Sanguiin H-11 from Sanguisorbae radix protects HT22 murine hippocampal cells against glutamate-induced death

Excessive glutamate level induces neuronal death in acute brain injuries and chronic neurodegenerative diseases. Natural compounds from medicinal and food plants have been attracting interest as a treatment for neurological disorders. Sanguiin H-11 (SH-11), a hydrolysable ellagitannin, inhibits neutrophil movement and nitric oxide -production. However, its neuroprotective effect has not been studied. Therefore, the present study examined the protective effect of SH-11 from Sanguisorbae radix and its mechanism against glutamate-induced death in HT22 cells. Our results showed that SH-11 possessed a strong antioxidant activity and prevented glutamate-induced death in HT22 cells. As a strong antioxidant, SH-11 significantly reduced glutamate-induced increases in intracellular reactive oxygen species accumulation and calcium ion influx. Western blotting analysis showed that glutamate-induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinases 1/2, c-Jun N-terminal kinase, and p38, was significantly decreased by SH-11. Furthermore, SH-11 significantly decreased the number of annexin V-positive HT22 cells, which is indicating apoptotic cell death. In conclusion, our results suggested that SH-11 exerted a potent neuroprotective activity against glutamate-mediated apoptotic cell death by inhibiting oxidative stress-mediated MAPK activation.     

Neuroprotective Effects of <Emphasis Type="Italic">Sigesbeckia pubescens</Emphasis> Extract on Glutamate-Induced Oxidative Stress in HT22 Cells via Downregulation of MAPK/caspase-3 Pathways

Sigesbeckia pubescens (SP) is a traditional Chinese medicine, possessing antioxidant and anti-inflammatory activities. In this study, we evaluate the neuroprotective activities of SP extract on glutamate-induced oxidative stress in HT22 cells and the molecular mechanism underlying neuroprotection. We applied 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), crystal violet, reactive oxygen species (ROS), lactate dehydrogenase (LDH), quantitative real-time polymerase chain reaction (qPCR), and western blot analyses for assessing the neuroprotective effects of SP extract. The experimental study revealed that SP considerably increased the cell viability, and reduced the oxidative stress promoted ROS and LDH generation in HT22 cells in a dose-dependent manner. Additionally, the morphology of HT22 cells was effectively improved by SP. Upregulated gene expressions of mitogen-activated protein kinase (MAPK) were markedly attenuated by SP. Similarly, SP notably suppressed the ROS-mediated phosphorylation of MAPK (pERK1/2, pJNK, and pp38) cascades and activation of apoptotic factor caspase-3 signaling pathway that overall contributed to the neuroprotection. Taken together, SP may exert neuroprotective effects via alteration of MAPK and caspase-3 pathways under oxidative stress condition. Therefore, SP is a potential agent for preventing oxidative stress-mediated neuronal cell death.     

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl¹-NH-Aryl² scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.     

Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels

In the present work, the potential hepatoprotective effects of five phenolic compounds against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) were evaluated in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damage in HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased levels of reduced glutathione (GSH). All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound's lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even greater importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damage induced in HepG2 cells. This could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.     

Design,Synthesis, and In Vitro Evaluation of a Novel Probucol Derivative: Protective Activity in Neuronal Cells Through GPx Upregulation

