Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H₂O₂-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H₂O₂-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.     

Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress

Antioxidants are likely potential pharmaceutical agents for the treatment of alcoholic liver disease. Metallothionein (MT) is a cysteine-rich protein and functions as an antioxidant. This study was designed to determine whether MT confers resistance to acute alcohol-induced hepatotoxicity and to explore the mechanistic link between oxidative stress and alcoholic liver injury. MT-overexpressing transgenic and wild-type mice were administrated three gastric doses of alcohol at 5 g/kg. Liver injury, oxidative stress, and ethanol metabolism-associated changes were determined. Acute ethanol administration in the wild-type mice caused prominent microvesicular steatosis, along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include glycogen and fat accumulation, organelle abnormality, and focal cytoplasmic degeneration. This acute alcohol hepatotoxicity was significantly inhibited in the MT-transgenic mice. Furthermore, ethanol treatment decreased hepatic-reduced glutathione, but increased oxidized glutathione along with lipid peroxidation, protein oxidation, and superoxide generation in the wild-type mice. This hepatic oxidative stress was significantly suppressed in the MT-transgenic mice. However, MT did not affect the ethanol metabolism-associated decrease in NAD(+)/NADH ratio or increase in cytochrome P450 2E1. In conclusion, MT is an effective agent in cytoprotection against alcohol-induced liver injury, and hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.     

Quercetin metabolites and protection against peroxynitrite-induced oxidative hepatic injury in rats

Quercetin has strong antioxidant potency. Quercetin-3′-O-sulphate (Q3′S) and quercetin-3-O-glucuronide (Q3GA) are the main circulating metabolites after consumption of quercetin-O-glucoside-rich diets by humans. However, information about how these quercetin metabolites function in vivo is limited. Hence, this study evaluated the efficacy of Q3′S and Q3GA for the protection of oxidative injury using in vitro and in vivo experiments. Peroxynitrite-mediated hepatic injury in rats was induced by administration of galactosamine/lipopolysaccharide (GalN/LPS). Twenty-four hours after GalN/LPS treatment, plasma ALT and AST levels δ increased significantly. However, pretreatment with 4^G-α-D-glucopyranosyl rutin, a quercetin glycoside (30 mg/kg body weight), prevented these increases and reduced nitrotyrosine formation, indicating that consumption of quercetin glycosides prevent oxidative hepatotoxicity. Moreover, physiological levels of Q3′S and Q3GA (1 µM) effectively prevented peroxynitrite-induced nitrotyrosine formation in human serum albumin in in vitro experiments. These findings indicate peroxynitrite-induced oxidative hepatotoxicity is protected by the in vivo metabolites of quercetin, Q3′S and Q3GA.     

Postanoxic oxidative injury in rat hepatocytes: lactate-dependent protection against tert-butylhydroperoxide.

Previous studies in this laboratory showed that hypoxia and anoxia enhance the susceptibility of hepatocytes to tert-butylhydroperoxide (TBH)-induced oxidative injury. To determine whether preceding exposure to anoxia affects postanoxic sensitivity to oxidative injury, viability was studied in hepatocytes incubated under anoxic conditions followed by reoxygenation without or with tert-butylhydroperoxide addition. Results showed that a preceding exposure to 60 min of anoxia substantially increased the vulnerability of cells to injury by the oxidant. Because substantial tissue lactate can accumulate during anoxia, the effect of increased lactate on postanoxic injury due to TBH was determined. Results showed that added lactate protected in a concentration-dependent manner. The TBH elimination rate was stimulated by lactate, and the pyruvate production rate approached the rate of TBH elimination. Thus, lactate protects against postanoxic oxidative injury by supplying reducing equivalents for peroxide reduction. This suggests that lactate accumulation during ischemia may be beneficial and that supplementation with lactate could be considered as a means to protect against postischemic injury.     

Upregulation of transcription factor NRF2-mediated oxidative stress response pathway in rat brain under short-term chronic hypobaric hypoxia

Exposure to high altitude (and thus hypobaric hypoxia) induces electrophysiological, metabolic, and morphological modifications in the brain leading to several neurological clinical syndromes. Despite the known fact that hypoxia episodes in brain are a common factor for many neuropathologies, limited information is available on the underlying cellular and molecular mechanisms. In this study, we investigated the temporal effect of short-term (0–12 h) chronic hypobaric hypoxia on global gene expression of rat brain followed by detailed canonical pathway analysis and regulatory network identification. Our analysis revealed significant alteration of 33, 17, 53, 81, and 296 genes (p < 0.05, <1.5-fold) after 0.5, 1, 3, 6, and 12 h of hypoxia, respectively. Biological processes like regulation, metabolic, and transport pathways are temporally activated along with anti- and proinflammatory signaling networks like PI3K/AKT, NF-κB, ERK/MAPK, IL-6 and IL-8 signaling. Irrespective of exposure durations, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress response pathway and genes were detected at all time points suggesting activation of NRF2-ARE antioxidant defense system. The results were further validated by assessing the expression levels of selected genes in temporal as well as brain regions with quantitative RT-PCR and western blot. In conclusion, our whole brain approach with temporal monitoring of gene expression patterns during hypobaric hypoxia has resulted in (1) deciphering sequence of pathways and signaling networks activated during onset of hypoxia, and (2) elucidation of NRF2-orchestrated antioxidant response as a major intrinsic defense mechanism. The results of this study will aid in better understanding and management of hypoxia-induced brain pathologies.     

