Glutamate-induced HT22 Cell Death Research Articles

Lumbricus Extract Prevents LPS-Induced Inflammatory Activation of BV2 Microglia and Glutamate-Induced Hippocampal HT22 Cell Death by Suppressing MAPK/NF-κB/NLRP3 Signaling and Oxidative Stress.

Microglia-induced inflammatory signaling and neuronal oxidative stress are mutually reinforcing processes central to the pathogenesis of neurodegenerative diseases. Recent studies have shown that extracts of dried Pheretima aspergillum (Lumbricus) can inhibit tissue fibrosis, mitochondrial damage, and asthma. However, the effects of Lumbricus extracts on neuroinflammation and neuronal damage have not been previously studied. Therefore, to evaluate the therapeutic potential of Lumbricus extract for neurodegenerative diseases, the current study assessed the extract's anti-inflammatory and antioxidant activities in BV2 microglial cultures stimulated with lipopolysaccharide (LPS) along with its neuroprotective efficacy in mouse hippocampal HT22 cell cultures treated with excess glutamate. Lumbricus extract dose-dependently inhibited the LPS-induced production of multiple proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) and reversed the upregulation of proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Lumbricus also activated the antioxidative nuclear factor erythroid 2-relayed factor 2/heme oxygenase-1 pathway and inhibited LPS-induced activation of the nuclear factor-κB/mitogen-activated protein kinases/NOD-like receptor family pyrin domain containing 3 inflammatory pathway. In addition, Lumbricus extract suppressed the glutamate-induced necrotic and apoptotic death of HT22 cells, effects associated with upregulated expression of antiapoptotic proteins, downregulation of pro-apoptotic proteins, and reduced accumulation of reactive oxygen species. Chromatography revealed that the Lumbricus extract contained uracil, hypoxanthine, uridine, xanthine, adenosine, inosine, and guanosine. Its effects against microglial activation and excitotoxic neuronal death reported herein support the therapeutic potential of Lumbricus for neurodegenerative diseases.

Extract of Styrax japonica attenuates glutamate-induced apoptosis via regulating MAPK signaling pathway in HT22 hippocampal cells

Two-thirds of people with dementia suffer from Alzheimer’s disease, and there is a need to develop treatments with fewer side effects. Cholinergic and glutamate-induced brain damage occurs in the early stages of Alzheimer’s disease, so substances that suppress these symptoms may be potential candidates for the treatment. Ethanol extracts of 40 kinds of oriental medicine plants were examined whether they have acetylcholine esterase (Ache) inhibitory properties. We next investigated whether the ethanol extracts of six oriental medicine plants showing Ache inhibitory activity could inhibit glutamate-induced HT22 cell death. The ethanol extract of Styrax japonica (EESJ) was found to be relatively superior in both inhibitory activities. MTT and annexin V/PI staining assays confirmed that EESJ inhibited glutamate-induced apoptosis in the HT22 mouse hippocampal cells. EESJ also suppressed glutamate-mediated ROS production and attenuated the phosphorylation levels of MAPK members including ERK, JNK, and p38 kinases. Therefore, EESJ is a suitable candidate for developing a substance of Alzheimer’s disease treatment.

The Neuroprotective Effects of Arecae Pericarpium against Glutamate-Induced HT22 Cell Cytotoxicity.

