidative damage via adjusting the levels of intracellular ROS, MDA, SOD, GSH and GSH-Px. Total of 18 differentially expressed microRNAs in rat heart tissue caused by DOX were screened out using microRNA microarray assay, especially showing that miR-140-5p was significantly increased by DOX which was selected as the target miRNA. Double-luciferase reporter assay showed that miR-140-5p directly targeted Nrf2 and Sirt2, as a result of affecting the expression levels of HO-1, NQO1, Gst, GCLM, Keap1 and FOXO3a, and thereby increasing DOX-caused myocardial oxidative damage. In addition, the levels of intracellular ROS were significantly increased or decreased in H9C2 cells treated with DOX after miR-140-5p mimic or miR-140-5p inhibitor transfection, respectively, as well as the changed expression levels of Nrf2 and Sirt2. Furthermore, DOX- induced myocardial oxidative damage was worsened in mice treated with miR-140-5p agomir, and however the injury was alleviated in the mice administrated with miR-140-5p antagomir. Therefore, miR-140-5p plays an important role in DOX-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2. Our data provide novel insights for investigating DOX-induced heart injury. In addition, miR-140-5p/ Nrf2 and miR-140-5p/Sirt2 may be the new targets to treat DOX-induced cardiotoxicity.

Role of gut microbiota and oxidative stress in the progression of non-alcoholic fatty liver disease to hepatocarcinoma: Current and innovative therapeutic approaches.

Abstract: Non-alcoholic fatty liver disease (NAFLD) represents the most common chronic liver disease in industrialized countries. NAFLD progresses through the inflammatory phase of non-alcoholic steatohepatitis (NASH) to fibrosis and cirrhosis, with some cases developing liver failure or hepatocellular carcinoma (HCC). Liver biopsy remains the gold standard approach to a definitive diagnosis of NAFLD and the distinction between simple steatosis and NASH. The pathogenesis of NASH is still not clear. Several theories have been proposed ranging from the "Two Hit Theory" to the "Multiple Hit Theory". However, the general consensus is that the gut microbiota, oxidative stress, and mitochondrial damage play key roles in the pathogenesis of NASH. The interaction between the gut epithelia and some commensal bacteria induces the rapid generation of reactive oxygen species (ROS). The main goal of any therapy addressing NASH is to reverse or prevent progression to liver fibrosis/cirrhosis. This problem represents the first "Achilles' heel" of the new molecules being evaluated in most ongoing clinical trials. The second is the inability of these molecules to reach the mitochondria, the primary sites of energy production and ROS generation. Recently, a variety of non-pharmacological and pharmacological treatment approaches for NASH have been evaluated including vitamin E, the thiazolidinediones, and novel molecules related to NASH pathogenesis (including obeticholic acid and elafibranor). Recently, a new isoform of human manganese superoxide dismutase (MnSOD) was isolated and obtained in a synthetic recombinant form designated rMnSOD. This protein has been shown to be a powerful antioxidant capable of mediating ROS dismutation, penetrating biological barriers via its uncleaved leader peptide, and reducing portal hypertension and fibrosis in rats affected by liver cirrhosis. Based on these distinctive characteristics, it can be hypothesized that this novel recombinant protein (rMnSOD) potentially represents a new and highly efficient adjuvant therapy to counteract the progression from NASH to HCC.

8-Oxoguanine DNA glycosylase 1: Beyond repair of the oxidatively modified base lesions

Abstract: Oxidative stress and the resulting damage to genomic DNA are inevitable consequences of endogenous physiological processes, and they are amplified by cellular responses to environmental exposures. One of the most frequent reactions of reactive oxygen species with DNA is the oxidation of guanine to pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG). Despite the vulnerability of guanine to oxidation, vertebrate genes are primarily embedded in GC-rich genomic regions, and over 72% of the promoters of human genes belong to a class with a high GC content. In the promoter, 8-oxoG may serve as an epigenetic mark, and when complexed with the oxidatively inactivated repair enzyme 8-oxoguanine DNA glycosylase 1, provide a platform for the coordination of the initial steps of DNA repair and the assembly of the transcriptional machinery to launch the prompt and preferential expression of redox-regulated genes. Deviations/variations from this artful coordination may be the etiological links between guanine oxidation and various cellular pathologies and diseases during ageing processes.

