NADH-quinone Oxidoreductase Research Articles

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.

Biochemical study on the protective effect of curcumin on acetaminophen and gamma-irradiation induced hepatic toxicity in rats.

Acetaminophen (APAP) is one of the few recommended analgesic and antipyretic drugs in some critical cases such as viral disease COVID-19. However, the unrestricted use of APAP develops liver disorders. Hepatotoxicity and liver injury can also be induced by ionizing radiation (IR) during radiotherapy. The data of the current study represents that treatment of rats with either APAP-overdose, or gamma-irradiation (R) induces hepatotoxicity, results in significant increases of the hepatic-enzymes activities (ALT, AST, ALP, GGT, LDH, and MDH), as well as enhancement of triglycerides, total cholesterol levels, combined with declines in albumin and total protein contents. An enhancement of the lipid peroxides (malondialdehyde; MDA), and nitric oxide levels along with a decline of reduced glutathione contents and suppression of superoxide dismutase, catalase, and glutathione peroxidase activities are also observed within the liver tissues of intoxicated animals. TNF-α, IL-1β, IL-6, iNOS, Cytochrome P450 2E1 (CYP2E1), miR-802 gene expression, NF-κB, and calcium levels are up-regulated, while Nuclear factor erythroid-related factor-2 (Nrf2), Hemoxygenase-1 (HO-1) protein and gene expressions, as well as, glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H-Quinone oxidoreductase (NQO1), and miR-122 gene expressions are down-regulated in the livers of intoxicated animals. All these parameters show significant improvement in R/APAP intoxicated animals. Curcumin pretreatment develops an amelioration of these effects in APAP-overdose, R-exposure, or R/APAP treatments. In conclusion, oral administration of curcumin shows hepatoprotective effects against APAP-overdose induced hepatic damage in normal and gamma-irradiated rats through prospective regulation of the therapeutic targets CYP2E1, Nrf2, and NF-κB, via organizing the miR-122 and miR-802 gene expression.

Regulation of secondary metabolite biosynthesis in Monascus purpureus via cofactor metabolic engineering strategies

This study investigated the effects of cofactor metabolism on secondary metabolite production in M. purpureus through the application of different cofactor engineering strategies. Total pigment production dramatically increased by 39.08% and 40.89%, and yellow pigment production increased by 74.62% and 114.06% after the addition of 1.0 mg/L of the exogenous cofactor reagents methyl viologen and rotenone, respectively, in submerged batch-fermentation. The extracellular red pigment tone changed to yellow with the application of electrolytic stimulation at 800 mV/cm2, but almost no citrinin production was detected. In addition, the total pigment, yellow pigment and citrinin production increased by 35.46%, 54.89% and 6.27% after disruption of the nuoⅠ gene that encodes NADH-quinone oxidoreductase, respectively. Thus, cofactor metabolic engineering strategies could be extended to the industrial production of Monascus pigment or high yellow pigment with free citrinin production.

Sex-, Age-, and Race/Ethnicity-Dependent Variations in Drug-Processing and NRF2-Regulated Genes in Human Livers.

