How a calculated oxidative stress can yield multiple therapeutic effects

It is proposed to discuss how ozonetherapy acts on patients affected by vascular and degenerative diseases. Ozone is a strong oxidant but, if used in small dosages on human blood ex vivo, acts as an acceptable stressor. By instantly reacting with PUFA bound to albumin, ozone is entirely consumed but generates two messengers acting in an early and in a late phase: the former is due to hydrogen peroxide, which triggers biochemical pathways on blood cells and the latter is due to alkenals which are infused into the donor patient. After undergoing a partial catabolism, alkenals enter into a great number of body's cells, where they react with Nrf2-Keap1 protein: the transfer of activated Nrf2 into the nucleus and its binding to antioxidant response element (ARE) is the crucial event able to upregulate the synthesis of antioxidant proteins, phase II enzymes and HO-1. With the progress of ozonetherapy, these protective enzymes are able to reverse the oxidative stress induced by chronic inflammation. Consequently, the repetition of graduated stresses induces a multiform adaptive response able to block the progress of the disease and to improve the quality of life.     

The cardioprotective effect of rosmarinic acid on acute myocardial infarction and genes involved in Ca2+ homeostasis

Acute myocardial infarction (AMI) is a common cause of hospitalisation and high mortality due to lethal arrhythmias. Sarcoplasmic reticulum Ca²⁺ ATPase (SERCA₂) and ryanodine receptor (RyR₂) regulate the cytosolic Ca²⁺ ion concentration. Rosmarinic acid (RA) is one of the most common caffeic esters in Rosmarinus officinalis. The present study was conducted to test the hypothesis whether RA can protect cardiac function against AMI and arrhythmias induced by isoproterenol through the regulatory effect of SERCA₂ and RyR₂ gene expression. To this aim, male Sprague–Dawley rats were allocated into in vivo and ex vivo studies and received RA (10, 15, and 30?mg/kg; 14 days). AMI was induced by two consecutive subcutaneous injections of 100?mg/kg isoproterenol. Blood pressure (BP), heart rate, electrocardiography (ECG) parameters, plasma levels of cardiac biomarkers, and antioxidative enzymes were evaluated (in vivo study). Cardiac functions were measured in isolated hearts using Langendorff set up. Gene expressions of SERCA₂ and RyR₂ were measured in left ventricular heart. Isoproterenol administration showed a significant decline in BP, QRS voltage, activities of antioxidant enzymes, cardiac function, and gene expressions of SERCA₂ and RyR₂. The results also indicated a significant increase in heart rate, ST-elevation, cardiac biomarkers, and antioxidant enzymes. RA at 30?mg/kg dosage showed the best effect on the improvement of the mentioned factors. This study suggests that RA has potent cardioprotective effects against AMI and arrhythmia, which may be due to its ability to enhance expression of plasma antioxidant enzymes and genes involved in Ca²⁺ homeostasis SERCA₂ and RyR₂. The protective role of RA is also possibly related to its antiadrenergic effects.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

15-Deoxy-Δ12,14-prostaglandin J2 prevents oxidative injury by upregulating the expression of aldose reductase in vascular smooth muscle cells

Abstract

The omega-6 fatty acid derivative 15-Deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is believed to play a role in cellular protection against oxidative stress in diverse cell systems. However, the cellular mechanisms by which protection is afforded by 15d-PGJ₂ are not fully elucidated in vascular smooth muscle cells (VSMCs). In this study, we report the finding that 15d-PGJ₂ elicited a time and concentration- dependent increase in aldose reductase (AR) expression. This induction was independent of the activation of peroxisome proliferator- activated receptor γ. Inhibition of phosphatidylinositol 3-kinase (PI3K) significantly suppressed the increase in expression and promoter activity of AR induced by 15d-PGJ₂. Luciferase reporter assays demonstrated that 15d-PGJ₂ targets the multiple stress response regions comprising the antioxidant response element in the promoter of the AR gene. 15d-PGJ₂-mediated induction of AR promoter activity was potentiated in the presence of nuclear factor-erythroid 2-related factor 2 (Nrf2), but not in cells expressing dominant negative Nrf2. Cells treated with 15d-PGJ₂ were resistant to oxidant-induced apoptotic cell death by inhibiting production of reactive oxygen species. These effects were significantly attenuated in the presence of an AR inhibitor or small interfering RNA against AR, indicating that AR plays a protective role against oxidative injury. Taken together, these findings demonstrate that activation of PI3K by 15d-PGJ₂ increases the expression of AR through Nrf2, and increased AR activity may function as an important cellular response against oxidative injury.     

Nrf2 regulates the alternative first exons of CD36 in macrophages through specific antioxidant response elements

We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARγ specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.     

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes

Objective: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.

Methods: Human keratinocytes were pretreated with Pter (5 and 10?μM) for 24?h prior to UVB irradiation (300?mJ/cm²). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.

Results: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.

Conclusion: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.     