A review of the basics of mitochondrial bioenergetics, metabolism, and related signaling pathways in cancer cells: Therapeutic targeting of tumor mitochondria with lipophilic cationic compounds.

Abstract: The present review is a sequel to the previous review on cancer metabolism published in this journal This review focuses on the selective antiproliferative and cytotoxic effects of mitochondria-targeted therapeutics (MTTs) in cancer cells Emerging research reveals a key role of mitochondrial respiration on tumor proliferation Previously, a mitochondria-targeted nitroxide was shown to selectively inhibit colon cancer cell proliferation at submicromolar levels This review is centered on the therapeutic use of MTTs and their bioenergetic profiling in cancer cells Triphenylphosphonium cation conjugated to a parent molecule (eg, vitamin-E or chromanol, ubiquinone, and metformin) via a linker alkyl chain is considered an MTT MTTs selectively and potently inhibit proliferation of cancer cells and, in some cases, induce cytotoxicity MTTs inhibit mitochondrial complex I activity and induce mitochondrial stress in cancer cells through generation of reactive oxygen species MTTs in combination with glycolytic inhibitors synergistically inhibit tumor cell proliferation This review discusses how signaling molecules traditionally linked to tumor cell proliferation affect tumor metabolism and bioenergetics (glycolysis, TCA cycle, and glutaminolysis)

Teaching the basics of reactive oxygen species and their relevance to cancer biology: Mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies.

Abstract: Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiol peroxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed.

Regulation of platelet activation and thrombus formation by reactive oxygen species.

Abstract: Reactive oxygen species (ROS) are generated within activated platelets and play an important role in regulating platelet responses to collagen and collagen-mediated thrombus formation. As a major collagen receptor, platelet-specific glycoprotein (GP)VI is a member of the immunoglobulin (Ig) superfamily, with two extracellular Ig domains, a mucin domain, a transmembrane domain and a cytoplasmic tail. GPVI forms a functional complex with the Fc receptor γ-chain (FcRγ) that, following receptor dimerization, signals via an intracellular immunoreceptor tyrosine-based activation motif (ITAM), leading to rapid activation of Src family kinase signaling pathways. Our previous studies demonstrated that an unpaired thiol in the cytoplasmic tail of GPVI undergoes rapid oxidation to form GPVI homodimers in response to ligand binding, indicating an oxidative submembranous environment in platelets after GPVI stimulation. Using a redox-sensitive fluorescent dye (H2DCF-DA) in a flow cytometric assay to measure changes in intracellular ROS, we showed generation of ROS downstream of GPVI consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation. In this review, we will discuss recent findings on the regulation of platelet function by ROS, focusing on GPVI-dependent platelet activation and thrombus formation.

Polydatin prevents fructose-induced liver inflammation and lipid deposition through increasing miR-200a to regulate Keap1/Nrf2 pathway.

Abstract: Oxidative stress is a critical factor in nonalcoholic fatty liver disease pathogenesis. MicroRNA-200a (miR-200a) is reported to target Kelch-like ECH-associated protein 1 (Keap1), which regulates nuclear factor erythroid 2-related factor 2 (Nrf2) anti-oxidant pathway. Polydatin (3,4',5-trihydroxy-stilbene-3-β-D-glucoside), a polyphenol found in the rhizome of Polygonum cuspidatum, have anti-oxidative, anti-inflammatory and anti-hyperlipidemic effects. However, whether miR-200a controls Keap1/Nrf2 pathway in fructose-induced liver inflammation and lipid deposition and the blockade of polydatin are still not clear. Here, we detected miR-200a down-regulation, Keap1 up-regulation, Nrf2 antioxidant pathway inactivation, ROS-driven thioredoxin-interacting protein (TXNIP) over-expression, NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome activation and dysregulation of peroxisome proliferator activated receptor-α (PPAR-α), carnitine palmitoyl transferase-1 (CPT-1), sterol regulatory element binging protein 1 (SREBP-1) and stearoyl-CoA desaturase-1 (SCD-1) in rat livers, BRL-3A and HepG2 cells under high fructose induction. Furthermore, the data from the treatment or transfection of miR-200a minic, Keap1 and TXNIP siRNA, Nrf2 activator and ROS inhibitor demonstrated that fructose-induced miR-200a low-expression increased Keap1 to block Nrf2 antioxidant pathway, and then enhanced ROS-driven TXNIP to activate NLRP3 inflammasome and disturb lipid metabolism-related proteins, causing inflammation and lipid deposition in BRL-3A cells. We also found that polydatin up-regulated miR-200a to inhibit Keap1 and activate Nrf2 antioxidant pathway, resulting in attenuation of these disturbances in these animal and cell models. These findings provide a novel pathological mechanism of fructose-induced redox status imbalance and suggest that the enhancement of miR-200a to control Keap1/Nrf2 pathway by polydatin is a therapeutic strategy for fructose-associated liver inflammation and lipid deposition.