Arecae Pericarpium has been found to exert anti-migraine, antidepressant, and antioxidative effects. However, the mechanisms involved are unclear. This study explored the possibility that Arecae Pericarpium ethanol extract (APE) exerts neuroprotective effects against oxidative stress-induced neuronal cell death. Since glutamate excitotoxicity has been implicated in the pathogenesis and development of several neurodegenerative disorders, we explored the mechanisms of action of APE on oxidative stress-induced by glutamate. Our results revealed that pretreatment with APE prevents glutamate-induced HT22 cell death. APE also reduced both the levels of intracellular reactive oxygen species and the apoptosis of cells, while maintaining glutamate-induced mitochondrial membrane potentials. Western blotting showed that pretreatment with APE facilitates the upregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) phosphorylation; the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2); and the production of antioxidant enzymes, including catalase, glutamate-cysteine ligase catalytic subunits, NAD(P)H quinone oxidoreductase 1, and heme oxygenase (HO)-1. The administration of LY294002, a PI3K/Akt inhibitor, attenuated the neuroprotective effects of APE on oxidative stress-induced neuronal cell damage. This allowed us to infer that the protective effects of APE on oxidative damage to cells can be attributed to the PI3K/Akt-mediated Nrf-2/HO-1 signaling pathway.

Chemical Constituents from the Aerial Parts of Elsholtzia ciliata and Their Protective Activities on Glutamate-Induced HT22 Cell Death

A new phenolic glucoside, (7E,9E)-3-hydroxyavenalumic acid-3-O-[6'-O-(E)-caffeoyl]-β-d-glucopyranoside (1), and three new acetylated flavone glycosides, acacetin-7-O-[β-d-glucopyranosyl(1″″→2″)-4‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (3), acacetin-7-O-[6″″-O-acetyl-β-d-glucopyranosyl(1″″→2″)-3‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (5), and acacetin-7-O-[3″″,6″″-di-O-acetyl-β-d-glucopyranosyl(1″″→2″)-4‴-O-acetyl-α-l-rhamnopyranosyl(1‴→6″)]-β-d-glucopyranoside (7), as well as 34 known compounds (2, 4, 6, and 8-38) were isolated from the aerial parts of Elsholtzia ciliata. The chemical structures of the new compounds were determined by spectroscopic/spectrometric data interpretation using NMR and HRESIMS. The neuroprotective effect of the isolated compounds was evaluated by a cell viability assay on HT22 murine hippocampal neuronal cells. Among them, 23 compounds, including new substances 1 and 3, exhibited neuroprotective effects against glutamate-induced HT22 cell death. In particular, compounds 2, 16, 17, 20, 22, 28, 29, and 31 presented potent neuroprotective effects with EC50 values of 1.5-8.3 μM.

Neuroprotective Effects of Tetrahydrocurcumin against Glutamate-Induced Oxidative Stress in Hippocampal HT22 Cells.

In the central nervous system, glutamate is a major excitable neurotransmitter responsible for many cellular functions. However, excessive levels of glutamate induce neuronal cell death via oxidative stress during acute brain injuries as well as chronic neurodegenerative diseases. The present study was conducted to examine the effect of tetrahydrocurcumin (THC), a major secondary metabolite of curcumin, and its possible mechanism against glutamate-induced cell death. We prepared THC using curcumin isolated from Curcuma longa (turmeric) and demonstrated the protective effect of THC against glutamate-induced oxidative stress in HT22 cells. THC abrogated glutamate-induced HT22 cell death and showed a strong antioxidant effect. THC also significantly reduced intracellular calcium ion increased by glutamate. Additionally, THC significantly reduced the accumulation of intracellular oxidative stress induced by glutamate. Furthermore, THC significantly diminished apoptotic cell death indicated by annexin V-positive in HT22 cells. Western blot analysis indicated that the phosphorylation of mitogen-activated protein kinases including c-Jun N-terminal kinase, extracellular signal-related kinases 1/2, and p38 by glutamate was significantly diminished by treatment with THC. In conclusion, THC is a potent neuroprotectant against glutamate-induced neuronal cell death by inhibiting the accumulation of oxidative stress and phosphorylation of mitogen-activated protein kinases.

Procyanidin B2 3″-O-gallate Isolated from Reynoutria elliptica Prevents Glutamate-Induced HT22 Cell Death by Blocking the Accumulation of Intracellular Reactive Oxygen Species.

