Neuroprotective Effects of Butanol Fraction of Cordyceps cicadae on Glutamate‐Induced Damage in PC12 Cells Involving Oxidative Toxicity

The current study was aimed at investigating the neuroprotective effects of the butanol fraction from Cordyceps cicadae (C_BU), which was responsible for the anti‐aging effect of this medicine. Glutamate‐induced PC12 cells were used as a model to determine the neuroprotective effect against oxidative cell death. Cell viability, cytotoxicity, flow cytometry, mitochondrial transmembrane potential (MMP), reactive oxygen species (ROS), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) levels were analyzed to assess neuronal cell survival or death. The results obtained from the above evaluations showed that C_BU was the most effective fraction and even better than pure compounds present in C. cicadae in terms of suppressing glutamate‐induced damage in PC12 cells, increasing cell viability, decreasing lactase dehydrogenase (LDH) release, and reduction of apoptosis induced by exposure to glutamate. Furthermore, C_BU protected cells against mitochondrial dysfunction and oxidative stress as indicated by the suppression of ROS accumulation and up regulation of the levels of GSH‐Px and SOD. In summary, the above results showed that C_BU exerted neuroprotective effect against oxidative damage, and this activity could be partly due to the action of nucleosides present in the C_BU.     

Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA‐induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA‐induced degradation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA‐induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole‐5,6‐quinone‐2‐carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole‐2,3,5‐tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA‐melanin was confirmed by direct measurements of singlet oxygen phosphorescence.     

Mitochondrial peroxiredoxin-3 protects hippocampal neurons from excitotoxic injury in vivo

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium-buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin-3 (Prx-3), to investigate the mechanism by which toxic properties of ROS were up-regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx-3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx-3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx-3 completely inhibited protein nitration and markedly reduced gliosis, a post-neuronal cell death event. Since mitochondrial Prx-3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx-3 up-regulation might be a useful novel approach for the management of neurodegenerative diseases.     

Antioxidants reveal an inverted U‐shaped dose‐response relationship between reactive oxygen species levels and the rate of aging in Caenorhabditis elegans

Reactive oxygen species (ROS) are potentially toxic, but they are also signaling molecules that modulate aging. Recent observations that ROS can promote longevity have to be reconciled with the numerous claims about the benefits of antioxidants on lifespan. Here, three antioxidants [N‐acetylcysteine (NAC), vitamin C, and resveratrol (RSV)] were tested on Caenorhabditis elegans mutants that alter drug uptake, mitochondrial function, and ROS metabolism. We observed that like pro‐oxidants, antioxidants can both lengthen and shorten lifespan, dependent on concentration, genotypes, and conditions. The effects of antioxidants thus reveal an inverted U‐shaped dose–response relationship between ROS levels and lifespan. In addition, we observed that RSV can act additively to both NAC and paraquat, to dramatically increase lifespan. This suggests that the effect of compounds that modulate ROS levels can be additive when their loci of action or mechanisms of action are sufficiently distinct.     

The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

Naked mole‐rats (NMRs) are mouse‐sized mammals that exhibit an exceptionally long lifespan (>30 vs. <4 years for mice), and resist aging‐related pathologies such as cardiovascular and pulmonary diseases, cancer, and neurodegeneration. However, the mechanisms underlying this exceptional longevity and disease resistance remain poorly understood. The oxidative stress theory of aging posits that (a) senescence results from the accumulation of oxidative damage inflicted by reactive oxygen species (ROS) of mitochondrial origin, and (b) mitochondria of long‐lived species produce less ROS than do mitochondria of short‐lived species. However, comparative studies over the past 28 years have produced equivocal results supporting this latter prediction. We hypothesized that, rather than differences in ROS generation, the capacity of mitochondria to consume ROS might distinguish long‐lived species from short‐lived species. To test this hypothesis, we compared mitochondrial production and consumption of hydrogen peroxide (H₂O₂; as a proxy of overall ROS metabolism) between NMR and mouse skeletal muscle and heart. We found that the two species had comparable rates of mitochondrial H₂O₂ generation in both tissues; however, the capacity of mitochondria to consume ROS was markedly greater in NMRs. Specifically, maximal observed consumption rates were approximately two and fivefold greater in NMRs than in mice, for skeletal muscle and heart, respectively. Our results indicate that differences in matrix ROS detoxification capacity between species may contribute to their divergence in lifespan.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.     