Trehalose protects against oxidative stress by regulating the Keap1-Nrf2 and autophagy pathways.

Abstract: Dysfunction of autophagy, which regulates cellular homeostasis by degrading organelles and proteins, is associated with pathogenesis of various diseases such as cancer, neurodegeneration and metabolic disease. Trehalose, a naturally occurring nontoxic disaccharide found in plants, insects, microorganisms and invertebrates, but not in mammals, was reported to function as a mechanistic target of the rapamycin (mTOR)-independent inducer of autophagy. In addition, trehalose functions as an antioxidant though its underlying molecular mechanisms remain unclear. In this study, we showed that trehalose not only promoted autophagy, but also increased p62 protein expression, in an autophagy-independent manner. In addition, trehalose increased nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in a p62-dependent manner and enhance expression of its downstream antioxidant factors, heme oxygenase-1 (Ho-1) and nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (Nqo1). Moreover, treatment with trehalose significantly reduced amount of reactive oxygen species. Collectively, these results suggested that trehalose can function as a novel activator of the p62-Keap1/Nrf2 pathway, in addition to inducing autophagy. Therefore, trehalose may be useful to treat many chronic diseases involving oxidative stress and dysfunction of autophagy.

Protective effect of dioscin against doxorubicin-induced cardiotoxicity via adjusting microRNA-140-5p-mediated myocardial oxidative stress.

Abstract: Clinical application of doxorubicin (DOX) is limited because of its cardiotoxicity. Thus, exploration of effective lead compounds against DOX-induced cardiotoxicity is necessary. The aim of the present study was to investigate the effects and possible mechanisms of dioscin against DOX-induced cardiotoxicity. The in vitro model of DOX- treated H9C2 cells and the in vivo models of DOX-treated rats and mice were used in this study. The results showed that discoin markedly increased H9C2 cell viability, decreased the levels of CK, LDH, and improved histopathological and electrocardio- gram changes in rats and mice to protect DOX-induced cardiotoxicity. Furthermore, dioscin significantly inhibited myocardial oxidative insult through adjusting the levels of intracellular ROS, MDA, SOD, GSH and GSH-Px in vitro and in vivo. Our data also indicated that dioscin activated Nrf2 and Sirt2 signaling pathways, and thereby affected the expression levels of HO-1, NQO1, Gst, GCLM, Keap1 and FOXO3a through decreasing miR-140-5p expression level. In addition, the level of intracellular ROS was significantly increased in H9C2 cells treated by DOX after miR-140-5p mimic transfection, as well as the down-regulated expression levels of Nrf2 and Sirt2, which were markedly reversed by dioscin. In conclusion, our data suggested that dioscin alleviated DOX-induced cardiotoxicity through modulating miR-140-5p-mediated myocardial oxidative stress. This natural product should be developed as a new candidate to alleviate cardiotoxicity caused by DOX in the future.

Chicoric acid prevents PDGF-BB-induced VSMC dedifferentiation, proliferation and migration by suppressing ROS/NFκB/mTOR/P70S6K signaling cascade.