In this study, we examined the neuroprotective effects of MeOH extract and bioactive compounds obtained from Reynoutria elliptica seeds using HT22 cells from the murine hippocampal cell line as its underlying molecular basis, which has not yet been elucidated. Our study showed that the MeOH extract of R. elliptica seeds strongly protected HT22 cells from glutamate toxicity. To clarify the responsible compound for the neuroprotective effects, we took an interest in procyanidins of R. elliptica since procyanidins are known to exhibit high structural diversity and neuroprotective activity. To isolate the procyanidins efficiently, a phytochemical investigation of the MeOH extract from R. elliptica seeds using the LC/MS-guided isolation approach was applied, and procyanidin B2 3″-O-gallate (1) was successfully isolated. The structure of 1 was elucidated by analyzing the nuclear magnetic resonance spectroscopic data and LC/MS analysis. The neuroprotective activities of 1 were thoroughly examined using HT22 cells. Compound 1 exhibited a strong antioxidant efficacy and blocked glutamate-mediated increase in the reactive oxygen species (ROS) accumulation. Furthermore, compound 1 significantly inhibited the phosphorylation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase, which were increased by glutamate. These findings prove that the extract of R. elliptica seeds containing procyanidin B2 3″-O-gallate, which is a strong neuroprotective component, can be used as a functional food forattenuating and regulating neurological disorders.

Neuroprotective effect of a dietary supplement against glutamate-induced excitotoxicity in retina.

To evaluate the neuroprotective effect of a dietary supplement (ClearVision EX®; CV) against glutamate-induced excitotoxicity in retina. We evaluated the protective effects CV on glutamate-induced cell toxicity of an immortalized mouse hippocampal cell line (HT-22) in vitro and N-methyl-D-aspartate (NMDA) induced retinal injury in vivo. Once-daily oral administration of CV or vehicle (5% Arabic gum) was started the day before the NMDA injection and continued until the end of the study. Electroretinograms (ERGs) were recorded to evaluate the retinal function at 2d after NMDA injection. Furthermore, a histological evaluation, Western blot analysis, and immunohistochemistry were performed for assessing the signal transduction pathway. HT-22 cell death was induced by the addition of glutamate and co-incubation with CV protected against it. Oral administration of CV inhibited the decrease in scotopic threshold response amplitudes induced by the intravitreal injection of NMDA and those of the thickness of the inner retinal layer in the histological evaluation. The increased phosphorylated levels of extracellular signal-regulated kinase (ERK) but not cAMP response element binding protein (CREB) or Akt were observed 1h after NMDA injection in both the vehicle- and CV-treated rats; however, pERK activation was no more upregulated at 3h after NMDA injection. pERK upregulation was observed in Müller cells. CV shows a protective effect against both glutamate-induced HT-22 cell death and NMDA-induced retinal damage. pERK upregulation in the Müller cells plays a key role in the protective effect of CV against glutamate-induced retinal toxicity.

Procyanidin C1 Activates the Nrf2/HO-1 Signaling Pathway to Prevent Glutamate-Induced Apoptotic HT22 Cell Death.

Natural sources are very promising materials for the discovery of novel bioactive compounds with diverse pharmacological effects. In recent years, many researchers have focused on natural sources as a means to prevent neuronal cell death in neuropathological conditions. This study focused on identifying neuroprotective compounds and their underlying molecular mechanisms. Procyanidin C1 (PC-1) was isolated from grape seeds and assessed for biological effects against glutamate-induced HT22 cell death. The results showed that PC-1 strongly prevented glutamate-induced HT22 cell death. Moreover, PC-1 was also found to prevent glutamate-induced chromatin condensation and reduce the number of annexin V-positive cells indicating apoptotic cell death. Procyanidin C1 possessed a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and inhibited glutamate-induced accumulation of intracellular reactive oxygen species and protein carbonylation. Additionally, PC-1 mediated nuclear translocation of nuclear factor erythroid-derived 2-related factor 2 and increased the expression levels of heme oxygenase (HO-1). Inhibition of HO-1 by tin protoporphyrin, a synthetic inhibitor, reduced the protective effect of PC-1. Furthermore, PC-1 also blocked glutamate-induced phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38, but not JNK. This study is the first experimental report to demonstrate the neuroprotective effects of PC-1 against glutamate-induced cytotoxicity in HT22 cells. Therefore, our results suggest that PC-1, as a potent bioactive compound of grape seeds, can prevent neuronal cell death in neuropathological conditions.