Individual variations in xenobiotic metabolism affect the sensitivity to diseases. In this study, the impacts of sex, age, and race/ethnicity on drug-processing genes and nuclear factor erythroid 2-related factor 2 (NRF2) genes in human livers were examined via QuantiGene multiplex suspension array (226 samples) and quantitative polymerase chain reaction (qPCR) (247 samples) to profile the expression of nuclear receptors, cytochrome P450s, conjugation enzymes, transporters, bile acid metabolism, and NRF2-regulated genes. Sex differences were found in expression of about half of the genes, but in general the differences were not large. For example, females had higher transcript levels of catalase, glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase 1, and thioredoxin reductase-1 compared with males via qPCR. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit (GCLM) and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of HO-1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, SULT2A1, small heterodimer partner, and bile salt export pump. An examination of 34 diseased and control human liver samples showed that compared with disease-free livers, fibrotic livers had higher NAD(P)H-quinone oxidoreductase 1 (NQO1), GCLC, GCLM, and NRF2; hepatocellular carcinoma had higher transcript levels of NQO1 and KEAP1; and steatotic livers had lower GCLC, GCLM, and HO-1 expression. In summary, in drug-processing gene and NRF2 genes, sex differences were the major findings, and there were no apparent age differences, and race/ethnicity differences occurred for a few genes. These descriptive findings could add to our understanding of the sex-, age-, and race/ethnicity-dependent differences in drug-processing genes as well as NRF2 genes in normal and diseased human livers. SIGNIFICANCE STATEMENT: In human liver drug-processing and nuclear factor erythroid 2-related factor 2 genes, sex differences were the main finding. There were no apparent differences due to age, except children had higher glutamate-cysteine ligase modifier subunit, and elderly had higher multidrug resistance protein 3. African Americans had lower expression of farnesoid X receptor (FXR) but higher expression of heme oxygenase 1, Caucasians had higher expression of organic anion transporter 2, and Hispanics had higher expression of FXR, small heterodimer partner, SULT2A1, and bile salt export pump.

Comparative transcriptome analysis to identify candidate genes involved in 2-methoxy-1,4-naphthoquinone (MNQ) biosynthesis in Impatiens balsamina L.

Impatiens balsamina L. is a tropical ornamental and traditional medicinal herb rich in natural compounds, especially 2-methoxy-1,4-naphthoquinone (MNQ) which is a bioactive compound with tested anticancer activities. Characterization of key genes involved in the shikimate and 1,4-dihydroxy-2-naphthoate (DHNA) pathways responsible for MNQ biosynthesis and their expression profiles in I. balsamina will facilitate adoption of genetic/metabolic engineering or synthetic biology approaches to further increase production for pre-commercialization. In this study, HPLC analysis showed that MNQ was present in significantly higher quantities in the capsule pericarps throughout three developmental stages (early-, mature- and postbreaker stages) whilst its immediate precursor, 2-hydroxy-1,4-naphthoquinone (lawsone) was mainly detected in mature leaves. Transcriptomes of I. balsamina derived from leaf, flower, and three capsule developmental stages were generated, totalling 59.643 Gb of raw reads that were assembled into 94,659 unigenes (595,828 transcripts). A total of 73.96% of unigenes were functionally annotated against seven public databases and 50,786 differentially expressed genes (DEGs) were identified. Expression profiles of 20 selected genes from four major secondary metabolism pathways were studied and validated using qRT-PCR method. Majority of the DHNA pathway genes were found to be significantly upregulated in early stage capsule compared to flower and leaf, suggesting tissue-specific synthesis of MNQ. Correlation analysis identified 11 candidate unigenes related to three enzymes (NADH-quinone oxidoreductase, UDP-glycosyltransferases and S-adenosylmethionine-dependent O-methyltransferase) important in the final steps of MNQ biosynthesis based on genes expression profiles consistent with MNQ content. This study provides the first molecular insight into the dynamics of MNQ biosynthesis and accumulation across different tissues of I. balsamina and serves as a valuable resource to facilitate further manipulation to increase production of MNQ.

Corrigendum.

Corrigendum.

Icaritin activates Nrf2/Keap1 signaling to protect neuronal cells from oxidative stress.

Our earlier study indicated that icaritin (ICT) protected mice from cerebral ischemic injury by inhibiting oxidative stress, and this study aimed to investigate its mechanism using a H2 O2 -treated SH-SY5Y cells model. Cell viability was assessed by cell counting kit 8 (CCK-8). Oxidative stress parameters were detected by flow cytometry, and signaling pathways were analyzed by immunoblotting. We found that ICT alleviated apoptosis and intracellular and mitochondrial reactive oxygen species (ROS) levels, decreased the expressions of Bax and cleaved caspase-3, and increased the expressions of Bcl-2 compared to H2 O2 group. ICT increased mitochondrial membrane potential (ΔΨm) and blocked the opening of mitochondrial membrane permeability transporter (MPT), and increased the activity of glutathione peroxidase (GSH-px), catalase (CAT), and superoxide dismutase (SOD), meanwhile, decreased the activity of malondialdehyde (MDA) compared to H2 O2 group. Further investigation revealed that ICT significantly up-regulated the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO-1). The anti-apoptosis and antioxidative effects of ICT were blocked bay ML385, a Nrf2/Keap1 signaling pathway inhibitor. These results indicate that ICT can play a neuroprotective role against oxidative stress injury by activating Nrf2/Keap1 signaling pathway.

