Protection of coumarins against linoleic acid hydroperoxide-induced cytotoxicity

Coumarins comprise a group of natural phenolic compounds found in a variety of plant sources. Protective effects of coumarins against cytotoxicity induced by linoleic acid hydroperoxide were examined in cultured human umbilical vein endothelial cells. When the cells were incubated in medium supplemented with linoleic acid hydroperoxide and coumarins, esculetin (6,7-dihydroxycoumarin) and 4-methylesculetin protected cells from injury by linoleic acid hydroperoxide. Fraxetin and caffeic acid showed weak, but significant, protection. Esculin as well as esculetin and 4-methylesculetin were effective for protecting cells against linoleic acid hydroperoxide-induced cytotoxicity in the case of pretreatment for 24 h, however fraxetin and caffeic acid showed no protection. Since esculetin was detected after 24 h treatment with esculin, a sugar moiety in the esculin molecule appears to be hydrolyzed during pretreatment. Coumarins such as umbelliferone containing only one hydroxyl group showed no protective effect in pretreatment or concurrent treatment. Esculetin and 4-methylesculetin provided synergistic protection against cytotoxicity induced by linoleic acid hydroperoxide with alpha-tocopherol. Furthermore, the radical-scavenging ability of coumarins was examined in electron spin resonance spectrometry. Esucletin, 4-methylesculetin, fraxetin, and caffeic acid showed the quenching effect on the 1,1-diphenyl-2-picrylhydrazyl radical. These results indicate that the presence of an ortho catechol moiety in the coumarin molecules plays an important role in the protective activities against linoleic acid hydroperoixde-induced cytotoxicity.     

Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity.

Heme oxygenase (HO)-1 is a stress-inducible enzyme protecting cells against oxidative stress, and mechanisms have been considered to depend exclusively on its enzyme activity. This study aimed to examine if the protein lacking catalytic activities could also display such resistance against oxidative stress. Stable transfectants of rat wild type HO-1 cDNA (HO-1-U937) and those of its H25A mutant gene (mHO-1-U937) were established using human monoblastic lymphoma cell U937. HO-1-U937 and mHO-1-U937 used in the study exhibited similar levels of the protein expression, while only the former increased enzyme activities. HO-1- and mHO-1 U937 cells became more and less sensitive to H(2)O(2) than mock transfectants, respectively; such distinct susceptibility between the cells was ascribable to differences in the capacity to scavenge H(2)O(2) through catalase and to execute iron-mediated oxidant propagation. On the other hand, both cell lines exhibited greater resistance to tert-butyl hydroperoxide than mock transfectants. The resistance of HO-1-U937 to hydroperoxides appeared to result from antioxidant properties of bilirubin, an HO-derived product, while that of mHO-1-U937 was ascribable to increased contents of catalase and glutathione. These results provided evidence that gene transfection of the activity-lacking mutant HO-1 protects cells against oxidative stress through multiple mechanisms involving up-regulation of catalase and glutathione contents.     

The activation of HO-1/Nrf-2 contributes to the protective effects of diallyl disulfide (DADS) against ethanol-induced oxidative stress

Background

Diallyl disulfide (DADS) is a garlic-derived organosulfur compound. The current study is designed to evaluate the protective effects of DADS against ethanol-induced oxidative stress, and to explore the underlying mechanisms by examining the HO-1/Nrf-2 pathway.

Methods

We investigated whether or not DADS could activate the HO-1 in normal human liver cell LO2, and then evaluated the protective effects of DADS against ethanol-induced damage in LO2 cells and in acute ethanol-intoxicated mice. The biochemical parameters were measured using commercial kits. HO-1 mRNA level was determined by RT-PCR. Histopathology and immunofluorescence assay were performed with routine methods. Protein levels were measured by western blot.

Results

DADS significantly increased the mRNA and protein levels of HO-1, stimulated the nuclear translocation of Nrf-2 and increased the phosphorylation of MAPK in LO2 cells. The nuclear translocation of Nrf-2 was abrogated by MAPK inhibitors. DADS significantly suppressed ethanol-induced elevation of lactate dehydrogenase (LDH) and aspartate transaminase (AST) activities, decrease of glutathione (GSH) level, increase of malondialdehyde (MDA) levels, and apoptosis of LO2 cells, which were all blocked by ZnPPIX. In mice, DADS effectively suppressed acute ethanol-induced elevation of aminotransferase activities, and improved liver histopathological changes, which might be associated with HO-1 activation.

Conclusion

These results demonstrate that DADS could induce the activation of HO-1/Nrf-2 pathway, which may contribute to the protective effects of DADS against ethanol-induced liver injury.

General significance

DADS may be beneficial for the prevention and treatment of ALD due to significant activation of HO-1/Nrf-2 pathway.     

Protective mechanisms of Mg-gluconate against oxidative endothelial cytotoxicity.

