Effect of Inducers and Inhibitors of the Keap1/Nrf2/ARE System on the Viability and Functional Activity of Model Neuronal-Like and Glial Cells.

On mouse neuroblastoma (Neuro-2a) and human glioblastoma (U-87 MG) cell lines, we studied the effect of inducers and inhibitors of redox-sensitive signaling system of the antioxidant-responsive element Keap1/Nrf2/ARE on the main processes that determine nerve cell viability and vital activity (proliferative activity, apoptosis, autophagy, and activation of the Keap1/Nrf2/ARE system). Inhibitors of the Keap1/Nrf2/ARE system stimulate apoptosis more pronouncedly than inducers, have a weaker effect on autophagy, and do not change the nuclear to cytoplasmic Nrf2 ratio. In general, the revealed effects testify in favor of the potential effectiveness of stimulating the Keap1/Nrf2/ARE system for the prevention and adjuvant therapy of neurodegenerative diseases.

Protective Effect of a New Monophenolic Antioxidant TS-13 in a Mouse Model of Parkinson's Disease.

The development of means of the prevention and treatment of age-related neurodegenerative diseases, as well as geroprotectors, among other things, is based on the inflammatory and free radical theories of aging. In this context, we studied the effect of sodium monophenol 3-(3'-tert-butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13) on the behavioral and locomotor activity of C57BL/6 mice in modeling Parkinson's disease by MPTP neurotoxin injection. TS-13 administration significantly improved orientation and exploratory activity and emotional response of the animals in the open field test, but did not affect the increase in anxiety caused by MPTP injection. Long-term (6 months) TS-13 administration did not suppress spontaneous motor activity in BALB/c mice and slightly increased their exploratory activity.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

Mazes of Nrf2 Regulation.

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

Plant Phenols and Autophagy.

Many plant phenols (stilbenes, curcumins, catechins, flavonoids, etc.) are effective antioxidants and protect cells during oxidative stress. Extensive clinical studies on the potential of phenolic compounds for treatment of cardiovascular, neurodegenerative, oncological, and inflammatory diseases are now being conducted. In addition to direct antioxidant effect, plant phenols may provide a protective effect via activation of the Keap1/Nrf2/ARE redox-sensitive signaling system and regulation of autophagy. In this review, mechanisms of effects of the most common plant phenols on autophagy are presented.

Protective effect of ARE-inducing phenol antioxidant TS-13 in chronic inflammation.

The protective effect of partially substituted monophenol TS-13 inducing the Nrf2/Keap1/ARE signaling system was studied on the model of chronic inflammation in vivo. It was found that during simulation of inflammation in an air pouch lined with synovial-like membrane, TS-13 did not affect the exudate volume, protein content, and cell count, but significantly reduced the intensity of oxidative metabolism in leukocytes of the exudate. In rheumatoid polyarthritis induced by heterologous collagen, TS-13 reduced the severity of clinical signs of inflammation only at the early stages, but inhibited H2O2 generation by monocytes and, partially, by blood neutrophils. These results suggest that the phlogolytic effect of the redox sensitive Nrf2/Keap1/ARE signaling system is less pronounced in chronic immune-mediated inflammatory processes than in acute inflammation.

Anti-inflammatory activity of TS-13, ARE-inducing phenol antioxidant.

The protective effect of water-soluble TS-13 monophenol inducing the antioxidant-responsive element (ARE) system was studied on the models of acute inflammation. Intragastric administration of TS-13 to rats significantly reduced the severity of acute aseptic inflammation induced by intravenous injection of zymosan particles: granulocyte blood count and volume density of infiltrates in the liver decreased on day 3, spontaneous production of activated oxygen metabolites and respiratory burst in blood granulocytes decreased on days 2 and 3. A single dose of TS-13 improved survival of mice with endotoxin shock induced by intraperitoneal injection of E. coli LPS. These results confirmed high anti-inflammatory activity of TS-13.

Keap1/Nrf2/ARE redox-sensitive signaling system as a pharmacological target.

The redox-sensitive signaling system Keap1/Nrf2/ARE plays a key role in maintenance of cellular homeostasis under stress, inflammatory, carcinogenic, and proapoptotic conditions, which allows us to consider it as a pharmacological target. Here we review the basic regulatory mechanisms of the Keap1/Nrf2/ARE system, key targets for pharmacological intervention, and interconnection of this system with other redox-sensitive transcriptional factors. We also discuss the range of currently available pharmaceuticals. Finally, we promote "indirect" antioxidants as a promising strategy for prevention and treatment of wide range of diseases associated with oxidative stress.

Changes in activity of free radical oxidation processes in the early stages of BCG granulomatosis.

We studied the intensity of free radical oxidation in the liver and activity of oxidative metabolism in mouse peritoneal exudate phagocytes at the early stages of chronic generalized BCG-induced granulomatosis (days 3 and 30 after a single intraperitoneal or intravenous administration of 0.5 mg of BCG vaccine). It was found that both methods of injection did not change the intensity of free radical lipid peroxidation in the liver in comparison with the control, but activity of free radical oxidation mediated by production of hydrogen peroxide was increased in the liver and peritoneal exudate at the stages of mature granuloma formation (day 30). At the same time, intraperitoneal injection contributed to more pronounced activation of lipid peroxidation and synthesis of hydrogen peroxide in the liver.