Epidermal growth factor protects against myocardial ischaemia reperfusion injury through activating Nrf2 signalling pathway

Alleviating the oxidant stress associated with myocardial ischaemia reperfusion has been demonstrated as a potential therapeutic approach to limit ischaemia reperfusion (I/R)-induced cardiac damage. It is reported that EGFR/erbB2 signalling is an important cardiac survival pathway in cardiac function and activation of EGFR has a cardiovascular effect in global ischaemia. Epidermal growth factor (EGF), a typical EGFR ligand, was considered to have a significant role in activating EGFR. However, no evidence has been published whether exogenous EGF has protective effects on myocardial ischaemia reperfusion. This study aims to investigate the effects of EGF in I/R-induced heart injury and to demonstrate its mechanisms. H9c2 cells challenged with H₂O₂ were used for in vitro biological activity and mechanistic studies. The malondialdehyde (MDA) and Superoxide Dismutase (SOD) levels in H9c2 cells were determined, and the cell viability was assessed by MTT assay. Myocardial I/R mouse administrated with or without EGF were used for in vivo studies. Pretreatment of H9c2 cells with EGF activated Nrf2 signalling pathway, attenuated H₂O₂-increased MDA and H₂O₂-reduced SOD level, followed by the inhibition of H₂O₂-induced cell death. In in vivo animal models of myocardial I/R, administration of EGF reduced infarct size and myocardial apoptosis. These data support that EGF decreases oxidative stress and attenuates myocardial ischaemia reperfusion injury via activating Nrf2.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Regulatory role of Sirt1 on the gene expression of fatty acid‐binding protein 3 in cultured porcine adipocytes

To investigate whether Sirt1 could modulate fatty acid‐binding protein 3 (FABP3), we treated porcine adipocytes either with the Sirt1 inhibitor nicotinamide (NAM), with the Sirt1 activator resveratrol (RES), or by knockdown of Sirt1 by Sirt1‐siRNA. NAM or knockdown with Sirt1‐siRNA significantly inhibited Sirt1 mRNA expression, while increasing FABP3 mRNA levels. RES or RES + Sirt1‐siRNA treatments further proved that Sirt1 negatively regulated FABP3 gene expression in adipocytes. We also found a similar Sirt1 regulation pattern for PPARγ to that of FABP3 in adipocytes. Furthermore, NAM/RES + PPARγ‐siRNA treatments showed that Sirt1 may regulate the FABP3 gene expression partly through the PPARγ‐mediated signals. In summary, Sirt1 regulates the expression of FABP3 gene in adipocytes, and PPARγ apparently plays an important role in this process. J. Cell. Biochem. 107: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Bromide alleviates fatty acid‐induced lipid accumulation in mouse primary hepatocytes through the activation of PPARα signals

Increased plasma free fatty acids (FFAs) and liver triglyceride (TG) accumulations have been implicated in the pathogenesis of hepatic steatosis. On the other hand, trace elements function as essential cofactors that are involved in various biochemical processes in mammals, including metabolic homeostasis. Notably, clinical and animal studies suggest that the plasma levels of bromide negatively correlate with those of TG, total cholesterol (TC) and high‐density lipoprotein‐cholesterol (HDL‐C). However, the effect of bromide on lipid accumulation and the direct molecular target responsible for its action remains unknown. Oil red O (ORO) and Nile red staining were used to detect the effect of bromide on lipid accumulation in mouse primary hepatocytes (PHs) treated with different doses of sodium bromide (NaBr) in the presence of FFAs (0.4 mM oleate/palmitic acid 1:1). Spectrophotometric and fluorometric analyses were performed to assess cellular TG concentrations and rates of fatty acid oxidation (FAO), respectively, in mouse PHs. We found that bromide decreased FFA‐induced lipid accumulation and increased FFA‐inhibited oxygen consumptions in mouse PHs in a dose‐dependent manner via activation of PPARα. Mechanical studies demonstrated that bromide decreased the phosphorylation levels of JNK. More importantly, the PPARα‐specific inhibitor GW6471 partially abolished the beneficial effects of bromide on mouse PHs. Bromide alleviates FFA‐induced excessive lipid storage and increases rates of FAO through the activation of PPARα/JNK signals in mouse PHs. Therefore, bromide may serve as a novel drug in the treatment of hepatic steatosis.     

Expression of a dominant‐negative AtNEET‐H89C protein disrupts iron–sulfur metabolism and iron homeostasis in Arabidopsis

Iron–sulfur (Fe–S) clusters play an essential role in plants as protein cofactors mediating diverse electron transfer reactions. Because they can react with oxygen to form reactive oxygen species (ROS) and inflict cellular damage, the biogenesis of Fe–S clusters is highly regulated. A recently discovered group of 2Fe–2S proteins, termed NEET proteins, was proposed to coordinate Fe–S, Fe and ROS homeostasis in mammalian cells. Here we report that disrupting the function of AtNEET, the sole member of the NEET protein family in Arabidopsis thaliana, triggers leaf‐associated Fe–S‐ and Fe‐deficiency responses, elevated Fe content in chloroplasts (1.2–1.5‐fold), chlorosis, structural damage to chloroplasts and a high seedling mortality rate. Our findings suggest that disrupting AtNEET function disrupts the transfer of 2Fe–2S clusters from the chloroplastic 2Fe–2S biogenesis pathway to different cytosolic and chloroplastic Fe–S proteins, as well as to the cytosolic Fe–S biogenesis system, and that uncoupling this process triggers leaf‐associated Fe–S‐ and Fe‐deficiency responses that result in Fe over‐accumulation in chloroplasts and enhanced ROS accumulation. We further show that AtNEET transfers its 2Fe–2S clusters to DRE2, a key protein of the cytosolic Fe–S biogenesis system, and propose that the availability of 2Fe–2S clusters in the chloroplast and cytosol is linked to Fe homeostasis in plants.