New pectic polysaccharides from Codonopsis pilosula and Codonopsis tangshen: structural characterization and cellular antioxidant activities.

BACKGROUND: Codonopsis pilosula and Codonopsis tangshen are plants widely used in traditional Chinese medicine. Two pectic polysaccharides from the roots of C. pilosula and C. tangshen named as CPP-1 and CTP-1 were obtained by boiling water extraction and column chromatography. RESULTS: The core structures of both CPP-1 and CTP-1 comprise the long homogalacturonan region (HG) as the backbone and the rhamnogalacturonan I (RG-I) region as the side chains. CPP-1 has methyl esterified galacturonic acid units and a slightly lower molecular weight than CTP-1. Biological testing suggested that CPP-1 and CTP-1 can protect IPEC-J2 cells against the H2 O2 -induced oxidative stress by up-regulating nuclear factor-erythroid 2-related factor 2 and related genes in IPEC-J2 cells. The different antioxidative activities of polysaccharides from different source of C. pilosula may be result of differences in their structures. CONCLUSION: All of the results indicated that pectic polysaccharides CPP-1 and CTP-1 from different species of C. pilosula roots could be used as a potential natural antioxidant source. These findings will be valuable for further studies and new applications of pectin-containing health products. © 2021 Society of Chemical Industry.

Citrus reticulata Blanco peel extract ameliorates hepatic steatosis, oxidative stress and inflammation in HF and MCD diet-induced NASH C57BL/6 J mice.

Excessive lipid deposition, oxidative stress and inflammation in liver tissues are regarded as crucial inducers of nonalcoholic steatohepatitis (NASH), which is the most frequent chronic liver disease and closely related to obesity and insulin resistance. In this work, the preventive and therapeutic effects of Citrus reticulata Blanco (Jizigan) peel extract (JZE) on NASH induced by high fat (HF) diet and methionine choline-deficient (MCD) diet in C57BL/6 mice were investigated. We found that daily supplementation of JZE with an HF diet effectively ameliorated glucose tolerance and insulin resistance. In addition, the key indexes of lipid profiles, oxidative stress, hepatic steatosis and inflammatory factors were also ameliorated in both NASH mouse models. Furthermore, JZE treatment activated nuclear factor erythroid-2-related factor 2 (Nrf2) in the livers of diet- induced NASH mice. Our study suggests that JZE might alleviate NASH via the activation of Nrf2 signaling and that citrus Jizigan could be used as a dietary therapy for NASH and related metabolic syndrome.

The treatment of Goji berry (Lycium barbarum) improves the neuroplasticity of the prefrontal cortex and hippocampus in aged rats.

The main characteristic of brain aging is an exacerbated inflammatory and oxidative response that affects dendritic morphology and the function of the neurons of the prefrontal cortex (PFC) and the hippocampus. This consequently causes memory loss. Recently, the use of the Goji berry (Lycium barbarum) as an antioxidant extract has provided neuroprotection and neuroplasticity, however, its therapeutic potential has not been demonstrated in aging conditions. The objective of this study was to evaluate the effect of Goji administration on memory recognition, as well as the changes in the dendritic morphology of the PFC and Hippocampus pyramidal neurons in old rats. Goji (3 g/kg) was administrated for 60 days in 18-month-old rats. After the treatment, recognition memory was evaluated using the new object recognition task (NORt). The changes in the neuron morphology of the PFC and hippocampus pyramidal neurons in old rats were evaluated by Golgi-cox stain and immunoreactivity for synaptophysin, glial fibrillary acidic protein (GFAP), caspase-3, 3-nitrotyrosine (3-NT) and nuclear factor erythroid 2-related factor 2 (Nrf2). The rats treated with Goji showed a significant increase in dendritic morphology in the PFC and hippocampus neurons, a greater immunoreactivity to synaptophysin and a decrease in reactive astrogliosis and also in caspase-3, in 3-NT and in Nrf2 in these brain regions was also observed. Goji administration promotes the plasticity processes in the PFC and in the hippocampus of old rats, critical structures in the brain aging process.

Farrerol Directly Targets GSK-3ß to Activate Nrf2-ARE Pathway and Protect EA.hy926 Cells against Oxidative Stress-Induced Injuries.

