Novel lipoic acid analogues that inhibit nitric oxide synthase

The synthesis and biological activity of novel lipoic acid analogues are reported. Lipoic acid and structural homologues coupled to arylthiophene amidine via carboxamide linkers are metabolic antioxidants capable of protecting neuronal cells against glutamate cytotoxicity, preventing loss of intracellular glutathione, and inhibit nitric oxide synthase.     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of alpha-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents alpha-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.     

Pharmacological and physiological properties of serofendic acid, a novel neuroprotective substance isolated from fetal calf serum

Excess activation of glutamate receptors and production of large amount of free radicals including nitric oxide (NO) may be responsible for neuronal death associated with neurodegenerative disorders, but endogenous defense systems that protect neurons from these insults are poorly understood. In the course of studies to explore neuroprotective substance in mammalian origin, we isolated a neuroprotective factor from an ether extract of fetal calf serum based on the ability to protect rat primary cortical neurons against NO-induced cytotoxicity. A novel lipophilic low-molecular-weight substance that exerted potent neuroprotective actions at submicromolar concentrations was named "serofendic acid". Mass spectrometry and nuclear magnetic resonance spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a NO donor as well as of glutamate, although it did not affect glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO but was capable of inhibiting the generation of hydroxyl radical, a presumed 'executor' radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight neuroprotective factor that attenuates free radical-mediated damage triggered by excessive stimulation of neuronal glutamate receptors.     

Chebulinic acid attenuates glutamate-induced HT22 cell death by inhibiting oxidative stress,calcium influx and MAPKs phosphorylation

Glutamate-induced excitotoxicity and oxidative stress is a major causative factor in neuronal cell death in acute brain injuries and chronic neurodegenerative diseases. The prevention of oxidative stress is a potential therapeutic strategy. Therefore, in the present study, we aimed to examine a potential therapeutic agent and its protective mechanism against glutamate-mediated cell death. We first found that chebulinic acid isolated from extracts of the fruit of Terminalia chebula prevented glutamate-induced HT22 cell death. Chebulinic acid significantly reduced intracellular reactive oxygen species (ROS) production and Ca²⁺ influx induced by glutamate. We further demonstrated that chebulinic acid significantly decreased the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, as well as inhibiting pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 protein expression. Moreover, we demonstrated that chebulinic acid significantly reduced the apoptosis induced by glutamate in HT22 cells. In conclusion, our results in this study suggest that chebulinic acid is a potent protectant against glutamate-induced neuronal cell death via inhibiting ROS production, Ca²⁺ influx, and phosphorylation of MAPKs, as well as reducing the ratio of Bax to Bcl-2, which contribute to oxidative stress-mediated neuronal cell death.     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of α-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol®) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents α-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.     

Novel osmotin attenuates glutamate-induced synaptic dysfunction and neurodegeneration via the JNK/PI3K/Akt pathway in postnatal rat brain

The glutamate-induced excitotoxicity pathway has been reported in several neurodegenerative diseases. Molecules that inhibit the release of glutamate or cause the overactivation of glutamate receptors can minimize neuronal cell death in these diseases. Osmotin, a homolog of mammalian adiponectin, is a plant protein from Nicotiana tabacum that was examined for the first time in the present study to determine its protective effects against glutamate-induced synaptic dysfunction and neurodegeneration in the rat brain at postnatal day 7. The results indicated that glutamate treatment induced excitotoxicity by overactivating glutamate receptors, causing synaptic dysfunction and neuronal apoptosis after 4 h in the cortex and hippocampus of the postnatal brain. In contrast, post-treatment with osmotin significantly reversed glutamate receptor activation, synaptic deficit and neuronal apoptosis by stimulating the JNK/PI3K/Akt intracellular signaling pathway. Moreover, osmotin treatment abrogated glutamate-induced DNA damage and apoptotic cell death and restored the localization and distribution of p53, p-Akt and caspase-3 in the hippocampus of the postnatal brain. Finally, osmotin inhibited glutamate-induced PI3K-dependent ROS production in vitro and reversed the cell viability decrease, cytotoxicity and caspase-3/7 activation induced by glutamate. Taken together, these results suggest that osmotin might be a novel neuroprotective agent in excitotoxic diseases.     

