This study aims to explore the specific mechanism by which puerarin inhibits ferroptosis and improves the myocardial contractile function in myocardial hypertrophy through the nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)/heme oxygenase-1(HO-1) signaling pathway. The hypertrophic cardiomyocyte model was established using phenylephrine, and H9c2 cells were divided into control group, model group, puerarin group, and puerarin+ML385 group. Cell viability and surface area were detected by cell counting kit-8(CCK-8) and immunofluorescence experiments. The mitochondrial membrane potential and Ca~(2+) concentration were measured. The ferroptosis-related indicators were detected by biochemical and fluorescence staining methods. The expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway was detected by Western blot. A myocardial hypertrophy model was established, and 40 rats were randomly divided into sham group, model group, puerarin group, and puerarin+Nrf2 inhibitor(ML385) group, with 10 rats in each group. Echocardiogram, hemodynamic parameters, and myocardial hypertrophy parameters were measured. Histopathological changes of myocardial tissues were observed by hematoxylin and eosin(HE) staining and Masson staining. Biochemical methods, enzyme-linked immunosorbent assay(ELISA), and fluorescence staining were used to detect inflammatory factors and ferroptosis-related indicators. Immunohistochemistry was used to detect the expression of proteins related to ferroptosis and the Nrf2/ARE/HO-1 signaling pathway. Cell experiments showed that puerarin intervention significantly enhanced the viability of hypertrophic cardiomyocytes, reduced their surface area, and restored mitochondrial membrane potential and Ca~(2+) homeostasis. Mechanism studies revealed that puerarin promoted Nrf2 nuclear translocation, upregulated the expression of HO-1, solute carrier family 7 member 11(SLC7A11), and glutathione peroxidase 4(GPX4), and decreased malondialdehyde(MDA), reactive oxygen species(ROS), and iron levels. These protective effects were reversed by ML385. In animal experiments, puerarin improved cardiac function in rats with myocardial hypertrophy, alleviated myocardial hypertrophy and fibrosis, inhibited inflammatory responses and ferroptosis, and promoted nuclear Nrf2 translocation and HO-1 expression. However, combined intervention with ML385 led to deterioration of hemodynamics and a rebound in ferroptosis marker levels. In conclusion, puerarin may inhibit cardiomyocyte ferroptosis through the Nrf2/ARE/HO-1 signaling pathway, thereby improving myocardial contractile function in myocardial hypertrophy.
Animals ; NF-E2-Related Factor 2/genetics* ; Rats ; Ferroptosis/drug effects* ; Signal Transduction/drug effects* ; Isoflavones/pharmacology* ; Male ; Rats, Sprague-Dawley ; Cardiomegaly/genetics* ; Myocytes, Cardiac/metabolism* ; Antioxidant Response Elements/drug effects* ; Myocardial Contraction/drug effects* ; Heme Oxygenase-1/genetics* ; Cell Line

OBJECTIVE: To explore the protective effects and underlying mechanisms of H ₂S against lipid peroxidation-mediated carbonyl stress in the uranium-treated NRK-52E cells. METHODS: Cell viability was evaluated using CCK-8 assay. Apoptosis was measured using flow cytometry. Reagent kits were used to detect carbonyl stress markers malondialdehyde, 4-hydroxynonenal, thiobarbituric acid reactive substances, and protein carbonylation. Aldehyde-protein adduct formation and alcohol dehydrogenase, aldehyde dehydrogenase 2, aldo-keto reductase, nuclear factor E2-related factor 2 (Nrf2), and cystathionine β-synthase (CBS) expression were determined using western blotting or real-time PCR. Sulforaphane (SFP) was used to activate Nrf2. RNA interference was used to inhibit CBS expression. RESULTS: GYY4137 (an H ₂S donor) pretreatment significantly reversed the uranium-induced increase in carbonyl stress markers and aldehyde-protein adducts. GYY4137 effectively restored the uranium-decreased Nrf2 expression, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2, accompanied by a reversal of the uranium-decreased expression of CBS and aldehyde-metabolizing enzymes. The application of CBS siRNA efficiently abrogated the SFP-enhanced effects on the expression of CBS, Nrf2 activation, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2 and concomitantly reversed the SFP-enhanced effects of the uranium-induced mRNA expression of aldehyde-metabolizing enzymes. Simultaneously, CBS siRNA reversed the SFP-mediated alleviation of the uranium-induced increase in reactive aldehyde levels, apoptosis rates, and uranium-induced cell viability. CONCLUSION H ₂S induces Nrf2 activation and nuclear translocation, which modulates the expression of aldehyde-metabolizing enzymes and the CBS/H ₂S axis. Simultaneously, the Nrf2-controlled CBS/H ₂S axis may at least partially promote Nrf2 activation and nuclear translocation. These events form a cycle-regulating mode through which H ₂S attenuates the carbonyl stress-mediated NRK-52E cytotoxicity triggered by uranium.
NF-E2-Related Factor 2/genetics* ; Animals ; Hydrogen Sulfide/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Cell Line ; Uranium/toxicity* ; Antioxidant Response Elements ; Kidney/metabolism* ; Oxidative Stress/drug effects* ; Cell Survival/drug effects* ; Apoptosis/drug effects*