Abstract: Phenotypic switch of vascular smooth muscle cells (VSMCs) is characterized by increased expressions of VSMC synthetic markers and decreased levels of VSMC contractile markers, which is an important step for VSMC proliferation and migration during the development and progression of cardiovascular diseases including atherosclerosis. Chicoric acid (CA) is identified to exert powerful cardiovascular protective effects. However, little is known about the effects of CA on VSMC biology. Herein, in cultured VSMCs, we showed that pretreatment with CA dose-dependently suppressed platelet-derived growth factor type BB (PDGF-BB)-induced VSMC phenotypic alteration, proliferation and migration. Mechanistically, PDGF-BB-treated VSMCs exhibited higher mammalian target of rapamycin (mTOR) and P70S6K phosphorylation, which was attenuated by CA pretreatment, diphenyleneiodonium chloride (DPI), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) and nuclear factor-κB (NFκB) inhibitor Bay117082. PDGF-BB-triggered ROS production and p65-NFκB activation were inhibited by CA. In addition, both NAC and DPI abolished PDGF-BB-evoked p65-NFκB nuclear translocation, phosphorylation and degradation of Inhibitor κBα (IκBα). Of note, blockade of ROS/NFκB/mTOR/P70S6K signaling cascade prevented PDGF-BB-evoked VSMC phenotypic transformation, proliferation and migration. CA treatment prevented intimal hyperplasia and vascular remodeling in rat models of carotid artery ligation in vivo. These results suggest that CA impedes PDGF-BB-induced VSMC phenotypic switching, proliferation, migration and neointima formation via inhibition of ROS/NFκB/mTOR/P70S6K signaling cascade.

Developing the next generation of graphene-based platforms for cancer therapeutics: The potential role of reactive oxygen species.

Abstract: Graphene has a promising future in applications such as disease diagnosis, cancer therapy, drug/gene delivery, bio-imaging and antibacterial approaches owing to graphene's unique physical, chemical and mechanical properties alongside minimal toxicity to normal cells, and photo-stability. However, these unique features and bioavailability of graphene are fraught with uncertainties and concerns for environmental and occupational exposure. Changes in the physicochemical properties of graphene affect biological responses including reactive oxygen species (ROS) production. Lower production of ROS by currently available theranostic agents, e.g. magnetic nanoparticles, carbon nanotubes, gold nanostructures or polymeric nanoparticles, restricts their clinical application in cancer therapy. Oxidative stress induced by graphene accumulated in living organs is due to acellular factors which may affect physiological interactions between graphene and target tissues and cells. Acellular factors include particle size, shape, surface charge, surface containing functional groups, and light activation. Cellular responses such as mitochondrial respiration, graphene-cell interactions and pH of the medium are also determinants of ROS production. The mechanisms of ROS production by graphene and the role of ROS for cancer treatment, are poorly understood. The aim of this review is to set the theoretical basis for further research in developing graphene-based theranostic platforms.

miR-200a-5p regulates myocardial necroptosis induced by Se deficiency via targeting RNF11

Abstract: Necroptosis has been discovered as a new paradigm of cell death and may play a key role in heart disease and selenium (Se) deficiency. Hence, we detected the specific microRNA (miRNA) in response to Se-deficient heart using microRNAome analysis. For high-throughput sequencing using Se-deficient chicken cardiac tissue, we selected miR-200a-5p and its target gene ring finger protein 11 (RNF11) based on differential expression in cardiac tissue and confirmed the relationship between miR-200a-5p and RNF11 by dual luciferase reporter assay and real-time quantitative PCR (qRT-PCR) in cardiomyocytes. We further explored the function of miR-200a-5p and observed that overexpression of miR-200a-5p spark the receptor interacting serine/threonine kinase 3 (RIP3)-dependent necroptosis in vivo and in vitro. To understand whether miR-200a-5p and RNF11 are involved in the RIP3-dependent necroptosis pathway, we presumed that oxidative stress, inflammation response and the mitogen-activated protein kinase (MAPK) pathway might trigger necroptosis. Interestingly, necroptosis trigger, z-VAD-fmk, failed to induce necroptosis but enhanced cell survival against necrosis in cardiomyocytes with knockdown of miR-200a-5p. Our present study provides a new insight that the modulation of miR-200a-5p and its target gene might block necroptosis in the heart, revealing a novel myocardial necrosis regulation model in heart disease.

Sorting cells alters their redox state and cellular metabolome.