The potential anti-radical properties and cytoprotective effects of Mg-gluconate were studied. When microsomal membranes were peroxidized by a *O2- driven, Fe-catalyzed oxy-radical system (R* = dihydroxyfumarate + Fe2+), Mg-gluconate inhibited lipid peroxidation (TBARS formation) in a concentration-dependent manner with IC50 being 2.3 mM. For the entire range of .25-2 mM, MgSO4 or MgCl2 were < or = 20% effective compared to Mg-gluconate. When cultured bovine aortic endothelial cells were incubated with the R* for 50 min. at 37 degrees C, 56% loss of total glutathione occurred. Pre-treatment (10 min.) of the cells with 0.25-4 mM Mg-gluconate before R* exposure significantly (p<0.05) prevented the GSH loss to varying degrees; the EC50 was 1.1 mM. In separate experiments, with 30 min. of free radical incubation of endothelial monolayers (approximately 65% confluent), cell survival/proliferation determined by the tetrazolium salt MTT assay, decreased to 38% of control at 24 hrs; Mg-gluconate concentration-dependently attenuated the lost cell survival with EC50 of approximately 1.3 mM. For comparison, the effects provided by MgSO4 or MgCl2 were significantly lower and were < or = 1/3 as potent as that produced by Mg-gluconate. In a Fenton-reaction system consisting of Fe(II)+ H2O2, Mg-gluconate but not other Mg-salts, significantly inhibited the formation of OH radicals as determined by the ESR DMPO-OH signal intensity. Mg-gluconate also dose-dependently inhibited the 'Fe-catalyzed' deoxyribose degradation suggesting that Mg-gluconate could displace Fe from 'catalytic sites' of oxidative damage. These data suggest that Mg-gluconate may serve as a more advantageous Mg-salt for clinical use due to its additional anti-radical and cytoprotective activities.     

Dual protective mechanisms of matrix metalloproteinases 2 and 9 in immune defense against Streptococcus pneumoniae

A localized and effective innate immune response to pathogenic bacterial invasion is central to host survival. Identification of the critical local innate mediators of lung defense against such pathogens is essential for a complete understanding of the mechanism(s) underlying effective host defense. In an acute model of Streptococcus pneumoniae lung infection, deficiency in matrix metalloproteinase (MMP)2 and MMP9 (Mmp2/9(-/-)) conferred a survival disadvantage relative to wild-type mice treated under the same conditions. S. pneumoniae-infected Mmp2/9(-/-) mice recruited more polymorphonuclear leukocytes to the lung but had higher bacterial burdens. Mmp2/9(-/-) mice showed significantly higher levels of IL-17A, IP-10, and RANTES in the lung. Although MMP2-dependent cleavage partially inactivated IL-17A, MMP9 was critical for effective bacterial phagocytosis and reactive oxygen species generation in polymorphonuclear neutrophils. These data demonstrate critical nonredundant and protective roles for MMP2 and MMP9 in the early host immune response against S. pneumoniae infection.     

Distinct mechanisms for Ctr1-mediated copper and cisplatin transport

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with multimer assembly of Ctr1. Copper near the K(m) value of Ctr1 enhanced FRET in a manner that correlated with cellular copper transport. In vitro cross-linking of Ctr1 confirmed that copper-induced FRET reflects conformational changes within pre-existing Ctr1 complexes. FRET assays in membrane-disrupted cells and protein extracts showed that intact cell structure is necessary for Ctr1 activity. Despite Ctr1-dependent cellular accumulation, cisplatin did not change Ctr1 FRET nor did it attenuate copper-induced FRET. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs that are dispensable for copper transport play a critical role for cisplatin uptake. Taken together, our data reveal functional roles for structural remodeling of the Ctr1 multimeric complex in copper transport and suggest distinct mechanisms employed by Ctr1 for copper and cisplatin transport.     

Molecular mechanisms of the protective effect of vitamin e against atherosclerosis

Oxidation of low-density lipoproteins constitutes the first step of a very complex process leading to atherosclerosis. Vitamin E, and principally a-tocopherol, is considered as the principal inhibitor of lipid peroxidation. Some studies showed the beneficial role of vitamin E in the prevention and reduction of atherosclerosis and its associated pathologies. However, other in vitro studies advance a prooxidant role of vitamin E. The results of the epidemiologic studies are difficult to generalize without taking account of the clinical randomized tests. In this work, we reviewed the principal studies devoted to the role of vitamin E and discussed the assumption of a prooxidant effect of this molecule.     