ARE-inducing phenol antioxidant TC-13 improves survival of Drosophila melanogaster in oxidative stress.

The effects of hydrophilic synthetic antioxidant TC-13 sodium (3'-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate on survival of various strains of Drosophila melanogaster were studied under conditions of oxidative stress induced with H(2)O(2)and paraquat. In a concentration of 1%, TC-13 significantly improved survival of Canton S males treated with H(2)O(2)and in a concentration of 0.2% it improved survival of H(2)O(2)-stressed Oregon R females. The protective effect of the antioxidant under conditions of paraquat-induced stress was observed in Canton S females and Oregon R flies of both genders. Addition of T-13 to diets led to prolongation of the maximum lifespan of insects in the majority of the experiment variants. A relationship between the protective effects of TC-13 and the genotype, gender, and environmental conditions was detected.

Mechanism of the Nrf2/Keap1/ARE signaling system.

Nrf2 regulates expression of genes containing antioxidant-respons(iv)e element (ARE) in their promoters and plays a pivotal role among all redox-sensitive transcription factors. Nrf2 is constitutively controlled by repressor protein Keap1, which acts as a molecular sensor of disturbances in cellular homeostasis. These molecular patterns are in close interconnection and function as parts of the integrated redox-sensitive signaling system Nrf2/Keap1/ARE. Depending on cellular redox balance, activity of this signaling system changes at the levels of transcription, translation, posttranslational modification, nuclear translocation of transcription factor, and its binding to ARE-driven gene promoters. This review summarizes current conceptions of Nrf2/Keap1/ARE induction and inactivation.

Effect of phenol inducing the antioxidant responsive element on Drosophila melanogaster lifespan.

The effect of hydrophilic synthetic antioxidant TC-13 (3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate sodium) inducing the antioxidant-responsive element on the lifespan of Drosophila melanogaster was studied. Addition of 1% TC-13 to diets prolonged the lifespan of long-lived D. melanogaster Canton S strain females and males, but not of short-lived Oregon R insects and reduced the mean lifespan of D. melanogaster males of the lgl558OR/Cy strain containing a recessive lethal mutation of tumor suppressor in the heterozygotic state. The geroprotective effects of TC-13 synthetic phenol antioxidant depended on D. melanogaster genotype and gender.

Synthetic water-soluble phenolic antioxidant regulates l-arginine metabolism in macrophages: a possible role of Nrf2/ARE.

Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150-1000 microM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1-100 microM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.

Combination of methods for in vitro study of antioxidant properties of chemical compounds.

We proposed a combination of methods to study antioxidant properties of compounds, including evaluation of the capacity of the test preparations to inhibit peroxidation of unsaturated lipids (in model systems with oxidation of ethyl oleate; aqueous emulsion medium) and low-density lipoproteins (in the presence of transition metal ions), generation of superoxide anion radical (system of lucigenin, xanthine oxidase, and xanthine) and NO(*)/ONOO(-) (system of SIN-1 and lucigenin), and induction of respiratory burst in blood granulocytes (luminol-induced and lucigenin-induced reaction after zymosan stimulation). In vitro study showed that the antioxidant properties of synthetic water-soluble phenols depend strongly on masking of the phenol OH group and nature of the ionogenic fragment in the p-propyl substituent.

Antioxidant and antiinflammatory activity of new water-soluble sulfur-containing phenolic compounds.

We synthesized a series of structurally related water-soluble alkyl phenols - sodium 4-hydroxyphenyl propyl sulfonates and thiosulfonates with different number of tert-butyl groups at the ortho-position. In experimental systems of transient metal-induced ethyl oleate and low-density lipoprotein oxidation the antioxidant activity of the compounds increased when the tert-butyl group number at the ortho-position increased and when the sulfonate group was replaced with thiosulfonate. Compounds containing thiosulfonate group in para-propyl substituent also more effectively inhibited reactive oxygen metabolites generated in xanthine-xanthine oxidase system and during morpholinosydnonimine decomposition compared to sulfonate-containing analogs. Phenols with one tert-butyl group at the ortho-position have been shown to exhibit the highest antiinflammatory activity in the model of carrageenan-induced rat paw inflammation, as well as with regard to the expression of the glutathione S-transferase P1-1 gene in HepG2 human hepatoma cell line. Thus, it can be reasonably speculated that the antiinflammatory activity of sulfur-containing phenolic antioxidants in vivo is mediated by their effect on redox-sensitive transcription factors.

Active defense under oxidative stress. The antioxidant responsive element.

This review considers the mechanisms and factors that stimulate transcription of genes regulated by the antioxidant responsive element (ARE). The latter is important for cell defense under conditions of oxidative stress and also for detoxification of electrophilic xenobiotics. There are differences in regulation of intracellular homeostasis involving Nrf2-mediated activation of ARE and other redox-sensitive factors (NF-kappaB and AP-1).