We attempted to examine anti-inflammatory and anti-oxidant effects of 4′-O-β-D-glucosyl-5-O-methylvisamminol (GOMV), the first epigenetic inhibitor of histone phosphorylation at Ser10. While GOMV did not affect the viability of murine macrophage RAW 264.7 cells, it significantly suppressed lipopolysaccharide (LPS)-induced generation of prostaglandin E₂ (PGE₂) and nitric oxide (NO) through transcriptional inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). GOMV also scavenged free radicals in vitro, increased NF-E2-related factor 2 (NRF2), and activated antioxidant response element (ARE), thereby resulting in the induction of phase II cytoprotective enzymes in human keratinocyte HaCaT cells. Finally, GOMV significantly protected HaCaT cells against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative intracellular damages. Together, our results illustrate that GOMV possesses anti-inflammatory and anti-oxidant activity.
Antioxidant Response Elements ; Antioxidants ; Cyclooxygenase 2 ; Epigenomics ; Free Radicals ; Histones ; Humans ; In Vitro Techniques ; Keratinocytes ; Macrophages ; NF-E2-Related Factor 2 ; Nitric Oxide ; Nitric Oxide Synthase Type II ; Phosphorylation ; RAW 264.7 Cells

BACKGROUND: NAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form. METHODS: Expression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2′,7′-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit. RESULTS: Z-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of nqo1-promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation. CONCLUSIONS: Z-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.
Acetylcysteine ; Adenine ; Antioxidant Response Elements ; Azo Compounds ; Blotting, Western ; Breast ; Consensus Sequence ; Epithelial Cells ; Flavoproteins ; Garlic ; Genes, Reporter ; Glutathione ; Humans ; Luciferases ; NF-E2-Related Factor 2 ; Oxidation-Reduction ; Phosphotransferases ; Quinones ; Reactive Oxygen Species ; RNA, Small Interfering ; Up-Regulation

Minor ginsenosides Rh1 and Rg2 were isolated from Korean red ginseng and reported to have various biological effects on anti-inflammatory and anti-stress activities. However, the effects of Rh1 and Rg2 on antioxidant activity and their regulatory effects on the antioxidant enzymes have not been studied. Since oxidative stress is one of the major toxic inflammatory responses stimulated by lipopolysaccharides (LPS), the present study investigated the role of minor ginsenosides Rh1 and Rg2 on antioxidant effects in LPS-treated RAW 264.7 cells. In this study, we found that treatment with ginsenosides Rh1 and Rg2 strongly inhibited LPS-stimulated intracellular ROS production in cells. Luciferase assay showed that treatment with LPS reduced antioxidant response element (ARE) encoding the pARE-luc promoter activity, while ginsenosides inhibited the pARE-luc promoter activity. Moreover, ginsenosides Rh1 and Rg2 exhibited anti-oxidative activity in LPS-induced cells by upregulating antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase. Our results suggest that minor ginsenosides Rh1 and Rg2 may be potential bio-active compounds for antioxidative effects by inhibiting the generation of ROS in RAW 264.7 cells.
Antioxidant Response Elements ; Antioxidants ; Catalase ; Ginsenosides* ; Glutathione Peroxidase ; Lipopolysaccharides ; Luciferases ; Oxidative Stress* ; Panax ; RAW 264.7 Cells* ; Reactive Oxygen Species ; Superoxide Dismutase

Soy isoflavones exhibit various biological activities, such as antioxidant, anti-tumor, anti-inflammatory, and cardiovascular protective effects. The present study was designed to investigate the effects of sixteen synthesized 3-amino-2-hydroxypropoxy genistein derivatives on cell proliferation and activation of Nrf2 (Nuclear factor erythroid 2-related factor 2)/ARE (antioxidant response elements) pathway in human cancer cell lines. Most of the tested compounds exerted greater cytotoxic activity than genistein, as measured by MTT assay. Moreover, compound 8c showed the highest ARE-luciferase reporter activity among the test compounds. It strongly promoted Nrf2 nuclear translocation and up-regulated the expression of total Nrf2 and downstream targets NQO-1 and HO-1 at protein level. The present study may provide a basis for the application of isoflavone derivatives as Nrf2/ARE pathway inducers for cancer therapy and cancer prevention.
Antioxidant Response Elements ; Cell Line, Tumor ; Cell Proliferation ; Genistein ; chemical synthesis ; pharmacology ; therapeutic use ; Heme Oxygenase-1 ; metabolism ; Humans ; Isoflavones ; NF-E2-Related Factor 2 ; metabolism ; Neoplasms ; drug therapy ; metabolism ; Signal Transduction ; Soybeans ; chemistry ; Up-Regulation

Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress.
Animals ; Antioxidant Response Elements ; Astrocytes* ; Catalase ; Cell Death ; Cell Survival ; DNA ; Gene Expression* ; Heme Oxygenase-1 ; Hydrogen ; Neurodegenerative Diseases ; Neurons ; Oxidative Stress ; Phosphotransferases* ; Rats* ; Reactive Oxygen Species ; Superoxides ; Up-Regulation

Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H:quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression.
Aging ; Antioxidant Response Elements* ; Cirsium* ; DNA Damage* ; DNA* ; Ethanol* ; Gene Expression ; Glutathione ; Humans* ; Keratinocytes* ; Korea ; Oxidants ; Phosphorylation ; Reactive Oxygen Species ; Stress, Mechanical ; Transcription Factors ; Xenobiotics

OBJECTIVETo explore a new method of establishing HepG2 cell model of steatosis and observe the expression and significance of nuclear factor erythroid-2p45-related factor 2(Nrf2)/antioxidative response element (ARE) pathway related factors in HepG2 cells of steatosis.

METHODSHepG2 cells were induced with DMEM containing 25% fetal bovine serum, 0.1% MCT/LCT Fat Emulsion and 0.1 mmol/L free fatty acid (FFA) at different stages and the control group cells were cultured with normal DMEM medium. After the cell models were successfully established, lipid droplets in cytoplasm were observed with Oil Red 0 staining, and the triglyceride (TG) accumulation in HepG2 cells were tested by biochemical assay. Intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Nitric oxide (NO), superoxide dismutase(SOD), malonyldialdehyde(MDA) and glutathione peroxidase(GSH-Px) were tested by biological reagent kit, while the protein expression of nuclear factor erythroid-2p45-related factor 2(Nrf2), heme oxygenase-1 (HO-1) and

NAD(P)Hquinone oxidoreductase-1(NQO1) were analyzed by Western blot.

RESULTSCompared with that in the control group, red cytoplasmic lipid droplets were visible in model group; TG,ROS, NO, MDA concentration (P < 0.05, P < 0.01) and the protein expression of Nrf2, HO-1 and NQO1 (P < 0.05, P < 0.01)were significantly higher in model group, while SOD, GSH-Px concentration reduced significantly (P < 0.01).

CONCLUSIONThe in vitro cell model of steatosis and oxidative stress was successfully established. The activation of Nrf2/ARE pathway related factors maybe relevant to the overreaction of oxidative stress in HepG2 cells of steatosis.

Antioxidant Response Elements ; Culture Media ; Fatty Acids, Nonesterified ; Fatty Liver ; metabolism ; GA-Binding Protein Transcription Factor ; Glutathione Peroxidase ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hep G2 Cells ; Humans ; Malondialdehyde ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Nitric Oxide ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Triglycerides ; metabolism

The cytoprotective enzyme heme oxygenase-1 (HO-1) influences endothelial cell survival, proliferation, inflammatory response, and angiogenesis in response to various angiogenic stimuli. In this study, we investigate the involvement of HO-1 in the angiogenic activity of orexin-A. We showed that orexin-A stimulates expression and activity of HO-1 in human umbilical vein endothelial cells (HUVECs). Furthermore, we showed that inhibition of HO-1 by tin (Sn) protoporphryin-IX (SnPP) reduced orexin-A-induced angiogenesis in vivo and ex vivo. Orexin-A-stimulated endothelial tube formation and chemotactic activity were also blocked in SnPP-treated vascular endothelial cells. Orexin-A treatment increased the expression of nuclear factor erythroid-derived 2 related factor 2 (Nrf2), and antioxidant response element (ARE) luciferase activity, leading to induction of HO-1. Collectively, these findings indicate that HO-1 plays a role as an important mediator of orexin-A-induced angiogenesis, and provide new possibilities for therapeutic approaches in pathophysiological conditions associated with angiogenesis.
Antioxidant Response Elements ; Endothelial Cells* ; Heme Oxygenase-1* ; Human Umbilical Vein Endothelial Cells ; Luciferases ; Tin ; Orexins