Glutathione biosynthesis via activation of the nuclear factor E2-related factor 2 (Nrf2)--antioxidant-response element (ARE) pathway is essential for neuroprotective effects of sulforaphane and 6-(methylsulfinyl) hexyl isothiocyanate

Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress-induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H(2)O(2)- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2-related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H(2)O(2) and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.     

Hepatic ischemia-reperfusion induces renal heme oxygenase-1 via NF-E2-related factor 2 in rats and mice

Hepatic ischemia-reperfusion (IR) results in Kupffer cell activation and subsequent tumor necrosis factor (TNF) alpha release, leading to localized hepatic injury and remote organ dysfunction. Heme oxygenase (HO)-1 is an enzyme that is induced by various stimuli, including proinflammatory cytokines, and exerts antioxidative and anti-inflammatory functions. Up-regulation of HO-1 is known to protect against hepatic IR injury, but the effects of hepatic IR on the kidney are poorly understood. Thus, the purpose of this study was to determine whether hepatic IR and resultant Kupffer cell activation alters renal HO-1 expression. Male Sprague-Dawley rats and wild-type and NF-E2-related factor 2 (Nrf2)-null mice were subjected to 60 min of partial hepatic ischemia, and at various times thereafter, blood, liver, and kidneys were collected. After reperfusion, 1) creatinine clearance decreased; 2) HO-1 mRNA and protein expression in liver and kidney markedly increased; 3) renal NAD(P)H: quinone oxidoreductase 1 mRNA expression was induced; 4) serum TNFalpha levels increased; 5) Nrf2 translocation into the nucleus of renal tissue increased; and 6) renal and urinary 15-deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) levels increased. Kupffer cell depletion by pretreating with gadolinium chloride 1) attenuated increased mRNA expression of HO-1 in kidney; 2) attenuated the increase in TNFalpha; 3) inhibited the increase in Nrf2 nuclear translocation; and 4) tended to attenuate renal 15-d-PGJ2 levels. Whereas renal HO-1 mRNA expression increased in wild-type mice, it was attenuated in Nrf2-null mice. These results suggest that renal HO-1 is induced via Nrf2 to protect the kidney from remote organ injury after hepatic IR.     

Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphane-treated human mesothelioma MSTO-211H cells

The nuclear factor erythroid-derived 2 related factor 2 (Nrf2)/heme oxygenase (HO)-1 induction plays cytoprotective roles against oxidative injury, apoptosis, and anticancer therapy; however, little is known about its regulation in human mesothelioma MSTO-211H cells. In this study, we investigated Nrf2/HO-1 induction in response to sulforaphane and determined the signaling pathways involved in this process. Sulforaphane treatment decreased cell viability and triggered a rapid and transient increase in the intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) prevented sulforaphane-induced cytotoxicity. Erk1/2 was activated within 1h of sulforaphane addition, whereas Akt phosphorylation was suppressed until the first 8h, and was then maintained at an elevated level until 72h, displaying a biphasic regulatory feature. Nrf2 protein levels in both nuclear and whole cell lysates were increased after sulforaphane treatment and were decreased by pretreatment with NAC, actinomycin D and cycloheximide. Activation of the Nrf2/HO-1 system after sulforaphane treatment was suppressed by pretreatment with NAC or Ly294002, a PI3K inhibitor. Knockdown of Nrf2 with siRNA decreased cell viability and attenuated sulforaphane-induced HO-1 up-regulation. Overall, our results indicate that ROS generation and/or activation of PI3K/Akt signaling regulate cell survival and Nrf2-driven HO-1 expression in sulforaphane-treated MSTO-211H cells.     

The role of Nrf2 signaling in the regulation of antioxidants and detoxifying enzymes after traumatic brain injury in rats and mice

Aim:

To determine whether Nrf2 signaling pathway activation could attenuate oxidative stress and neuronal damage following traumatic brain injury (TBI).

Methods:

Controlled cortical impact (CCI) injury was performed in Sprague-Dawley rats and Nrf2-knockout or control mice. Sulforaphane (SFN), a potent Nrf2 activator, was used to activate Nrf2. Oxidative stress, lesion volume, neuron degeneration, and neurologic dysfunction were determined using biochemical, histopathological and neuroethologic approaches. Protein and mRNA levels of Nrf2 and the antioxidant enzymes heme oxygenase 1 (HO-1) and NAD(P)H:quinine oxidoreductase 1 (NQO1) were assessed using Western blot analysis and RT-PCR.