Neuroprotective Effects of Alpha Lipoic Acid on Haloperidol-Induced Oxidative Stress in the Rat Brain

Haloperidol is an antipsychotic drug that exerts its' antipsychotic effects by inhibiting dopaminergic neurons. Although the exact pathophysiology of haloperidol extrapyramidal symptoms are not known, the role of reactive oxygen species in inducing oxidative stress has been proposed as one of the mechanisms of prolonged haloperidol-induced neurotoxicity. In the present study, we evaluate the protective effect of alpha lipoic acid against haloperidol-induced oxidative stress in the rat brain. Sprague Dawley rats were divided into control, alpha lipoic acid alone (100 mg/kg p.o for 21 days), haloperidol alone (2 mg/kg i.p for 21 days), and haloperidol with alpha lipoic acid groups (for 21 days). Haloperidol treatment significantly decreased levels of the brain antioxidant enzymes super oxide dismutase and glutathione peroxidase and concurrent treatment with alpha lipoic acid significantly reversed the oxidative effects of haloperidol. Histopathological changes revealed significant haloperidol-induced damage in the cerebral cortex, internal capsule, and substantia nigra. Alpha lipoic acid significantly reduced this damage and there were very little neuronal atrophy. Areas of angiogenesis were also seen in the alpha lipoic acid-treated group. In conclusion, the study proves that alpha lipoic acid treatment significantly reduces haloperidol-induced neuronal damage.     

Propofol hemisuccinate protects neuronal cells from oxidative injury

Oxidative stress contributes to the neuronal death observed in neurodegenerative disorders and neurotrauma. Some antioxidants for CNS injuries, however, have yet to show mitigating effects in clinical trials, possibly due to the impermeability of antioxidants across the blood-brain barrier (BBB). Propofol (2,6-diisopropylphenol), the active ingredient of a commonly used anesthetic, acts as an antioxidant, but it is insoluble in water. Therefore, we synthesized its water-soluble prodrug, propofol hemisuccinate sodium salt (PHS), and tested for its protective efficacy in neuronal death caused by non-receptor-mediated, oxidative glutamate toxicity. Glutamate induces apoptotic death in rat cortical neurons and the mouse hippocampal cell line HT-22 by blocking cystine uptake and causing the depletion of intracellular glutathione, resulting in the accumulation of reactive oxygen species (ROS). PHS has minimal toxicity and protects both cortical neurons and HT-22 cells from glutamate. The mechanism of protection is attributable to the antioxidative property of PHS because PHS decreases the ROS accumulation caused by glutamate. Furthermore, PHS protects HT-22 cells from oxidative injury induced by homocysteic acid, buthionine sulfoximine, and hydrogen peroxide. For comparison, we also tested alpha-tocopherol succinate (TS) and methylprednisolone succinate (MPS) in the glutamate assay. Although TS is protective against glutamate at lower concentrations than PHS, TS is toxic to HT-22 cells. In contrast, MPS is nontoxic but also nonprotective against glutamate. Taken together, PHS, a water-soluble prodrug of propofol, is a candidate drug to treat CNS injuries owing to its antioxidative properties, low toxicity, and permeability across the BBB.     

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.     