Neuroprotective effects of myristargenol A against glutamate-induced apoptotic HT22 cell death.

Glutamate is an important neurotransmitter in the central nervous system; however, at high concentrations, it causes excitotoxicity and many neurological disorders. Excitotoxicity induces cell death by apoptosis. Thus, factors that can inhibit the apoptotic pathways are a target of drug development for the treatment and prevention of neurodegenerative diseases. Herein, the antioxidative and neuroprotective effects of myristargenol A were examined in glutamate-induced mouse hippocampal neuronal HT22 cells. When the HT22 cells were stressed with glutamate, cell viability decreased to 44.4 ± 5.6% when compared with the case of the control cells (100 ± 4.8%); however, when these cells were treated with myristargenol A (10 μM), the cell viability was increased by 113.6 ± 2.3%. The protective effect of myristargenol A against the apoptosis of glutamate-induced HT22 cells was also confirmed using FITC-annexin V/propidium iodide double staining. In addition, myristargenol A protected the mitochondrial membrane potential (ΔΨm). Subsequently, the expression levels of proteins in the caspase pathway related with the induction of apoptosis were decreased. Moreover, the expression levels of mitochondrial-related proteins, such as Bcl-2 and Bax, were examined, and it was found that the expression ratio of Bax/Bcl-2 decreased. In addition, myristargenol A inhibited the activity of mitogen-activated protein kinases, including p38 and c-Jun N-terminal kinase, for an oxidative stress protection effect but increased the activity of the extracellular signal-regulated kinases 1 and 2 for cell proliferation. These results reveal that myristargenol A possesses a neuroprotective effect against the neuronal cell damage caused by glutamate.

Chemical Identification of Isoflavonoids from a Termite-Associated Streptomyces sp. RB1 and Their Neuroprotective Effects in Murine Hippocampal HT22 Cell Line.

Insect-associated bacteria have been recognized as a very promising natural resource for discovering bioactive secondary metabolites with diverse pharmacological effects. One new isoflavonoid glycoside, termisoflavone D (1), together with seven known isoflavonoids (2–8), were identified from MeOH extracts of the fungus-growing termite-associated Streptomyces sp. RB1. The chemical structure of the new compound 1 was elucidated using comprehensive spectroscopic methods including 1D and 2D NMR, along with LC/MS analysis. The existence of two rhamnose moieties in 1 was determined with comparative NMR analysis, and the absolute configuration was elucidated using chemical reactions. The neuroprotective activities of compounds 1–8 were thoroughly investigated using the murine hippocampal HT22 cell line. Compound 5 prevented glutamate-induced HT22 cell death by blocking intracellular reactive oxygen species (ROS) accumulation. The present study provides the first experimental evidence for the potential use of isoflavonoids from termite-associated bacteria as lead compounds that can prevent neuronal damage induced by glutamate.

Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor.

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of PARP can lead to NAD+ depletion, mitochondrial energy failure, and cell death. Since PARP activation facilitates genomic but impedes mitochondrial DNA repair, nonselective PARP inhibitors are likely to have opposing effects in these cellular compartments. Herein, we describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100 nM) and more potent than veliparib in protection from oxygen-glucose deprivation (OGD) in primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial NAD+ after OGD; however, only XJB-veliparib prevented release of NAD+ into cytosol. XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria but not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in HT22 cells exposed to H2O2 to produce DNA damage, XJB-veliparib (10 μM) had no effect on nuclear DNA repair, in contrast to veliparib (10 μM) where DNA repair was retarded. XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g., ischemia, oxidative stress, and radiation exposure.

