Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30 min of ischemia and 120 min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6 ± 2.4 vs. 39.2 ± 2.1%, p < 0.05), but this protection was abolished in DN-Trx1-PostC group (49.7 ± 1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9 ± 2.1 and 38.4 ± 1.3%, p < 0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3β phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3β survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.     

Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine,as pharmacological post-conditioning agent

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 μmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.     

Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated Human keratinocytes

UVA irradiation-induced skin damage and redox imbalance have been shown to be ameliorated by ergothioneine (EGT), a naturally occurring sulfur-containing amino acid. However, the responsible molecular mechanism with nanomolar concentrations of EGT remains unclear. We investigated the dermato protective efficacies of EGT (125–500 nM) against UVA irradiation (15 J/cm²), and elucidated the underlying molecular mechanism in human keratinocyte-derived HaCaT cells. We found that EGT treatment prior to UVA exposure significantly increased the cell viability and prevented lactate dehydrogenase release into the medium. UVA-induced ROS and comet-like DNA formation were remarkably suppressed by EGT with a parallel inhibition of apoptosis, as evidenced by reduced DNA fragmentation (TUNEL), caspase-9/-3 activation, and Bcl-2/Bax dysregulation. Furthermore, EGT alleviated UVA-induced mitochondrial dysfunction. Dose-dependent increases of antioxidant genes, HO-1, NQO-1, and γ-GCLC and glutathione by EGT were associated with upregulated Nrf2 and downregulated Keap-1 expressions. This was confirmed by increased nuclear accumulation of Nrf2 and inhibition of Nrf2 degradation. Notably, augmented luciferase activity of ARE may explain Nrf2/ARE-mediated signaling pathways behind EGT dermato-protective properties. We further demonstrated that Nrf2 translocation was mediated by PI3K/AKT, PKC, or ROS signaling cascades. This phenomenon was confirmed with suppressed nuclear Nrf2 activation, and consequently diminished antioxidant genes in cells treated with respective pharmacological inhibitors (LY294002, GF109203X, and N-acetylcysteine). Besides, increased basal ROS by EGT appears to be crucial for triggering the Nrf2/ARE signaling pathways. Silencing of Nrf2 or OCTN1 (EGT carrier protein) signaling with siRNA showed no such protective effects of EGT against UVA-induced cell death, ROS, and apoptosis, which is evidence of the vitality of Nrf2 translocation and protective efficacy of EGT in keratinocytes. Our findings conclude that EGT at nanomolar concentrations effectively ameliorated UVA-induced skin damage, and may be considered as a desirable food supplement for skin protection and/or preparation of skin care products.     

Melatonin stimulates antioxidant enzymes and reduces oxidative stress in experimental traumatic brain injury: the Nrf2–ARE signaling pathway as a potential mechanism

The goal of this study was to evaluate the potential involvement of melatonin in the activation of the nuclear factor erythroid 2-related factor 2 and antioxidant-responsive element (Nrf2–ARE) signaling pathway and the modulation of antioxidant enzyme activity in an experimental model of traumatic brain injury (TBI). In experiment 1, ICR mice were divided into four groups: sham group, TBI group, TBI + vehicle group, and TBI + melatonin group (n = 38 per group). Melatonin (10 mg/kg) was administered via an intraperitoneal (ip) injection at 0, 1, 2, 3, and 4 h post-TBI. In experiment 2, Nrf2 wild-type (Nrf2^+/+ group) and Nrf2-knockout (Nrf2^−/− group) mice received a TBI insult followed by melatonin administration (10 mg/kg, ip) at the corresponding time points (n = 35 per group). The administration of melatonin after TBI significantly ameliorated the effects of the brain injury, such as oxidative stress, brain edema, and cortical neuronal degeneration. Melatonin markedly promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus; increased the expression of Nrf2–ARE pathway-related downstream factors, including heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1; and prevented the decline of antioxidant enzyme activities, including superoxide dismutase and glutathione peroxidase. Furthermore, knockout of Nrf2 partly reversed the neuroprotection of melatonin after TBI. In conclusion, melatonin administration may increase the activity of antioxidant enzymes and attenuate brain injury in a TBI model, potentially via mediation of the Nrf2–ARE pathway.     

Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

CpG-ODN,the TLR9 agonist,attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling

BackgroundToll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.MethodsMale C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1 h prior to myocardial ischemia (60 min) followed by reperfusion. Untreated mice served as I/R control (n = 10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14 days.ResultsCpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14 days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.ConclusionCpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (α, β₁, β₂, γ, δ and ɛ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and ɛ isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while α isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by α, β₁, γ, δ and ɛ were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC α isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and ɛ isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and ɛ isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.     

PKC mediates fluctuant ERK-paxillin signaling for hepatocyte growth factor-induced migration of hepatoma cell HepG2

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24 h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24 h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.     