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

Insights into the New Cancer Therapy through Redox Homeostasis and Metabolic Shifts.

Modest levels of reactive oxygen species (ROS) are necessary for intracellular signaling, cell division, and enzyme activation. These ROS are later eliminated by the body’s antioxidant defense system. High amounts of ROS cause carcinogenesis by altering the signaling pathways associated with metabolism, proliferation, metastasis, and cell survival. Cancer cells exhibit enhanced ATP production and high ROS levels, which allow them to maintain elevated proliferation through metabolic reprograming. In order to prevent further ROS generation, cancer cells rely on more glycolysis to produce ATP and on the pentose phosphate pathway to provide NADPH. Pro-oxidant therapy can induce more ROS generation beyond the physiologic thresholds in cancer cells. Alternatively, antioxidant therapy can protect normal cells by activating cell survival signaling cascades, such as the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway, in response to radio- and chemotherapeutic drugs. Nrf2 is a key regulator that protects cells from oxidative stress. Under normal conditions, Nrf2 is tightly bound to Keap1 and is ubiquitinated and degraded by the proteasome. However, under oxidative stress, or when treated with Nrf2 activators, Nrf2 is liberated from the Nrf2-Keap1 complex, translocated into the nucleus, and bound to the antioxidant response element in association with other factors. This cascade results in the expression of detoxifying enzymes, including NADH-quinone oxidoreductase 1 (NQO1) and heme oxygenase 1. NQO1 and cytochrome b5 reductase can neutralize ROS in the plasma membrane and induce a high NAD+/NADH ratio, which then activates SIRT1 and mitochondrial bioenergetics. NQO1 can also stabilize the tumor suppressor p53. Given their roles in cancer pathogenesis, redox homeostasis and the metabolic shift from glycolysis to oxidative phosphorylation (through activation of Nrf2 and NQO1) seem to be good targets for cancer therapy. Therefore, Nrf2 modulation and NQO1 stimulation could be important therapeutic targets for cancer prevention and treatment.

Exploring the binding pocket of quinone/inhibitors in mitochondrial respiratory complex I by chemical biology approaches.

ATP: adenosine triphosphate; BODIPY: boron dipyrromethene; complex I: proton-translocating NADH-quinone oxidoreductase; DIBO: dibenzocyclooctyne; EM: electron microscopy; FeS: iron-sulfur; FMN: flavin adenine mononucleotide; LDT: ligand-directed tosylate; NADH: nicotinamide adenine dinucleotide; ROS: reactive oxygen species; SMP: submitochondrial particle; TAMRA: 6-carboxy-N,N,N',N'-tetramethylrhodamine; THF: tetrahydrofuran; TMH: transmembrane helix.

Genome Analysis of a Member of the Uncultured Phylum Riflebacteria Revealed Pathways of Organotrophic Metabolism and Dissimilatory Iron Reduction