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3ß- (GSK-3ß-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3ß and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3ß at Ser9 without affecting the expression level of total GSK-3ß protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3ß siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3ß inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3ß, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3ß and further activating the Nrf2-ARE signaling pathway.

Pretreatment with dexmedetomidine alleviates lung injury in a rat model of intestinal ischemia reperfusion.

The aim of the present study was to investigate the antioxidant mechanisms of dexmedetomidine against lung injury during intestinal ischemia reperfusion (IIR) in rats. The model of IIR­induced acute lung injury was established by occluding the superior mesenteric artery (SMA) for 1 h and reperfusing for 2 h using Sprague­Dawley rats. Pathological examination was used to assess the extent of the lung injury. Oxidative stress was evaluated by measuring malondialdehyde, myeloperoxidase and superoxide dismutase in the lung and plasma. The proinflammatory cytokines tumor necrosis factor­α and interleukin­6 were determined via an enzyme­linked immunosorbent assay. The mRNA and protein expression of nuclear factor­erythroid 2 related factor 2 (Nrf2) and heme oxygenase 1 (HO­1) were determined using a reverse transcription­quantitative polymerase chain reaction and western blotting. Pretreatment with dexmedetomidine significantly inhibited the oxidative stress response and proinflammatory factor release caused by IIR compared with the normal saline group (MDA and SOD in lung and plasma, P<0.05; MPO, IL­1ß and TNF­α in lung and plasma, P<0.05). Dexmedetomidine improved pulmonary pathological changes in IIR rats compared with the normal saline group. Investigations into the molecular mechanism revealed that dexmedetomidine increased the expression levels of Nrf2 and HO­1 via activating α2 adrenergic receptors compared with the normal saline group. The antagonism of α2 adrenergic receptors may reverse the protective effect of dexmedetomidine on lung injury during IIR, including decreasing the expression levels of Nrf2 and HO­1, elevating the oxidative stress response and increasing the proinflammatory factor release. In conclusion, pretreatment with dexmedetomidine demonstrated protective effects against lung injury during IIR via α2 adrenergic receptors. The Nrf2/HO­1 signaling pathway may serve a function in the protective effect of dexmedetomidine.

N-carbamylglutamate and l-arginine promote intestinal function in suckling lambs with intrauterine growth restriction by regulating antioxidant capacity via a nitric oxide-dependent pathway.

Data indicate that intrauterine growth restriction (IUGR) in newborns can be partly alleviated through the supply of l-arginine (Arg) and N-carbamylglutamate (NCG). The current work aimed to explore whether Arg and NCG promote intestinal function by regulating antioxidant capacity in suckling lambs with IUGR via a nitric oxide (NO)-dependent pathway. Forty eight newly born Hu lambs with normal weights at birth (CON) or suffering from IUGR were randomly divided into 4 groups (n = 12 per group), namely, the CON, IUGR, IUGR + 1% Arg, and IUGR + 0.1% NCG groups. The animals were used for experiments from the age of day 7 to 28. Compared with the lambs in the IUGR group, the lambs in the Arg or NCG group had higher (P < 0.05) final body weights. The plasma insulin, NO, and NO synthase (NOS) concentrations in the IUGR group were higher (P < 0.05) compared with those in IUGR + 1% Arg or IUGR + 0.1% NCG. The jejunal level of the tumor necrosis factor α (TNF-α) in the IUGR lambs was greater (P < 0.05) compared with that in IUGR + 1% Arg or IUGR + 0.1% NCG. The plasma and jejunal total antioxidant capacity (T-AOC) values for the IUGR + 1% Arg or IUGR + 0.1% NCG group were greater (P < 0.05) compared with those for the IUGR group. Compared with the IUGR + 1% Arg or IUGR + 0.1% NCG lambs, the IUGR lambs had lower (P < 0.05) abundance of mRNA and protein abundance of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), zonula occludens-1 (ZO-1), occludin, inducible NOS (iNOS), and epithelial NOS (eNOS). Overall, the data suggest that the Arg or NCG supplementation to suckling lambs with IUGR enhances the intestinal function by regulating the oxidant status via the NO-dependent pathway.

Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.

The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.

Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish.

The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in nfe2, we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and nfe2 null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in nfe2 mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.

Dietary supplementation with omega-3 polyunsaturated fatty acid-rich oils protects against visible-light-induced retinal damage in vivo.

The effects of administering omega-3 (ω-3) polyunsaturated fatty acid (PUFA)-rich oils on visible-light-induced retinal damage were investigated in rabbits. The mole percentages of α-linolenic acid in sea buckthorn berry oil, sea buckthorn oil (SO), sea buckthorn seed oil and flaxseed oil (FO) were 2.12%, 12.98%, 31.56% and 55.41%, respectively. Algal oil (AO) contains 33.34% docosahexaenoic acid. SO has the highest total phenolic content (63.42 ± 0.59 mg SAE per 100 g) amongst these oils. The administration of SO, FO and AO provided structural and functional protection to the retina. In the retina, we observed a significant increase in the levels of DHA in the AO group compared with the normal group. The mechanism of retinal protection by SO, FO and AO involves up-regulating the expression of nuclear factor erythroid-2 related factor 2 and haem oxygenase-1. The levels of interleukin-1 ß, tumour necrosis factor-alpha, interleukin-8, and cyclooxygenase 2 in the retina were significantly reduced with AO treatment. The administration of AO resulted in the down-regulation of nuclear factor kappa B mRNA expression. In addition, the treatment with AO significantly attenuated the light-induced apoptosis and angiogenesis in the retina. These results suggest that dietary ω-3 PUFA-rich oils protect against visible-light-induced retinal damage.

Role of resveratrol in regulation of cellular defense systems against oxidative stress.

Resveratrol, a natural polyphenolic compound, is found in various kinds of fruits, plants, and their commercial products such as red wine. It has been demonstrated to exhibit a variety of health-promoting effects including prevention and/or treatment of cardiovascular diseases, inflammation, diabetes, neurodegeneration, aging, and cancer. Cellular defensive properties of resveratrol can be explained through its ability of either directly neutralizing reactive oxygen species/reactive nitrogen species (ROS/RNS) or indirectly upregulating the expression of cellular defensive genes. As a direct antioxidant agent, resveratrol scavenges diverse ROS/RNS as well as secondary organic radicals with mechanisms of hydrogen atom transfer and sequential proton loss electron transfer, thereby protecting cellular biomolecules from oxidative damage. Resveratrol also enhances the expression of various antioxidant defensive enzymes such as heme oxygenase 1, catalase, glutathione peroxidase, and superoxide dismutase as well as the induction of glutathione level responsible for maintaining the cellular redox balance. Such defenses could be achieved by regulating various signaling pathways including sirtuin 1, nuclear factor-erythroid 2-related factor 2 and nuclear factor κB. This review provides current understanding and information on the role of resveratrol in cellular defense system against oxidative stress. © 2017 BioFactors, 44(1):36-49, 2018.

Isocitrate protects DJ-1 null dopaminergic cells from oxidative stress through NADP+-dependent isocitrate dehydrogenase (IDH).

DJ-1 is one of the causative genes for early onset familiar Parkinson's disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP+-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of IDH induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in Drosophila, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using Drosophila and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian DJ-1 null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with DJ-1 dysfunction.

Nuclear Factor Erythroid 2 Regulates Human HSC Self-Renewal and T Cell Differentiation by Preventing NOTCH1 Activation.

Nuclear factor erythroid-derived 2 (NF-E2) has been associated with megakaryocyte maturation and platelet production. Recently, an increased in NF-E2 activity has been implicated in myeloproliferative neoplasms. Here, we investigate the role of NF-E2 in normal human hematopoiesis. Knockdown of NF-E2 in the hematopoietic stem and progenitor cells (HSPCs) not only reduced the formation of megakaryocytes but also drastically impaired hematopoietic stem cell activity, decreasing human engraftment in immunodeficient (NSG) mice. This phenotype is likely to be related to both increased cell proliferation (p21-mediated) and reduced Notch1 protein expression, which favors HSPC differentiation over self-renewal. Strikingly, although NF-E2 silencing in HSPCs did not affect their myeloid and B cell differentiation in vivo, it almost abrogated T cell production in primary hosts, as confirmed by in vitro studies. This effect is at least partly due to Notch1 downregulation in NF-E2-silenced HSPCs. Together these data reveal that NF-E2 is an important driver of human hematopoietic stem cell maintenance and T lineage differentiation.

