Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro

We tested the hypothesis that stress responses mediated by the Nrf2-antioxidant responsive element (ARE) pathway are involved in the initiation of retinal neuroprotection provided by bright-cyclic-light rearing. Albino rats born and raised in dim (5 lux) or bright (400 lux) cyclic light were exposed to damaging light (3000 lux, 6 h). After exposure, the outer nuclear layer thickness and area and the electroretinogram a- and b-wave amplitudes were significantly reduced in the dim-light-reared rats compared to the bright-light-reared rats, demonstrating a light adaptation neuroprotection phenomenon. In bright-cyclic-light-reared rats, the retinal levels of thioredoxin (Trx) (2.4-fold), Trx reductase (TrxR) (2.9-fold), and proteins modified by 4-hydroxynonenal (4-HNE) (1.5-fold) were upregulated by Western blot analyses, and the nuclear translocation of Nrf2 (2.2-fold) and the DNA binding activity of Nrf2, small Maf, and cJun to the ARE were increased as determined by electrophoretic mobility shift assays. In mouse photoreceptor-derived 661W cells, pretreatment with a sublethal dose of 4-HNE protected against H₂O₂-induced cell damage. Treatment with 4-HNE upregulated cellular Trx, TrxR, and heme oxygenase-1 (HO-1) levels in addition to DNA binding activity of Nrf2, small Maf, and cJun to the ARE. Downregulation of Nrf2 using RNA interference technology diminished 4-HNE-mediated upregulation of Trx and Trx reductase but did not affect the upregulation of HO-1 by 4-HNE. Cytoprotection by 4-HNE pretreatment against H₂O₂-induced cell damage was not observed in 661W cells with a silenced Nrf2 gene. The results suggest that upregulation of the Trx system by 4-HNE via the Nrf2–ARE pathway may be involved in the molecular mechanism of the retinal neuroprotection phenomenon.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway. [BMB Reports 2014; 47(2): 98-103]     

Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells

Polysulfide is a bound sulfur species derived from endogenous H₂S. When mouse neuroblastoma, Neuro2A cells were exposed to tert-butyl hydroperoxide after treatment with polysulfide, a significant decline in cell toxicity was observed. Rapid uptake of polysulfides induced translocation of Nrf2 into the nucleus, resulting in acceleration of GSH synthesis and HO-1 expression. We demonstrated that polysulfide reversibly modified Keap1 to form oxidized dimers and induced the translocation of Nrf2. Moreover, polysulfide treatment accelerated Akt phosphorylation, which is a known pathway of Nrf2 phosphorylation. Thus, polysulfide may mediate the activation of Nrf2 signaling, thereby exerting protective effects against oxidative damage in Neuro2A cells.     

Quality and lipid composition of spermatozoa in rabbits fed DHA and vitamin E rich diets

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200 mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. α- and δ-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. δ-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.     

Low-dose triptolide in combination with idarubicin induces apoptosis in AML leukemic stem-like KG1a cell line by modulation of the intrinsic and extrinsic factors

Leukemia stem cells (LSCs) are considered to be the main reason for relapse and are also regarded as a major hurdle for the success of acute myeloid leukemia chemotherapy. Thus, new drugs targeting LSCs are urgently needed. Triptolide (TPL) is cytotoxic to LSCs. Low dose of TPL enhances the cytotoxicity of idarubicin (IDA) in LSCs. In this study, the ability of TPL to induce apoptosis in leukemic stem cell (LSC)-like cells derived from acute myeloid leukemia cell line KG1a was investigated. LSC-like cells sorted from KG1a were subjected to cell cycle analysis and different treatments, and then followed by in vitro methyl thiazole tetrazolium bromide cytotoxicity assay. The effects of different drug combinations on cell viability, intracellular reactive-oxygen species (ROS) activity, colony-forming ability and apoptotic status were also examined. Combination index-isobologram analysis indicates a synergistic effect between TPL and IDA, which inhibits the colony-forming ability of LSC-like cells and induces their apoptosis. We further investigated the expression of Nrf2, HIF-1α and their downstream target genes. LSC-like cells treated with both TPL and IDA have increased levels of ROS, decreased expression of Nrf2 and HIF-1α pathways. Our findings indicate that the synergistic cytotoxicity of TPL and IDA in LSCs-like cells may attribute to both induction of ROS and inhibition of the Nrf2 and HIF-1α pathways.     

Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative–nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis.     

Ox-PAPC activation of PMET system increases expression of heme oxygenase-1 in human aortic endothelial cell

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) has been demonstrated to accumulate in atherosclerotic lesions and regulates expression of more than 1,000 genes in human aortic endothelial cell (HAEC). Among the most highly induced is heme oxygenase-1 (HO-1), a cell-protective antioxidant enzyme, which is sensitively induced by oxidative stress. To identify the pathway by which Ox-PAPC induces HO-1, we focused on the plasma membrane electron transport (PMET) complex, which contains ecto-NADH oxidase 1 (eNOX1) and NADPH:quinone oxidoreductase 1 (NQO1) and affects cellular redox status by regulating levels of NAD(P)H. We demonstrated that Ox-PAPC and its active components stimulated electron transfer through the PMET complex in HAECs from inside to outside [as determined by extracellular 2-(4-iodophenyl)-3-(44-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) reduction] and from outside to inside of the cell (as determined by intracellular NBT reduction). Chemical inhibitors of PMET system and siRNAs to PMET components (NQO1 and eNOX1) significantly decreased HO-1 induction by Ox-PAPC. We present evidence that Ox-PAPC activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in HAEC plays an important role in the induction of HO-1 and PMET inhibitors blocked Nrf2 activation by Ox-PAPC. We hypothesized that PMET activation by Ox-PAPC causes intracellular NAD(P)H depletion, which leads to the increased oxidative stress and HO-1 induction. Supporting this hypothesis, cotreatment of cells with exogenous NAD(P)H and Ox-PAPC significantly decreased oxidative stress and HO-1 induction by Ox-PAPC. Taken together, we demonstrated that the PMET system in HAEC plays an important role in the regulation of cellular redox status and HO-1 expression by Ox-PAPC.     

Synthesis, crystal structures, cytotoxicity and qualitative structure-activity relationship (QSAR) of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes, (n)Bu2Sn(L)2

A series of cis-bis{5-[(E)-2-(aryl)-1-diazenyl]quinolinolato}di-n-butyltin(IV) complexes has been synthesized and characterized by ¹H-, ¹³C-, ¹¹⁹Sn NMR, ESI-MS (electrospray ionization mass spectrometry), IR and ^119mSn Mössbauer spectroscopic techniques in combination with elemental analyses. The structures of four di-n-butyltin(IV) complexes, viz., ⁿBu₂Sn(L³)₂ (3), ⁿBu₂Sn(L⁴)₂ (4), ⁿBu₂Sn(L⁵)₂ (5) and ⁿBu₂Sn(L⁷)₂ · 0.5C₆H₆ (7) (LH = 5-[(E)-2-(aryl)-1-diazenyl)quinolin-8-ol) were determined by single crystal X-ray diffraction. In general, the complexes were found to adopt a distorted cis-octahedral arrangement around the tin atom. These complexes retain their solid-state structure in non-coordinating solvent as evidenced by ¹¹⁹Sn and ¹³C NMR spectroscopic results. The in vitro cytotoxicity of di-n-butyltin(IV) complexes (3-8) is reported against seven well characterized human tumour cell lines. The basicity of the two quinolinolato donor N and O atoms of the ligands are discussed in relation to the cytotoxicity data.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.