The candidate phylum Riflebacteria was described based on analysis of genomes assembled from the metagenomes of various anaerobic ecosystems; however, to date, no member of Riflebacteria has been isolated in a pure culture. In this work, the genome of a new member of Riflebacteria was obtained from the metagenome of the microbial community of a deep subsurface thermal aquifer in Western Siberia. Phylogenetic analysis indicates that this bacterium, designated Ch46, represents a new genus of the phylum Riflebacteria, distinct from the previously described Candidatus Ozemobacter. The genome of Ch46 was found to encode putatively secreted alpha-amylases and various sugar and peptide transporters. Reconstruction of central metabolic pathways suggests that Ch46 is an anaerobic organotroph capable of fermenting sugars and proteinaceous substrates. The bacterium lacks the genes of the known pathways of autotrophic carbon fixation, as well as the aerobic respiratory chain. The transmembrane ion gradient can be generated by an Rnf complex, as well as by a sodium-transporting NADH‒quinone oxidoreductase. The presence of multiheme c-type cytochromes indicates the possibility of dissimilatory Fe(III) reduction. The pathways of dissimilatory reduction of sulfate, nitrate, nitrite, arsenate, and sulfur were not detected. In the subsurface aquifer, Riflebacteria probably utilize starch and similar polysaccharides, as well as low-molecular-weight organic compounds. Based on the results of phylogenetic and genomic analysis, it is proposed to classify the novel bacterium as ‘Candidatus Rifleibacterium amylolyticum.’

The protective effect of recombinant globular adiponectin on testis by modulating autophagy, endoplasmic reticulum stress and oxidative stress in streptozotocin-induced diabetic mice

This study was to investigate whether recombinant globular adiponectin produced its protective effect on the testis of diabetic mice by modulating autophagy, endoplasmic reticulum stress and oxidative stress. Male mice were randomly divided into control, diabetic, diabetic treated with low and high dose of adiponectin. Mice were killed at the termination after 4 weeks and 8 weeks of adiponectin treatment. Serum levels of glucose, lipids, testosterone, insulin, LH and FSH were measured. The protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), Caspase12, Beclin1, microtubule-associated protein light chain 3 (LC3) and p62 was determined by western blotting. The mRNA expression of adiponectin receptor 1 (AdipoR1), p22phox, p47phox, nuclear factor erythroid2-related factor 2 (Nrf2), NAD(P)H-quinone oxidoreductase 1(NQO1), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) were determined by real-time fluorescence quantitative PCR. The testicular weight, the sperm number and motility, and the serum levels of testosterone and insulin were significantly decreased in diabetic mice (P < 0.05). The expression of Beclin1, LC3, Nrf2, NQO1, HO-1, SOD and AdipoR1 were significantly decreased (P < 0.05), while the expression of GRP78, CHOP, Caspase12, p62, p22phox and p47phox were notably increased in the testes of diabetic mice (P < 0.05). Adiponectin treatment significantly reversed the above-mentioned changes in the testes of diabetic mice, some of which were dose- and time-dependent (P < 0.05). These data suggested that recombinant globular adiponectin may produce the protective effect on the testes of diabetic mice by inducing autophagy and inhibiting ER stress and oxidative stress.

Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM–1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

Cardiac Dysfunction after Burn Injury: Role of the AMPK-SIRT1-PGC1α-NFE2L2-ARE Pathway

Mitochondrial oxidative stress plays a prominent role in the development of burn-induced cardiac dysfunction. AMP-activated kinase (AMPK), an energy sensor, has a central role in the pathogenesis of heart failure. However, its role in cardiac dysfunction after burn injury is unclear. Our hypothesis is that burn injury acts through the AMPK-sirtuin 1-PGC1α-nuclear factor erythroid 2-related factor 2 (NFE2L2)-ARE signaling pathway, leading to cardiac mitochondrial impairment, resulting in cardiac dysfunction. Male Sprague-Dawley rats underwent sham procedure or 60% total body surface area full-thickness burn. Echocardiograms were performed 24 hours post burn. Heart tissue was harvested at 24 hours post burn for biochemistry/molecular biologic analysis. AC16 cardiomyocytes were treated with either sham or burned rat serum (±AMPK inhibitor/AMPK activator/PGC1α activator) for evaluation of cardiomyocyte mitochondrial function by using seahorse invitro. Burn injury-induced cardiac dysfunction was measured by echocardiogram. Burn injury suppressed cardiac AMPK, sirtuin 1, and PGC1 expression, leading to acetylation of cardiomyocyte proteins. In addition, burn injury caused NFE2L2 and NFE2L2 regulated antioxidants (heme oxygenase 1, NADH quinone oxidoreductase 1, glutamatecysteine ligase catalytic subunit, manganese superoxide dismutase, and glutathione peroxidase) to decrease, resulting in cardiac oxidative stress. Invitro, AMPK1 activator and PGC1α agonist treatment improved Ac16 cell mitochondrial dysfunction, and AMPK1 inhibitor treatment worsened Ac16 cellular damage. Burn-induced cardiac dysfunction and cardiac mitochondrial damage occur via the AMPK-sirtuin 1-PGC1α-NFE2L2-ARE signaling pathway. AMPK and PGC1α agonists might be promising therapeutic agents to reverse cardiac dysfunction after burn injury.

Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I

NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky "block" that is ∼13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.

Cytotoxic and antiproliferative effects of thymoquinone on rat C6 glioma cells depend on oxidative stress

Thymoquinone (TQ) is a highly perspective chemotherapeutic agent against gliomas and glioblastomas because of its ability to cross the blood-brain barrier and its selective cytotoxicity for glioblastoma cells compared to primary astrocytes. Here, we tested the hypothesis that TQ-induced mild oxidative stress provokes C6 glioma cell apoptosis through redox-dependent alteration of MAPK proteins. We showed that low concentrations of TQ (20-50μM) promoted cell-cycle arrest and induced hydrogen peroxide generation as a result of NADH-quinone oxidoreductase 1-catalyzed two-electron reduction of this quinone. Similarly, low concentrations of TQ efficiently conjugated intracellular GSH disturbing redox state of glioma cells and provoking mitochondrial dysfunction. We demonstrated that high concentrations of TQ (70-100μM) induced reactive oxygen species generation due to its one-electron reduction. TQ provoked apoptosis in C6 glioma cells through mitochondrial potential dissipation and permeability transition pore opening. The identified TQ modes of action on C6 glioma cells open up the possibility of considering it as a promising agent to enhance the sensitivity of cancer cells to standard chemotherapeutic drugs.

Bioinspired RGD-Engineered Bacteriophage Nanofiber Cues against Oxidative Stress.

Instructive tissue engineering biomaterials provide a vascular niche and protect oxidative stress in injured tissue. In this study, we exploited bioinspired bacteriophage nanofibers, previously recognized by their biochemical and structural cues inducing angiogenesis, as an antioxidant tissue engineering material. We demonstrated that topological cues of Arg-Gly-Asp (RGD)-engineered bacteriophage nanofibers provide angiogenic niches and cytoprotective functions against cellular oxidative stress with increased expression of antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1) via the extracellular-signal-regulated kinase (ERK)-nuclear factor erythroid 2-related factor2 (Nrf2)-mediated signaling pathway, where a high density of RGD cues on the phage body support efficient interaction of cells with phage cues. These bioinspired RGD-engineered bacteriophage nanofibers can serve as a novel therapeutic platform for curing ischemic diseases.

The role of ANXA5 in DBP-induced oxidative stress through ERK/Nrf2 pathway

Di-N-butylphthalate (DBP) have given rise to more and more attention due to its unique endocrine toxicity to male reproductive system. Our previous studies have demonstrated antioxidative Nrf2 (nuclear factor erythroid related factor 2) pathway play a vital role in DBP induced oxidative stress injury. ANXA5 (annexin A5), which is highly expressed in testicular Leydig and Sertoli cells, was found upregulated after DBP stimulation. Mouse Leydig and Sertoli cells were exposed to different concentration of DBP for 24 h to examine the ROS (Reactive oxygen species), MDA (Malondialdehyde), SOD (superoxide dismutase) level and ANXA5, Nrf2, NQO1 (NAD(P)H-quinone oxidoreductase 1), HO-1 (heme oxygenase 1) and ERK/P-ERK protein expression by DHE (Dihydroethidium) staining, ELISA (enzyme-linked immunosorbent assay) and Western blot respectively. Firstly, the oxidative stress injury induced by DBP was re-validated. Then, we confirmed the change of Nrf2 pathway and ANXA5 level after DBP exposure to testicular cells. Additionally, overexpressed ANXA5 could activate Nrf2/HO-1/NQO1 antioxidant pathway and significantly attenuate DBP-induced oxidative stress. Ultimately, we demonstrated ANXA5 could increase ERK phosphorylated level and the activated role of ANXA5 on ERK/Nrf2 pathway could be reversed by ERK inhibitor. Overall, this study illuminated that ANXA5 could defend testicle Leydig and Sertoli cells against DBP-induced oxidative stress injury through ERK/Nrf2 pathway.