Effects of aflatoxin B1 on mitochondrial respiration, ROS generation and apoptosis in broiler cardiomyocytes.

Aflatoxin B1 (AFB1) develops various toxic effects in the liver by impairing mitochondrial function, inducing cell apoptosis. However, little is focused on its toxicity to broiler cardiomyocytes (BCMs). Here, the mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, cardiac troponin T (cTnT) location, apoptosis induced by AFB1, and antioxidative genes were investigated in BCMs. It was found that AFB1 evoked intracellular ROS generation, and induced apoptosis in BCMs. AFB1 treatment resulted in increased percentage of apoptotic cells, increased location range of cTnT in cytoplasm, upregulated messenger RNA (mRNA) expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and downregulated mRNA expressions of Mn-superoxide dismutase in BCMs. These findings suggested AFB1 treatment caused significant cardiomyocyte damage and cardiotoxicity, impairment of mitochondrial functions, activated ROS generation, and induced apoptosis, and probably was involved in the Nrf2 signal pathway in BCMs.

RS9, a novel Nrf2 activator, attenuates light-induced death of cells of photoreceptor cells and Müller glia cells.

The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure.

Rosemary Extract-Mediated Lifespan Extension and Attenuated Oxidative Damage in Drosophila melanogaster Fed on High-Fat Diet.

Rosemary extract has a potent antioxidant activity and is widely used in the food industry. In this study, the lifespan prolonging and antioxidant activity of rosemary extract was evaluated by high-fat-induced oxidative damage in Drosophila melanogaster. The results revealed that the lifespan and climbing ability of fruit flies was enhanced significantly by feeding rosemary extract. Furthermore, feeding with rosemary extract significantly increased the enzyme activity of superoxide dismutase (SOD) and catalase (CAT), and significantly decreased the level of malonaldehyde. The gene expression of SOD, CAT, and nuclear factor erythroid-2 related factor 2 was enhanced and that for methuselah was significantly reduced. The comet assay showed that high-fat diet-induced DNA lesion was significantly reduced in larvae treated with the rosemary extract. Our results suggest that feeding with rosemary extract is effective to the extended lifespan in fruit flies by strengthening of the resistance to high-fat-induced oxidative stress and by stimulating, at least in part, the endogenous antioxidant response.

Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production.

Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H2O2 to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H2O2 concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H2O2 treatment, and Bacillus pre-protection + H2O2 treatment. Bacillus were co-cultured with IPEC-1 for 3 h in Bacillus and Bacillus pre-protection + H2O2 treatments. Then, based on OS model, 300 µmol/L H2O2 was added into medium of H2O2 and Bacillus pre-protection + H2O2 treatments for another 12 h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψm), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H2O2 induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Δψm was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.

p38 MAPK-SKN-1/Nrf signaling cascade is required for intestinal barrier against graphene oxide toxicity in Caenorhabditis elegans.

Biological barrier plays a crucial role for organisms against the possible toxicity from engineered nanomaterials (ENMs). Graphene oxide (GO) has been proven to cause potential toxicity on organisms. However, the molecular mechanisms for intestinal barrier of animals against GO toxicity are largely unclear. Using in vivo assay system of Caenorhabditis elegans, we found that mutation of genes encoding core p38 mitogen-activated protein kinase (MAPK) signaling pathway caused susceptible property to GO toxicity and enhanced translocation of GO into the body of nematodes. Genetic assays indicated that SKN-1/Nrf functioned downstream of p38 MAPK signaling pathway to regulate GO toxicity and translocation. Transcription factor of SKN-1 could regulate GO toxicity and translocation at least through function of its targeted gene of gst-4 encoding one of phase II detoxification proteins. Moreover, intestine-specific RNA interference (RNAi) assay demonstrated that the p38 MAPK-SKN-1/Nrf signaling cascade could function in intestine to regulate GO toxicity and intestinal permeability in GO exposed nematodes. Therefore, p38 MAPK-SKN-1/Nrf signaling cascade may act as an important molecular basis for intestinal barrier against GO toxicity in organisms. Exposure to GO induced significantly increased expression of genes encoding p38 MAPK-SKN-1/Nrf signaling cascade, which further implies that the identified p38 MAPK-SKN-1/Nrf signaling cascade may encode a protection mechanism for nematodes in intestine to be against GO toxicity.