Gene expression profiling of the synergy of 5-aza-2'-deoxycytidine and paclitaxel against renal cell carcinoma

ABSTRACT: We previously demonstrated that 5-Aza-2'-deoxycytidine (DAC) could significantly increase the susceptibility of renal cell carcinoma (RCC) cells to Paclitaxel (PTX) treatment in vitro, and showed the synergy of DAC and PTX against RCC. However, the gene expression profiling and the pathways involved in the synergy of these two agents remain unknown. In this study, we performed cDNA microarray, which was coupled with real-time PCR, to identify critical genes in the synergistic mechanism of the both agents against RCC cells. Various patterns of gene expression were observed by cluster analysis, and results indicated that lymphoid enhancer-binding factor 1 (LEF1), transforming growth factor beta-induced (TGFBI), C-X-C motif ligand 5 (CXCL5) and myelocytomatosis viral related oncogene (c-myc) may play a pivotal role in the synergy of DAC and PTX. In addition, network analysis using IPA software, suggested that the PI3K/Akt pathway and some other pathways associated with cyclins, DNA replication and cell cycle/mitotic regulation were also associated with the synergy of DAC and PTX against RCC.     

Mg-Gluconate provides superior protection against postischemic dysfunction and oxidative injury compared to Mg-sulfate

Cardioprotection by Mg Sulfate (MgSO4) during ischemia/reperfusion (I/R) is attributed largely to the Mg2+ cation. However, Mg-gluconate (MgGl2) may provide added benefit, possibly through its anion's antioxidant properties. Protective effects of both Mg-salts and their anions during 30 min global I and 50 min R were assessed in Langendorff-perfused (Krebs-Henseleit buffer) rat hearts. Recovery of function was compared between untreated hearts and those receiving supplement (2.4 mM MgGl2, MgSO4, or Na2SO4, or 4.8 mM NaGI) for 5 min prior to I and during the initial 30 min R. The final 20 min R was conducted without supplement. End diastolic pressure (EDP, mmHg) of the 50 min reperfused MgGl2 group (2.6) was lower than MgSO4 (16.2) and untreated (35.6) groups, and the NaGI group (25.2) was considerably lower than Na2SO4 (38.8). Recovery of developed pressure (% preischemic DP) at the onset of R for MgGl2 (74.9) was greater than MgSO4 (37.9) and untreated (33.2). After 50 min, MgGl2 (77.9) and MgSO4 (66.9) provided protection compared to untreated (51.8). In separate studies, ESR spin trapping with alpha-phenyl-N-tert-butylnitrone (3 mM PBN) showed that I/R alkoxyl radical production was reduced with MgGl2 (0.0 vs. 2.4 vs. 3.6 mM: 184 vs. 97 vs. 54.8 nM/g tissue x min) to a greater extent than seen with MgSO4 (3.6 mM: 108). Additional studies suggest that Gl(1-), unlike SO4(2-), may scavenge hydroxyl radicals, accounting for the added protection. MgGl2 treated hearts exhibited less postischemic dysfunction and oxidative injury compared to MgSO4, suggesting the contribution of Gl(1-) to cardioprotection.     

Complementary gene regulation by NRF1 and NRF2 protects against hepatic cholesterol overload

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LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.     

Gene expression profiling of scrapie-infected brain tissue

The underlying pathomechanisms in prion infections of the central nervous system are still insufficiently understood. The identification of genes with altered expression patterns in the diseased brain may provide insight into the disease development on the molecular level, which ultimately leads to neuronal loss. To provide a detailed analysis of changes in the molecular level in prion disease pathology we used a large-scale gene array based approach, which covers more than 11,000 functionally characterised sequences and expressed sequence tags, for the analysis of gene expression profile alterations in the cortex, medulla, and pons of scrapie-infected mice. The study identified in total 114 genes with altered mRNA levels, the majority of which were previously not known to be affected by the disease. Overall the gene array data demonstrate the presence of a strong inflammatory reaction and stress response, and show similarities to gene expression patterns found in brains affected by Alzheimer's disease and aging, respectively.     

Gene expression profiling of cardiovascular disease models

Recent development of gene expression profiling technologies has enabled the large-scale analysis of gene expression changes during disease progression. Frequently, cardiovascular diseases involve complex interactions of multiple cell types over prolonged periods of time. A better understanding of the pathology of cardiovascular diseases and the potential identification of underlying genetic defects are currently being explored by using profiling methodologies in a number of animal and tissue-culture models.     

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.