基于模式生物斑马鱼Nrf2的相关研究进展

Nrf2/ARE信号通路是大多数生物体内抗氧化应激反应、抵抗内外界刺激的关键通路,在抗炎症、免疫、抗肿瘤、抗凋亡、神经保护等方面起着重要的作用。斑马鱼作为一种常见的模式动物,广泛地应用于发育生物学、遗传学和毒理学等研究领域。研究表明转录因子NF_E2相关因子2(NF-E2-related factor 2,Nrf2)不仅在哺乳动物体内存在,也在斑马鱼体内存在并且高度保守,并在抗氧化应激反应中发挥着重要作用。本文通过对斑马鱼Nrf2的结构、生物学功能及其信号通路等方面的最新研究进行阐述,以期为Nrf2及其信号通路引发的相关疾病的预防和治疗提供新的思路。     

Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

Zeaxanthin (Zea) is a major carotenoid pigment contained in human retina, and its daily supplementation associated with lower risk of age-related macular degeneration. Despite known property of Zea as an antioxidant, its underlying molecular mechanisms of action remain poorly understood. In this study, we aim to study the regulation mechanism of Zea on phase II detoxification enzymes. In normal human retinal pigment epithelium cells, Zea promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and induced mRNA and protein expression of phase II enzymes, the induction was suppressed by specific knockdown of Nrf2. Zea also effectively protected against tert-butyl hydroperoxide-induced mitochondrial dysfunction and apoptosis. Glutathione (GSH) as the most important antioxidant was also induced by Zea through Nrf2 activation in a time- and dose-dependent manner, whereas the protective effects of Zea were decimated by inhibition of GSH synthesis. Finally, Zea activated the PI3K/Akt and MAPK/ERK pathway, whereas only PI3K/Akt activation correlated with phase II enzymes induction and Zea protection. In further in vivo analyses, Zea showed effects of inducing phase II enzymes and increased GSH content, which contributed to the reduced lipid and protein peroxidation in the retina as well as the liver, heart, and serum of the Sprague–Dawley rats. For the first time, Zea is presented as a phase II enzymes inducer instead of being an antioxidant. By activating Nrf2-mediated phase II enzymes, Zea could enhance anti-oxidative capacity and prevent cell death both in vivo and in vitro.     

Extract of Ginkgo biloba induces phase 2 genes through Keap1-Nrf2-ARE signaling pathway

The standard extract of Ginkgo biloba (EGb) has been demonstrated to possess remarkable antioxidant activity in both cell lines and animals. However, the molecular mechanism underlying this effect is not fully understood. Phase 2 enzymes play important roles in the antioxidant system by reducing electrophiles and reactive oxygen species (ROS). We demonstrated that EGb induced typical phase 2 genes: glutamate cysteine ligase catalytic subunit (GCLC) and glutathione-S-transferase subunit-P1 (GST-P1), by real-time PCR. To investigate the molecular mechanism of this induction, we used quinone oxidoreductase 1 (NQO1) -- Antioxidant response element (ARE) reporter assay and found that EGb activated the activity of the wild type but not the one with ARE mutated. It indicated that EGb induced these genes through ARE, a cis-acting motif located in the promoter region of nearly all phase 2 genes. Since nuclear factor erythroid 2-related factor 2 (Nrf2) binds ARE to enhance the expression of phase 2 genes, we detected the Nrf2 content in nucleus and found an accumulation of Nrf2 stimulated by EGb. In a further test of Kelch-like ECH-associated protein 1 (Keap1), the repression protein of Nrf2 in the cytosol under resting condition, we found that Keap1 content was inhibited by EGb and then more Nrf2 would be released to translocate into nucleus. Thus, EGb was testified for the first time to induce the phase 2 genes through the Keap1-Nrf2-ARE signaling pathway, which is (or part of) the antioxidant mechanism of EGb.     

Abscisic acid-induced apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H₂O₂ production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl₃ staining, respectively, and the relationship between ABA-induced H₂O₂ production and ABA-induced subcellular activities of antioxidant enzymes was studied. H₂O₂ generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2–4 h. In mesophyll and bundle sheath cells, ABA-induced H₂O₂ accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O ₂ ⁻ scavenger Tiron and the H₂O₂ scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H₂O₂ is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O ₂ ⁻ and then H₂O₂ in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H₂O₂ produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.     

Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression

Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product.     

Nrf2及其药理性活化剂在糖尿病心血管并发症中的作用

糖尿病(DM)导致的心脑血管并发症是危害人类健康的重大疾病。氧化应激被认为是DM相关心血管并发症发生、发展的重要机制,但通过补充外源性抗氧化剂并未能使心血管疾病患者远期获益。核因子E2相关因子2(Nrf2)可增加内源性抗氧化酶的活性从而提高机体的抗氧化应激能力,可能是治疗糖尿病心血管并发症的一个重要靶点,提示靶向Nrf2药物的开发可能获得防治糖尿病相关血管并发症的新一代药物。本文就Nrf2在糖尿病相关心血管并发症发生、发展中的作用及其药理性活化剂对糖尿病(DM)相关心血管病变的治疗作用进行综述。     

Influence of cis-[Re2GABA2Cl4]Cl2 on the antioxidant defense system parameters of normal human blood

The effect of the rhenium complex cis-[Re₂GABA₂Cl₄]Cl₂ on the antioxidant parameters of normal human blood in vitro have been studied. The results suggest that the complex influences various enzymes in the cascade of reactions utilizing active oxygen metabolites. However, the manifestation of this activity varies over the studied concentration range of the complex in the preincubation medium (10^–12-10^–4 M), so the effects appear to be concentration-dependent. The largest differences in antioxidant parameters in comparison with control were observed for the concentrations 10^–8, 10^–5, and 10^–4 M. Thus, correlations between the peroxidation level, superoxide dismutase (SOD) activity, antioxidant factor (F), and indexes of resistance of erythrocytes for hemolysis (TR) were found.