Rutaecarpine may improve neuronal injury, inhibits apoptosis, inflammation and oxidative stress by regulating the expression of ERK1/2 and Nrf2/HO-1 pathway in rats with cerebral ischemia-reperfusion injury.

BackgroundCerebral ischemia-reperfusion (CI/R) injury is a more serious brain injury caused by the recovery of blood supply after cerebral ischemia for a certain period of time. Rutaecarpine (Rut) is an alkaloid isolated from Evodia officinalis with various biological activities. Previous studies have shown that Rut has a certain protective effect on ischemic brain injury, but the specific molecular mechanism is still unknown.MethodsIn this study, a rat model of CI/R was established to explore the effects and potential molecular mechanisms of Rut on CI/R injury in rats.ResultsThe results showed that Rut alleviated neuronal injury induced by CI/R in a dose-dependent manner. Besides, Rut inhibited neuronal apoptosis by inhibiting the activation of caspase 3 and the expression of Bax. In addition, Rut alleviated the inflammatory response and oxidative stress caused by CI/R through inhibiting the production of pro-inflammatory factors (IL-6 and IL-1β), lactate dehydrogenase (LDH), malondialdehyde (MDA) and ROS, and increased the levels of anti-inflammatory factors (IL-4 and IL-10) and superoxide dismutase (SOD). Biochemically, Western blot analyses showed that Rut inhibited the phosphorylation of ERK1/2 and promoted the expression of nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway-related proteins (Nrf2, heme oxygenase 1 (HO-1) and NAD (P) H-quinone oxidoreductase 1) in a dose-dependent manner. These results show that Rut may alleviate brain injury induced by CI/R by regulating the expression of ERK1/2 and the activation of Nrf2/HO-1 pathway.ConclusionIn conclusion, these results suggest that Rut may be used as an effective therapeutic agent for damage caused by CI/R.

Physiological and proteomic analyses of Korean F1 maize (Zea mays L.) hybrids under water-deficit stress during flowering

Despite the relevance of drought stress, the regulation of gene expression, protein accumulation, and plant physiology under water-deficit stress is not well understood in Korean F1 maize (Zea mays L.) hybrids. In this study, we investigated the effect of water deficit on the F1 maize hybrids, Ilmichal (Ilmi) and Gwangpyeongok (GPOK), by withholding water for 10 days during flowering. Water deficit severely reduced the relative water content, area, SPAD values, and stomatal conductance of leaves, stem length, and the dry matter content of aerial tissues in drought-stressed plants of both hybrids. However, the dry matter content of roots was reduced only in GPOK. Two-dimensional gel electrophoresis identified 24 spots representing proteins accumulated to differential levels in well-watered and drought-stressed plants of both hybrids. Further analysis of protein spots using matrix assisted laser desorption ionization–time of flight mass spectrometry and protein database searches revealed that nine proteins were involved in carbohydrate metabolism, seven in stress response, and two in photosynthesis. Among these proteins, delta 3,5-delta 2,4-dienoyl-CoA isomerase (spot 8) and bifunctional 3-phosphoadenosine 5-phosphosulfate synthetase 2 (spot 23) were present only in GPOK, whereas NAD-dependent epimerase/dehydratase (spot 13), NAD(P)H-quinone oxidoreductase subunit 2 A (spot 24), and an uncharacterized protein (spot 19) were present only in Ilmi, in response to water-deficit stress. Semi-quantitative reverse transcription PCR analysis showed that the transcript levels of most of the genes encoding these proteins correlated well with their protein levels, suggesting that water deficit affects gene transcription in F1 maize hybrids at the flowering stage.