A novel role for nuclear factor-erythroid 2 in erythroid maturation by modulation of mitochondrial autophagy.

We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy.

Impaired Nrf2 regulation of mitochondrial biogenesis in rostral ventrolateral medulla on hypertension induced by systemic inflammation.

Oxidative stress in rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, is involved in the development of hypertension under systemic inflammation. Mitochondrial dysfunction contributes to tissue oxidative stress. In this study, we sought to investigate whether hypertension developed under systemic inflammation is attributable to impaired mitochondrial biogenesis in RVLM. In normotensive Sprague-Dawley rats, intraperitoneal infusion of a low dose Escherichia coli lipopolysaccharide (LPS) for 7 days promoted a pressor response, alongside a decrease in mitochondrial DNA (mtDNA) copy number, reductions in protein expression of nuclear DNA-encoded transcription factors for mitochondrial biogenesis, including mitochondrial transcription factor A (TFAM) and nuclear factor erythroid-derived 2-like 2 (Nrf2), and suppression of nuclear translocation of the phosphorylated Nrf2 (p-Nrf2) in RVLM neurons; all of which were abrogated by treatment with intracisternal infusion of an interleukin-1ß (IL-1ß) blocker, IL-1Ra, or a mobile mitochondrial electron carrier, coenzyme Q10 (CoQ10). Microinjection into RVLM of IL-1ß suppressed the expressions of p-Nrf2 and TFAM, and evoked a pressor response; conversely, the Nrf2 inducer, tert-butylhydroquinone, lessened the LPS-induced suppression of TFAM expression and pressor response. At cellular level, exposure of neuronal N2a cells to IL-1ß decreased mtDNA copy number, increased protein interaction of Nrf2 to its negative regulator, kelch-like ECH-associated protein 1 (Keap1), and reduced DNA binding activity of p-Nrf2 to Tfam gene. Together these results indicate that defect mitochondrial biogenesis in RVLM neurons entailing redox-sensitive and IL-1ß-dependent suppression of TFAM because of the increase in the formation of Keap1/Nrf2 complex, reductions in nuclear translocation of the activated Nrf2 and its binding to the Tfam gene promoter may underlie hypertension developed under the LPS-induced systemic inflammation.

Regulation and function of the NFE2 transcription factor in hematopoietic and non-hematopoietic cells.

The NFE2 transcription factor was identified over 25 years ago. The NFE2 protein forms heterodimers with small MAF proteins, and the resulting complex binds to regulatory elements in a large number of target genes. In contrast to other CNC transcription family members including NFE2L1 (NRF1), NFE2L2 (NRF2) and NFE2L3 (NRF3), which are widely expressed, earlier studies had suggested that the major sites of NFE2 expression are hematopoietic cells. Based on cell culture studies it was proposed that this protein acts as a critical regulator of globin gene expression. However, the knockout mouse model displayed only mild erythroid abnormalities, while the major phenotype was a defect in megakaryocyte biogenesis. Indeed, absence of NFE2 led to severely impaired platelet production. A series of recent data, also summarized here, shed new light on the various functional roles of NFE2 and the regulation of its activity. NFE2 is part of a complex regulatory network, including transcription factors such as GATA1 and RUNX1, controlling megakaryocytic and/or erythroid cell function. Surprisingly, it was recently found that NFE2 also has a role in non-hematopoietic tissues, such as the trophoblast, in which it is also expressed, as well as the bone, opening the door to new research areas for this transcription factor. Additional data showed that NFE2 function is controlled by a series of posttranslational modifications. Important strides have been made with respect to the clinical significance of NFE2, linking this transcription factor to hematological disorders such as polycythemias.