Recent studies have shown that probucol (PB), a hipocholesterolemic agent with antioxidant and anti-inflammatory properties, presents neuroprotective properties. On the other hand, adverse effects have limited PB’s clinical application. Thus, the search for PB derivatives with no or less adverse effects has been a topic of research. In this study, we present a novel organoselenium PB derivative (RC513) and investigate its potential protective activity in an in vitro experimental model of oxidative toxicity induced by tert-butyl hydroperoxide (tBuOOH) in HT22 neuronal cells, as well as exploit potential protective mechanisms. tBuOOH exposure caused a significant decrease in the cell viability, which was preceded by (i) increased reactive species generation and (ii) decreased mitochondrial maximum oxygen consumption rate. RC513 pretreatment (48 h) significantly prevented the tBuOOH-induced decrease of cell viability, RS generation, and mitochondrial dysfunction. Of note, RC513 significantly increased glutathione peroxidase (GPx) activity and mRNA expression of GPx1, a key enzyme involved in peroxide detoxification. The use of mercaptosuccinic acid, an inhibitor of GPx, significantly decreased the protective activity of RC513 against tBuOOH-induced cytotoxicity in HT22 cells, highlighting the importance of GPx upregulation in the observed protection. In summary, the results showed a significant protective activity of a novel PB derivative against tBuOOH-induced oxidative stress and mitochondrial dysfunction, which was related to the upregulation of GPx. Our results point to RC513 as a promising neuroprotective molecule, even though studies concerning potential beneficial effects and safety aspects of RC513 under in vivo conditions are well warranted.     

Protective activity of aromatic amines and imines against oxidative nerve cell death.

Oxidative stress is a widespread phenomenon in the pathology of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Neuronal cell death due to oxidative stress may causally contribute to the pathogeneses of these diseases. Therefore, neuroprotective antioxidants are considered to be a promising approach to slow down disease progression. We have investigated different aromatic amine and imine compounds for neuroprotective antioxidant functions in cell culture, and found that these compounds possess excellent cytoprotective potential in diverse paradigms of oxidative neuronal cell death, including clonal cell lines, primary cerebellar neurons, and organotypic hippocampal slice cultures. Aromatic amines and imines are effective against oxidative glutamate toxicity, glutathione depletion, and hydrogen peroxide toxicity. Their mode of action as direct antioxidants was experimentally confirmed by electron spin resonance spectroscopy, cell-free brain lipid peroxidation assays, and intracellular peroxide measurements. With half-maximal effective concentrations of 20-75 nM in different neuroprotection experiments, the aromatic imines phenothiazine, phenoxazine, and iminostilbene proved to be about two orders of magnitude more effective than common phenolic antioxidants. This remarkable efficacy could be directly correlated to calculated properties of the compounds by means of a novel, quantitative structure-activity relationship model. We conclude that bridged bisarylimines with a single free NH-bond, such as iminostilbene, are superior neuroprotective antioxidants, and may be promising lead structures for rational drug development.     

Opposing roles for ERK1/2 in neuronal oxidative toxicity: distinct mechanisms of ERK1/2 action at early versus late phases of oxidative stress

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.     

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G₂/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G₂/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.     

Hydroxytyrosol prevents dermal papilla cells inflammation under oxidative stress by inducing autophagy

Hydroxytyrosol (HT), a primary phenolic antioxidant in olive oil, can afford protection from oxidative stress (OS) in different cells, including skin cells. In particular, it regulates several inflammation‐associated processes as well as in improving the antioxidant defense system. However, there is no information about HT used in the treatment of hair loss. This work aimed at exploring the potential protective actions of HT against OS in rat dermal papilla cells. After treatment, the related expression of protein and messenger RNA were detected using morphological and molecular analyses. The results showed that HT significantly reduced intracellular reactive oxygen species level, apoptotic markers and inflammation induced by OS and enhanced cell survival by regulating autophagy. Furthermore, HT enhanced the secretion of hair growth factors in the anti‐inflammation process. These results suggest that HT has a significant protective ability against OS and encourage the use of this biological ingredient as a possible tool to prevent alopecia.     

Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity

Minocycline is neuroprotective in animal models of a number of acute CNS injuries and neurodegenerative diseases. While anti-inflammatory and anti-apoptotic effects of minocycline have been characterized, the molecular basis for the neuroprotective effects of minocycline remains unclear. We report here that minocycline and a number of antioxidant compounds protect mixed neuronal cultures in an oxidative stress assay. To evaluate the role of minocycline's direct antioxidant properties in neuroprotection, we determined potencies for minocycline, other tetracycline antibiotics, and reference antioxidant compounds using a panel of in vitro radical scavenging assays. Data from in vitro rat brain homogenate lipid peroxidation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays show that minocycline, in contrast to tetracycline, is an effective antioxidant with radical scavenging potency similar to vitamin E. Our findings suggest that the direct antioxidant activity of minocycline may contribute to its neuroprotective effects in some cell-based assays and animal models of neuronal injury.     

Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.     

N,N-Bis-(8-hydroxyquinoline-5-yl methyl)-benzyl substituted amines (HQNBA): peroxisome proliferator-activated receptor (PPAR-γ) agonists with neuroprotective properties

We report on the neuroprotective effects of N,N-bis-(8-hydroxyquinoline-5-yl methyl)-benzyl substituted amines (HQNBA) in a model of oxidative stress-induced nerve cell death using mouse hippocampal-derived HT22 cells. The four derivatives (JLK1472, JLK1486, JLK1522 and JLK1535) protected the HT22 cells from death at concentrations ranging from 0.1 to 1 μM. Their action is partially dependent on their ability to act as PPARγ agonists. These analogues also maintain GSH levels suggesting that they have indirect anti-oxidant effects.     

Oxidative nerve cell death in Alzheimer's disease and stroke: antioxidants as neuroprotective compounds

Many neurodegenerative disorders and syndromes are associated with an excessive generation of reactive oxygen species (ROS) and oxidative stress. The pathways to nerve cell death induced by diverse potential neurotoxins such as peptides, excitatory amino acids, cytokines or synthetic drugs commonly share oxidative downstream processes, which can cause either an acute oxidative destruction or activate secondary events leading to apoptosis. The pathophysiological role of ROS has been intensively studied in in vitro and in vivo models of chronic neurodegenerative diseases such as Alzheimer's disease (AD) and of syndromes associated with rapid nerve cell loss as occuring in stroke. In AD, oxidative neuronal cell dysfunction and cell death caused by protofibrils and aggregates of the AD-associated amyloid beta protein (Abeta) may causally contribute to pathogenesis and progression. ROS and reactive nitrogen species also take part in the complex cascade of events and the detrimental effects occuring during ischemia and reperfusion in stroke. Direct antioxidants such as chain-breaking free radical scavengers can prevent oxidative nerve cell death. Although there is ample experimental evidence demonstrating neuroprotective activities of direct antioxidants in vitro, the clinical evidence for antioxidant compounds to act as protective drugs is relatively scarce. Here, the neuroprotective potential of antioxidant phenolic structures including alpha-tocopherol (vitamin E) and 17beta-estradiol (estrogen) in vitro is summarized. In addition, the antioxidant and cytoprotective activities of lipophilic tyrosine- and tryptophan-containing structures are discussed. Finally, an outlook is given on the neuroprotective potential of aromatic amines and imines, which may comprise novel lead structures for antioxidant drug design.     

Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells

Neuronal cell death caused by oxidative stress is common in a variety of neural diseases and can be investigated in detail in cultured HT22 neuronal cells, where the amino acid glutamate at high concentrations causes glutathione depletion by inhibition of the glutamate/cystine antiporter system, intracellular accumulation of reactive oxygen species (ROS) and eventually oxidative stress-induced neuronal cell death. Using this paradigm, we have previously reported that resveratrol (3,5,4′-trans-trihydroxystilbene) protects HT22 neuronal cells from glutamate-induced oxidative stress by inducing heme oxygenase (HO)-1 expression. Piceatannol (3,5,4′,3′-trans-trihydroxystilbene), which is a hydroxylated resveratrol analog and one of the resveratrol metabolites, is estimated to exert neuroprotective effect similar to that of resveratrol. The aim of this study, thus, is to determine whether piceatannol, similarly to resveratrol, would protect HT22 neuronal cells from glutamate-induced oxidative stress. Glutamate at high concentrations induced neuronal cell death and ROS formation. Piceatannol reduced glutamate-induced cell death and ROS formation. The observed cytoprotective effect was much higher when HT22 neuronal cells were pretreated with piceatannol for 6 or 12 h prior to glutamate treatment than when pretreated for 0.5 h. Piceatannol also increased HO-1 expression and HO activity via its activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, neuroprotective effect of piceatannol was partly (but not completely) abolished by either down-regulation of HO-1 expression or blockage of HO-1 activity. Taken together, our results suggest that piceatannol, similar to resveratrol, is capable of protecting HT22 neuronal cells against glutamate-induced cell death, at least in part, by inducing Nrf2-dependent HO-1 expression.     