Antioxidative effects of Panax notoginseng saponins in brain cells

Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death associated with neurological disorders such as stroke, Alzheimer's disease and traumatic brain injury. Antioxidant compounds that improve endogenous antioxidant defenses have been proposed for neural protection. The purpose of this study was to investigate the potential protective effects of total saponin in leaves of Panax notoginseng (LPNS) on oxidative stress and cell death in brain cells in vitro. Lactate dehydrogenase (LDH) assay indicated that LPNS (5 μg/ml) reduced H₂O₂-induced cell death in primary rat cortical astrocytes (23 ± 8% reduction in LDH release vs. control). Similar protection was found in oxygen and glucose deprivation/reoxygenation induced SH-SY5Y (a human neuroblastoma cell line) cell damage (78 ± 7% reduction vs. control). The protective effects of LPNS in astrocytes were associated with attenuation of reactive oxygen species (ROS) accumulation. These effects involved activation of Nrf2 (nuclear translocation) and upregulation of downstream antioxidant systems including heme oxygenase-1 (HO-1) and glutathione S-transferase pi 1 (GSTP1). These results demonstrate for the first time that LPNS has antioxidative effects which may be neuroprotective in neurological disorders.     

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart

The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups (n = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.     

RISK pathway is involved in oxytocin postconditioning in isolated rat heart

The reperfusion injury salvage kinase (RISK) pathway is a fundamental signal transduction cascade in the cardioprotective mechanism of ischemic postconditioning. In the present study, we examined the cardioprotective role of oxytocin as a postconditioning agent via activation of the RISK pathway (PI3K/Akt and ERK1/2).Animals were randomly divided into 6 groups. The hearts were subjected under 30 minutes (min) ischemia and 100 min reperfusion. OT was perfused 15 min at the early phase of reperfusion. RISK pathway inhibitors (Wortmannin; an Akt inhibitor, PD98059; an ERK1/2 inhibitor) and Atosiban (an OT receptor antagonist) were applied either alone 10 min before the onset of the ischemia or in the combination with OT during early reperfusion phase. Myocardial infarct size, hemodynamic factors, ventricular arrhythmia, coronary flow and cardiac biochemical marker were measured at the end of reperfusion.OT postconditioning (OTpost), significantly decreased the infarct size, arrhythmia score, incidence of ventricular fibrillation, Lactate dehydrogenase and it increased coronary flow. The cardioprotective effect of OTpos was abrogated by PI3K/Akt, ERK1/2 inhibitors and Atosiban.Our data have shown that OTpost can activate RISK pathway mostly via the PI3K/Akt and ERK1/2 signaling cascades during the early phase of reperfusion.     

Carnosine ameliorates cognitive deficits in streptozotocin-induced diabetic rats: Possible involved mechanisms

Diabetic patients are at increased risk to develop cognitive deficit and senile dementia. This study was planned to assess the benefits of chronic carnosine administration on prevention of learning and memory deterioration in streptozotocin (STZ)-diabetic rats and to explore some of the involved mechanisms. Rats were divided into 5 groups: i.e., control, carnosine100-treated control, diabetic, and carnosine-treated diabetics (50 and 100 mg/kg). Carnosine was injected i.p. at doses of 50 or 100 mg/kg for 7 weeks, started 1 week after induction of diabetes using streptozotocin. Treatment of diabetic rats with carnosine at a dose of 100 mg/kg at the end of the study lowered serum glucose, improved spatial recognition memory in Y maze, improved retention and recall in elevated plus maze, and prevented reduction of step-through latency in passive avoidance task. Furthermore, carnosine at a dose of 100 mg/kg reduced hippocampal acetylcholinesterase (AChE) activity, lowered lipid peroxidation, and improved superoxide dismutase (SOD) activity and non-enzymatic antioxidant defense element glutathione (GSH), but not activity of catalase. Meanwhile, hippocampal level of nuclear factor-kappaB (NF-κB), tumor necrosis factor α (TNF-α), and glial fibrillary acidic protein (GFAP) decreased and level of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) increased upon treatment of diabetic group with carnosine at a dose of 100 mg/kg. Taken together, chronic carnosine treatment could ameliorate learning and memory disturbances in STZ-diabetic rats through intonation of NF-κB/Nrf2/HO-1 signaling cascade, attenuation of astrogliosis, possible improvement of cholinergic function, and amelioration of oxidative stress and neuroinflammation.     