Antioxidant activities of seabuckthorn (Hippophae rhamnoides) during hypoxia induced oxidative stress in glial cells

The present study reports the cytoprotective and antioxidant properties of alcoholic leaf extract of seabuckthorn (SBT) against hypoxia induced oxidative stress in C-6 glioma cells. Exposure of cells to hypoxia for 12 h resulted in a significant increase in cytotoxicity and decrease in mitochondrial transmembrane potential compared to the controls. Further an appreciable increase in nitric oxide and reactive oxygen species (ROS) production was noted which in turn was responsible for fall in intracellular antioxidant levels and GSH/GSSG ratio. There was a significant increase in DNA damage during hypoxia as revealed by comet assay. Pretreatment of cells with alcoholic leaf extract of SBT at 200 μg/ml significantly inhibited cytotoxicity, ROS production and maintained antioxidant levels similar to that of control cells. Further, the leaf extract restored the mitochondrial integrity and prevented the DNA damage induced by hypoxia. These results indicate that the leaf extract of SBT has strong antioxidant and cytoprotective activity against hypoxia induced oxidative injury. (Mol Cell Biochem 278: 9–14, 2005)     

Renoprotective Effects of Alpha Lipoic Acid on Iron Overload-Induced Kidney Injury in Rats by Suppressing NADPH Oxidase 4 and p38 MAPK Signaling

Biological Trace Element Research - We aimed to investigate the protective effect of alpha lipoic acid (ALA), a powerful antioxidant, against oxidative kidney damage induced by iron overload in...     

Protective effects of green tea polyphenols on human HepG2 cells against oxidative damage of fenofibrate

The aim of this work was to investigate the protective effects of green tea polyphenols on the cytotoxic effects of hypolipidemic agent fenofibrate (FF), a peroxisome proliferator (PP), in human HepG2 cells. The results showed that high concentrations of FF induced human HepG2 cell death through a mechanism involving an increase of reactive oxygen species (ROS) and intracellular reduced glutathione (GSH) depletion. These effects were partially prevented by antioxidant green tea polyphenols. The elevated expression of PP-activated receptors alpha (PPARalpha) in HepG2 cells induced by FF was also decreased by treatment with green tea polyphenols. In conclusion, this result demonstrates that oxidative stress and PPARalpha are involved in FF cytotoxicity and green tea polyphenols have a protective effect against FF-induced cellular injury. It may be beneficial for the hyperlipidemic patients who were administered the hypolipidemic drug fenofibrate to drink tea or use green tea polyphenols synchronously during their treatment.     

Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells

Dihydrocaffeic acid (DHCA) is a metabolite of caffeic acid with potent antioxidant properties. Since DHCA has been detected in human plasma following coffee ingestion, we tested the hypothesis that DHCA protects the endothelium from oxidative stress in a model in human-derived EA.hy926 endothelial cells. During culture for 16-24 hours, the cells accumulated DHCA against a concentration gradient to low millimolar concentrations. In alpha-tocopherol-loaded cells, DHCA spared alpha-tocopherol during overnight culture in a dose-dependent manner. In response to oxidant stress induced by a water-soluble free radical initiator, both alpha-tocopherol and DHCA diminished oxidation of cis-parinaric acid that had been incorporated into the cells, although their antioxidant activities were not additive. DHCA also decreased intracellular oxidation of dihydrofluorescein due to redox cycling by menadione. This suggests that the protective effects of DHCA were caused by scavenging of intracellular reactive oxygen species. DHCA also increased nitric oxide synthase activity in a dose-dependent manner in cultured cells, which was associated with a comparable increase in endothelial nitric oxide synthase protein. Although the DHCA concentrations required for these effects are higher than those likely to be present in plasma or the interstitium, these results indicate that DHCA can function as an intracellular antioxidant.     

Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.     

Mangiferin attenuates oxidative stress induced renal cell damage through activation of PI3K induced Akt and Nrf-2 mediated signaling pathways