Reversible oxidation of ERK-directed protein phosphatases drives oxidative toxicity in neurons

Oxidative stress links diverse neuropathological conditions that include stroke, Parkinson's disease, and Alzheimer's disease and has been modeled in vitro with various paradigms that lead to neuronal cell death following the increased accumulation of reactive oxygen species. For example, immortalized neurons and immature primary cortical neurons undergo cell death in response to depletion of the antioxidant glutathione, which can be elicited by administration of glutamate at high concentrations. We have demonstrated previously that this glutamate-induced oxidative toxicity requires activation of the mitogen-activated protein kinase member ERK1/2, but the mechanisms by which this activation takes place in oxidatively stressed neurons are still not fully known. In this study, we demonstrate that during oxidative stress, ERK-directed phosphatases of both the serine/threonine- and tyrosine-directed classes are selectively and reversibly inhibited via a mechanism that is dependent upon the oxidation of cysteine thiols. Furthermore, the impact of ERK-directed phosphatases on ERK1/2 activation and oxidative toxicity in neurons was tested in a neuronal cell line and in primary cortical cultures. Overexpression of the highly ERK-specific phosphatase MKP3 and its catalytic mutant, MKP3 C293S, were neuroprotective in transiently transfected HT22 cells and primary neurons. The neuroprotective effect of the MKP3 C293S mutant, which enhances ERK1/2 phosphorylation but blocks its nuclear translocation, demonstrates the necessity for active ERK1/2 nuclear localization for oxidative toxicity in neurons. Together, these data implicate the inhibition of endogenous ERK-directed phosphatases as a mechanism that leads to aberrant ERK1/2 activation and nuclear accumulation during oxidative toxicity in neurons.     

Antioxidant neuroprotection in Alzheimer's disease as preventive and therapeutic approach

Various neurodegenerative disorders and syndromes are associated with oxidative stress. The deleterious consequences of excessive oxidations and the pathophysiological role of reactive oxygen species (ROS) have been intensively studied in Alzheimer's disease (AD). Neuronal cell dysfunction and oxidative cell death caused by the AD-associated amyloid beta protein may causally contribute to the pathogenesis of AD. Antioxidants that prevent the detrimental consequences of ROS are consequently considered to be a promising approach to neuroprotection. While there is ample experimental evidence demonstrating neuroprotective activities of antioxidants in vitro, the clinical evidence that antioxidant compounds act as protective drugs is still relatively scarce. Nevertheless, antioxidants constitute a major part of the panel of clinical and experimental drugs that are currently considered for AD prevention and therapy. Here, focus is put mainly on phenolic antioxidant structures that belong to the class of direct antioxidants. Experimental and clinical evidence for the neuroprotective potential of alpha-tocopherol (vitamin E) and 17beta-estradiol (estrogen) is shortly summarized and an outlook is given on possible novel antioxidant lead structures with improved pharmacological features.     

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.     

Protection of Saccharomyces cerevisiae against oxidative and radiation-caused damage by alkyl hydroxybenzenes

The effects of C7-alkylhydroxybenzene (C7-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under gamma irradiation of cell suspensions using doses of 10-150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensibilized with chlorin e6 (10 micrograms/l). C7-AHB was found to exert a protective effect. The addition of 0.05-0.16 vol% of C7-AHB to cell suspensions 30 min before irradiation protected yeast cells from gamma radiation (50 krad). The protective effect of C7-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C7-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C7-AHB demonstrated that the protective mechanism of C7-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial C7-AHB are not related to their chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.