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt,AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr³⁺) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia + NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia + NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia + NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia + NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Bama miniature pigs’ liver possess great heat tolerance through upregulation of Nrf2-mediated antioxidative enzymes

The liver is one of the most crucial organs affected by high ambient temperature. Bama miniature pig show a heat tolerance in hot summer months. However, the physiological condition of liver under high ambient temperature has not been well elucidated in Bama miniature pig. Here we performed an experiment to investigate the effects of high ambient temperature on liver function, redox status and Nrf2 antioxidant pathway in Bama miniature pigs. Twelve pigs were randomly divided into two groups and separately exposed to the neutral temperature (NT, 25 °C) and high temperature (HT, 40 °C) for 8 days. The hepatic damage marker, such as total bilirubin (TBIL), alkaline phosphatase (AKP), γ-glutamyl transpeptidas (γ-GT), alanine transaminase (ALT) and aspartase transminase (AST), didn’t reach statistical significance between NT and HT group. Moreover, abnormal observation of hepatic histology and hepatocyte ultrastructure were not detected in HT group. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) as well as glutathione (GSH) content, were dramatically increased after heat exposure. Heat treatment didn’t increase hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations. The expression of Nrf2-regulated genes, such as nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H: quinine oxidoreductase 1 (NQO1), superoxide dismutase 1 (SOD1), heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC), were significantly increased in HT group. Nrf2 protein was accumulated in HT group through immunohistochemical analysis. The current data provide clear evidence that Bama miniature pigs’ liver possess great capacity of heat tolerance, which related to activation of Nrf2 antioxidant pathway.     

Minocycline inhibits 5-lipoxygenase expression and accelerates functional recovery in chronic phase of focal cerebral ischemia in rats

AimsWe previously reported that minocycline attenuates acute brain injury and inflammation after focal cerebral ischemia, and this is partly mediated by inhibition of 5-lipoxygenase (5-LOX) expression. Here, we determined the protective effect of minocycline on chronic ischemic brain injury and its relation with the inhibition of 5-LOX expression after focal cerebral ischemia.Main methodsFocal cerebral ischemia was induced by 90 min of middle cerebral artery occlusion followed by reperfusion for 36 days. Minocycline (45 mg/kg) was administered intraperitoneally 2 h and 12 h after ischemia and then every 12 h for 5 days. Sensorimotor function was evaluated 1–28 days after ischemia and cognitive function was determined 30–35 days after ischemia. Thereafter, infarct volume, neuron density, astrogliosis, and 5-LOX expression in the brain were determined.Key findingsMinocycline accelerated the recovery of sensorimotor and cognitive functions, attenuated the loss of neuron density, and inhibited astrogliosis in the boundary zone around the ischemic core, but did not affect infarct volume. Minocycline significantly inhibited the increased 5-LOX expression in the proliferated astrocytes in the boundary zone, and in the macrophages/microglia in the ischemic core.SignificanceMinocycline accelerates functional recovery in the chronic phase of focal cerebral ischemia, which may be partly associated with the reduction of 5-LOX expression.     

A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity

Dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72 h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200 μg/L) or 30 mM N-acetylcysteine (NAC)+200 μg/L PCB126. After 72 h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200 μg/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72 h exposure, 200 μg/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72 h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72 h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression.     

Participation of protein kinase C in the activation of Nrf2 signaling by ischemic preconditioning in the isolated rabbit heart

Activation of protein kinase C (PKC) is a critical intracellular signaling triggered by ischemic preconditioning (IPC), but the precise mechanisms underlying the actions of PKC in IPC-mediated cardioprotection remain unclear. Here, we investigated the role of PKC activation on the antioxidant activity by IPC in rabbit hearts. Isolated rabbit hearts were subjected to 60?min of global ischemia by cold cardioplegic arrest (4?°C) and 60?min of reperfusion (37?°C). IPC was induced by three cycles of 2-min ischemia following 3?min of reperfusion (37?°C) before cardioplegic arrest. IPC resulted in a better recovery of mechanical function, increased tissue reduced glutathione-to-oxidized glutathione ratio (GSH/GSSG), superoxide dismutase and catalase content, and decreased tissue malondialdehyde (MDA) content compared to control hearts subjected to 60?min of cardioplegic ischemia and 60?min of reperfusion. IPC also significantly induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the inductions of antioxidant genes heme oxygenase-1 (HO-1) and manganese superoxide dismutase (MnSOD). Injection of phorbol 12-myristate 13 acetate, an activator of PKC, before cardioplegic ischemia induced translocation of PKC-?? and -?? isoforms to membrane fraction, nuclear accumulation of Nrf2, and conferred cardioprotection similar to IPC. Polymyxin B, an inhibitor of PKC, blocked the membrane translocation of PKC-?? and -?? during IPC, inhibited Nrf2 nuclear accumulation, and significantly diminished the IPC-induced cardioprotection when administrated before IPC. These results indicate that the activation of PKC induces the translocation of Nrf2 and the enhancement of endogenous antioxidant defenses in the IPC hearts and suggest that PKC may target Nrf2 to confer cardioprotection.