Results:

Activation of Nrf2 by SFN( 5 mg/kg, ip) induced the nuclear translocation and activation of Nrf2, which resulted in an up-regulation of Nrf2-dependent antioxidant enzymes and a reduction of oxidative damage after TBI. In accordance with these biochemical changes, SFN also significantly reduced neuronal death, contusion volume, and neurological dysfunction after TBI. Furthermore, Nrf2-knockout mice showed more severe oxidative stress and neurologic deficits after TBI and did not benefit from the effects of SFN.

Conclusion:

Nrf2 plays a pivotal role in cell defenses against the oxidative stress of TBI. In addition, pharmacological activation of the Nrf2 signaling pathway by small molecule inducers such as SFN attenuated oxidative stress and neuronal damage following TBI.     

Study of inducibility and mechanism of phase 2 enzymes by quercetin and rutin

Objective The increasing recognition of the role for oxidative stress in hepatic disorders has led to extensive investigation on the protection by exogenous antioxidants against hepatic injury.In this study,we choose two typical polyphenol,quercetin and rutin,to investigate the mechanism of induction of cellular antioxidants and phase 2 enzymes in human HepG2 cells.Methods The HepG2 cells were treated with various concentrations of quercetin and rutin for 6 h and 24 h.The activities of NAD(P)H:quinone oxidoreductase(NQO1)in HepG2 cells were measured by 2,6-dichloroindophenol reduction method.The content of superoxide dismutase(SOD)was determined with the method of chemical colorimetry.The protein expressions of NQO1 and NF-E2-related factor 2(Nrf2)in HepG2 cells were detected by Western blotting.Results Incubation of HepG2 cells with quercetin and rutin resulted in a marked concentration-and time-dependent induction of a number of cellular antioxidants and phase 2 enzymes,including NQO1,SOD.Quercetin and rutin treatment of HepG2 cells also caused increase in protein expressions of NQO1 and Nrf2.Conclusions This study demonstrates that a series of phase 2 enzymes in HepG2 cells can be induced by quercetin and rutin in a concentration-and time-dependent fashion by upregulation the protein expression of nrf2.     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.     

Activated iRhom2 drives prolonged PM2.5 exposure-triggered renal injury in Nrf2-defective mice

Abstract

Research suggests that particulate matter (PM_2.5) is a predisposing factor for metabolic syndrome-related systemic inflammation and oxidative stress injury. TNF-α as a major pro-inflammatory cytokine was confirmed to participate in various diseases. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for shedding of TNF-α in immune cells. Importantly, kidney-resident macrophages are critical to inflammation-associated chronic renal injury. Podocyte injury can be induced by stimulants and give rise to nephritis, but how iRhom2 contributes to PM_2.5-induced renal injury is unclear. Thus, we studied whether PM_2.5 causes renal injury and characterized iRhom2 with respect to TNF-α release in mice macrophages and renal tissues in long-term PM_2.5-exposed mouse models. After long-term PM_2.5 exposures, renal injury was confirmed via inflammatory cytokine, chemokine expression, and reduced antioxidant activity. Patients with kidney-related diseases had increased TNF-α, which may contribute to renal injury. We observed up-regulation of serum creatinine, serum urea nitrogen, kidney injury molecule 1, uric acid, TNF-α, MDA, H₂O_2, and O₂^– in PM_2.5-treated mice, which was greater than that found in Nrf2^?/? mice. Meanwhile, increases in metabolic disorder-associated indicators were involved in PM_2.5-induced nephritis. In vitro, kidney-resident macrophages were observed to be critical to renal inflammatory infiltration and function loss via regulation of iRhom2/TACE/TNF-α signaling, and suppression of Nrf2-associated anti-oxidant response. PM_2.5 exposure led to renal injury partly by inflammation-mediated podocyte injury. Reduced SOD1, SOD2, Nrf2 activation, and increased XO, NF-κB activity, TACE, iNOS, IL-1β, TNF-α, IL-6, MIP-1α, Emr-1, MCP-1, and Cxcr4, were also noted. Long-term PM_2.5 exposure causes chronic renal injury by up-regulation of iRhom2/TACE/TNF-α axis in kidney-resident macrophages. Overexpression of TNF-α derived from macrophages causes podocyte injury and kidney function loss. Thus, PM_2.5 toxicities are related to exposure duration and iRhom2 may be a potential therapeutic renal target.     

The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 μM) esculetin for 8 h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 μM esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.