Abstract: A growing appreciation of the metabolic artifacts of cell culture has generated heightened enthusiasm for performing metabolomics on populations of cells purified from tissues and biofluids. Fluorescence activated cell sorting, or FACS, is a widely used experimental approach to purify specific cell types from complex heterogeneous samples. Here we show that FACS introduces oxidative stress and alters the metabolic state of cells. Compared to unsorted controls, astrocytes subjected to FACS prior to metabolomic analysis showed altered ratios of GSSG to GSH, NADPH to NADP+, and NAD+ to NADH. Additionally, a 50% increase in reactive oxygen species was observed in astrocytes subjected to FACS relative to unsorted controls. At a more comprehensive scale, nearly half of the metabolomic features that we profiled by liquid chromatography/mass spectrometry were changed by at least 1.5-fold in intensity due to cell sorting. Some specific metabolites identified to have significantly altered levels as a result of cell sorting included glycogen, nucleosides, amino acids, central carbon metabolites, and acylcarnitines. Although the addition of fetal bovine serum to the cell-sorting buffer decreased oxidative stress and attenuated changes in metabolite concentrations, fetal bovine serum did not preserve the metabolic state of the cells during FACS. We conclude that, irrespective of buffer components and data-normalization strategies we examined, metabolomic results from sorted cells do not accurately reflect physiological conditions prior to sorting.

Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 via stimulating the Akt/GSK-3ß/Fyn pathway.

Abstract: Aims Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by sulforaphane (SFN) protects from, and deletion of the Nrf2 gene exaggerates, diabetic cardiomyopathy. Angiotensin II (Ang II) plays a critical role in the development of diabetic cardiomyopathy. Therefore, whether SFN prevents Ang II-induced cardiomyopathy through activation of Nrf2 was examined using wild-type, global deletion of Nrf2 gene (Nrf2-KO) and cardiomyocyte-specific overexpression of Nrf2 gene (Nrf2-TG) mice. Methods and results Administration of a subpressor dose of Ang II to wild-type mice induced cardiac oxidative stress, inflammation, remodeling and dysfunction, all of which could be prevented by SFN treatment with Nrf2 up-regulation and activation. Nrf2-KO mice are susceptible, and Nrf2-TG mice are resistant, respectively, to Ang II-induced cardiomyopathy. Meanwhile, the ability of SFN to protect against Ang II-induced cardiac damage was lost in Nrf2-KO mice. Up-regulation and activation of Nrf2 by SFN is accompanied by activation of Akt, inhibition of glycogen synthase kinase (GSK)-3β, and accumulation of Fyn in nuclei. In vitro up-regulation of Nrf2 by SFN was abolished and nuclear Fyn accumulation was increased when cardiac cells were exposed to a PI3K inhibitor or GSK-3β-specific activator. Conclusion These results suggest that Nrf2 plays a central role in the prevention of Ang II-induced cardiomyopathy, and SFN prevents Ang II-induced cardiomyopathy partially via the Akt/GSK-3β/Fyn-mediated Nrf2 activation.

Exosomal transfer of mitochondria from airway myeloid-derived regulatory cells to T cells

Abstract: Chronic inflammation involving both innate and adaptive immune cells is implicated in the pathogenesis of asthma. Intercellular communication is essential for driving and resolving inflammatory responses in asthma. Emerging studies suggest that extracellular vesicles (EVs) including exosomes facilitate this process. In this report, we have used a range of approaches to show that EVs contain markers of mitochondria derived from donor cells which are capable of sustaining a membrane potential. Further, we propose that these participate in intercellular communication within the airways of human subjects with asthma. Bronchoalveolar lavage fluid of both healthy volunteers and asthmatics contain EVs with encapsulated mitochondria; however, the % HLA-DR+ EVs containing mitochondria and the levels of mitochondrial DNA within EVs were significantly higher in asthmatics. Furthermore, mitochondria are present in exosomes derived from the pro-inflammatory HLA-DR+ subsets of airway myeloid-derived regulatory cells (MDRCs), which are known regulators of T cell responses in asthma. Exosomes tagged with MitoTracker Green, or derived from MDRCs transduced with CellLight Mitochondrial GFP were found in recipient peripheral T cells using a co-culture system, supporting direct exosome-mediated cell-cell transfer. Importantly, exosomally transferred mitochondria co-localize with the mitochondrial network and generate reactive oxygen species within recipient T cells. These findings support a potential novel mechanism of cell-cell communication involving exosomal transfer of mitochondria and the bioenergetic and/or redox regulation of target cells.