BackgroundMangiferin is a polyphenolic xanthonoid with remarkable antioxidant activity. Oxidative stress plays the key role in tert-butyl hydroperoxide (tBHP) induced renal cell damage. In this scenario, we consider mangiferin, as a safe agent in tBHP induced renal cell death and rationalize its action systematically, in normal human kidney epithelial cells (NKE).MethodsNKE cells were exposed to 20 µM mangiferin for 2 h followed by 50 µM tBHP for 18 h. The effect on endogenous ROS production, antioxidant status (antioxidant enzymes and thiols), mitochondrial membrane potential, apoptotic signaling molecules, PI3K mediated signaling cascades and cell cycle progression were examined using various biochemical assays, FACS and immunoblot analyses.ResultstBHP exposure damaged the NKE cells and decreased its viability. It also elevated the intracellular ROS and other oxidative stress-related biomarkers within the cells. However, mangiferin dose dependently, exhibited significant protection against this oxidative cellular damage. Mangiferin inhibited tBHP induced activation of different pro-apoptotic signals and thus protected the renal cells against mitochondrial permeabilization. Further, mangiferin enhanced the expression of cell proliferative signaling cascade molecules, Cyclin d1, NFκB and antioxidant molecules HO-1, SOD2, by PI3K/Akt dependent pathway. However, the inhibitor of PI3K abolished mangiferin's protective activity.ConclusionsResults show Mangiferin maintains the intracellular anti-oxidant status, induces the expression of PI3K and its downstream molecules and shields NKE cells against the tBHP induced cytotoxicity.General significanceMangiferin can be indicated as a therapeutic agent in oxidative stress-mediated renal toxicity. This protective action of mangiferin primarily attributes to its potent antioxidant and antiapoptotic nature.     

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.     

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.     

Mangiferin Antagonizes Rotenone: Induced Apoptosis Through Attenuating Mitochondrial Dysfunction and Oxidative Stress in SK-N-SH Neuroblastoma Cells

In the present study, using a human neuroblastoma SK-N-SH cells, we explored antioxidant, mitochondrial protective and antiapoptotic properties of mangiferin against rotenone-mediated cytotoxicity. SK-N-SH cells are divided into four experimental groups based on 3-(4,5-dimethyl2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay—untreated cells, cells incubated with rotenone (100 nM), cells treated with mangiferin (20 μg) (pretreatment 4 h before) + rotenone (100 nM) and mangiferin alone treated. 24 h after treatment with rotenone and 28 h after treatment with mangiferin, levels of ATP thiobarbituricacid reactive substances and reduced glutathione and activities of enzymatic antioxidants including superoxide dismutase, catalase and glutathione peroxidise were measured. Finally mitochondrial transmembrane potential and expressions of apoptotic protein were also analysed. Pre-treatment with mangiferin significantly enhanced cell viability, ameliorated decrease in mitochondrial membrane potential and decreased rotenone-induced apoptosis in the cellular model of Parkinson’s disease. Moreover oxidative imbalance induced by rotenone was partially rectified by mangiferin. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone induced cytotoxicity.     

Induction by beta-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca(2+)-dependent formation of reactive oxygen species

The component of the venom of the Taiwanese banded krait Bungarus multicinctus, beta-bungarotoxin (beta-BuTx), acts as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. While induction of cell death is dependent on toxin binding to voltage-activated K+ channels and subsequent internalization, the pro-apoptotic signals triggered by picomolar concentrations of beta-BuTx are not understood. Following toxin binding, a dramatic increase in intracellular Ca2+ became detectable after 30 min, and in reactive oxygen species (ROS) after 3-4 h. Conversely, Ca2+ chelators, radical quenchers and antioxidants efficiently antagonized beta-BuTx induced apoptosis. As shown for the antioxidant 2,3-dihydroxybenzoic acid, analysis by matrix assisted laser desorbtion-time of flight (MALDI-TOF) mass spectrometry excluded the protective effects to be due to reductive cleavage of the toxic beta-BuTx dimer. Inhibitors of the intracellular antioxidant defence system enhanced neuronal susceptibility to beta-BuTx, supporting the essential role of ROS in beta-BuTx-initiated apoptosis. Cell damage was accompanied by an accumulation of markers of oxidative cell stress, phospholipid hydroxyperoxides and the lipid peroxidation product, malonyl dialdehyde. These observations indicate that beta-BuTx-induced cell death resulted from an intracellular signalling cascade involving subsequent stages of a dramatic rise in free Ca2+, the accumulation of ROS, membrane lipid peroxidation and, finally, apoptosis.