A role for Bach1 and HO-2 in suppression of basal and UVA-induced HO-1 expression in human keratinocytes

Ultraviolet A (UVA) radiation is an oxidizing agent that strongly induces the heme oxygenase 1 (HO-1) gene and expression of the protein in cultured human skin fibroblasts but weakly induces it in skin keratinocytes. Lower basal levels of HO-1 and much higher basal levels of HO-2 protein are observed in keratinocytes compared with fibroblasts. Using both overexpression and knockdown approaches, we demonstrate that HO-2 modulates basal and UVA-induced HO-1 protein levels, whereas HO-1 levels do not affect HO-2 levels in skin fibroblasts and keratinocytes. Silencing of Bach1 strongly increases HO-1 levels in transformed HaCaT keratinocytes and these HO-1 levels are not further increased by either UVA irradiation or silencing of HO-2. This is consistent with the conclusion that high constitutive levels of HO-2 expression in keratinocytes are responsible for the resistance of these cells to HO-1 induction by UVA radiation and that Bach1 plays a predominant role in influencing the lack of HO-1 expression in keratinocytes. Bach1 inhibition leading to HO-1 induction reduced UVA-irradiation-induced damage as monitored both by the extent of LDH release and by nuclear condensation, so that Bach1 inhibition seems to protect against UVA-irradiation-induced damage in keratinocytes.     

Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit

We report on the detection and characterization of two forms of heme oxygenase in rabbit tissues and provide data suggesting that heme oxygenases in rat and rabbit are not identical and constitute a group of heterogenous proteins. Certain molecular properties, however, are shared by the isozymes in rat and rabbit; the predominant form of the enzyme in control liver and testis is HO-2, in the liver HO-1 is the inducible form, and in the brain HO-1 is not detectable. HO-1 was purified from liver of rabbits treated with bromobenzene to near homogeneity with a specific activity of 8,270 nmol of bilirubin/mg/h and compared with a homogenous preparation of rat HO-1 with a specific activity of 6,220, also obtained from bromobenzene-treated animals. Rat and rabbit HO-1, on sodium dodecyl sulfate-polyacrylamide gel, had molecular weights of 30,000 and 30,700, respectively. Rabbit HO-2 was partially purified from testis to a specific activity of 386 nmol of bilirubin/mg/h and compared with a purified preparation of rat testis HO-2 with a specific activity of 5,700. Using Western immunoblotting, rabbit HO-2 displayed intense cross-reactivity with antibody raised in rabbit to sodium dodecyl sulfate-denatured rat HO-2, and had a substantially larger molecular weight than the rat HO-2 (42,000 versus 36,000). Rabbit HO-1 did not cross-react with antibody to rat HO-1 which was also raised in rabbit. Unlike the rat enzymes, rabbit HO-1 and HO-2 did not differ in thermolability. It is speculated that HO-1 in rat and rabbit, and possibly HO-2, have evolved from divergent evolution of a common ancestral gene(s).     

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.     

Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses

The capacity of mesenchymal stem cells (MSCs) to survive and engraft in the target tissue may lead to promising therapeutic effects. However, the fact that the majority of MSCs die during the first few days following transplantation complicates cell therapy. Hence, it is necessary to strengthen the stem cells to withstand the rigors of the microenvironment to improve the efficacy of cell therapy. In this study, we manipulated MSCs to express a cytoprotective factor, heme oxygenase-1 (HO-1), to address this issue. Full-length cDNA of human HO-1 was isolated and cloned into TOPO vector by TOPO cloning reaction. Then, the construct was ligated to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the recombinant virus expressing HO-1 was produced in appropriate mammalian cell line and used to infect MSCs. The HO-1 engineered MSCs were exposed to hypoxic and oxidative stress conditions followed by evaluation of the cells’ viability and apoptosis. Transient expression of HO-1 was detected within MSCs. It was observed that HO-1 expression could protect MSCs against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. The MSCs-HO-1 retained their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages. These findings could be applied as a strategy for prevention of graft cell death in MSCs-based cell therapy and is a good demonstration of how an understanding of cellular stress responses can be used for practical applications.     

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

Mutations of the genes encoding aminoacyl-t RNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5 AG, p.D2 G; c.1367 CT, p.S456 L; c.1535 GA, p.R512 Q and c.1846_1847 del, p.Y616 Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-t RNA synthetase(h Arg RS), are linked to Pelizaeus-Merzbacher-like disease(PMLD) with unclear pathogenesis. Among these mutations, c.5 AG is the most extensively reported mutation, leading to a p.D2 G mutation in the N-terminal extension of the long-form h Arg RS. Here, we showed the detrimental effects of R512 Q substitution and ΔC mutations on the structure and function of h Arg RS, while the most frequent mutation c.5 AG, p.D2 G acted in a different manner without impairing h Arg RS activity. The nucleotide substitution c.5 AG reduced translation of h Arg RS m RNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.     

The biochemical protective mechanisms against non-steroidal anti-inflammatory drug induced G-I mucosal damage

Conventional anti-ulcer agents give some protection against NSAID-induced GI injury, but they are by no means totally effective nor without some disadvantage for their side effects cost and for lack of efficacy. More selective protective effects may be achieved by use of agents which counteract the biochemical and cytological changes induced by the NSAIDs. The relative success of these approaches has yet to be fully evaluated but at least some of these procedures may prove cheaper and devoid of side effects encountered with traditional anti-ulcer therapies.