Protective effect of furosin isolated from Euphorbia helioscopia against glutamate-induced HT22 cell death

Protective effect of furosin isolated from Euphorbia helioscopia against glutamate-induced HT22 cell death

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.

Discovery of a novel multifunctional carbazole–aminoquinoline dimer for Alzheimer's disease: copper selective chelation, anti-amyloid aggregation, and neuroprotection

A novel multifunctional carbazole–aminoquinoline dimer PZ001 was designed, synthesized, and evaluated. The results indicated that PZ001 possessed selective copper chelation, and inhibited copper-induced Aβ1–42 aggregation. Furthermore, PZ001 exerted powerful neuroprotection against glutamate-induced HT22 cell death. These results suggest that PZ001 may be a promising multifunctional anti-AD compound.

The comparison of neuroprotective effects of isoliquiritigenin and its Phase I metabolites against glutamate-induced HT22 cell death

It is becoming increasingly important to investigate drug metabolites to evaluate their toxic or preventive effects after administration of the parent compound. In our previous study, isoliquiritigenin isolated from Glycyrrhizae Radix effectively protected mouse-derived hippocampal neuronal cells (HT22) against 5mM glutamate-induced oxidative stress. However, there is little information on the protective effects of the metabolites of isoliquiritigenin on HT22 cells. In this study, isoliquiritigenin and its Phase I metabolites were prepared and their neuroprotective activities on glutamate-treated HT22 cells were compared. The prepared metabolites were liquiritigenin (1), 2′,4,4′,5′-tetrahydroxychalcone (2), sulfuretin (3), butein (4), davidigenin (5), and cis-6,4′-dihydroxyaurone (6). Among the six metabolites, 4 showed better neuroprotective effects than the parent compound, isoliquiritigenin. Our study suggests that the neuroprotective effect of isoliquiritigenin could be elevated by its active metabolite 4, which is a chalcone containing a catechol group in the B ring.

Butein provides neuroprotective and anti-neuroinflammatory effects through Nrf2/ARE-dependent haem oxygenase 1 expression by activating the PI3K/Akt pathway.

Butein, 3,4,2',4'-tetrahydroxychalcone, has various pharmacological effects. However, no study has demonstrated the specific neurobiological mechanisms of the effects of butein in neuronal cells. The present study examined the role of butein as an antioxidative and anti-inflammatory inducer of haem oxygenase 1 (HO1) in mouse hippocampal HT22, BV2 microglial and primary mouse hippocampus neurons. We investigated the neuroprotective effects of butein on glutamate-induced HT22 cell and primary mouse hippocampal neuron death and its anti-neuroinflammatory effects on LPS-induced activation of BV2 cells. We elucidated the underlying mechanisms by assessing the involvement of NF-κB, HO1, nuclear factor-E2-related factor 2 (Nrf2) and Akt signalling. Butein decreased cellular oxidative injury and the production of ROS in glutamate-treated HT22 cells and primary mouse hippocampal neurons. Furthermore, butein suppressed LPS-induced pro-inflammatory enzymes and mediators in BV2 microglia. Butein inhibited IL-6, IL-1β and TNF-α production and mRNA expression. In addition, butein decreased NO and PGE2 production and inducible NOS and COX-2 expression through the NF-κB signalling pathway. Butein up-regulated Nrf2/ARE-mediated HO1 expression through the PI3K/Akt pathway and this was positively associated with its cytoprotective effects and anti-neuroinflammatory actions. Our results indicate that butein effectively prevents glutamate-induced oxidative damage and LPS-induced activation and that the induction of HO1 by butein through the PI3K/Akt pathway and Nrf2 activation appears to play a pivotal role in its effects on neuronal cells. Our results provide evidence for the neuroprotective properties of butein.

Natural Xanthones from Garcinia mangostana with Multifunctional Activities for the Therapy of Alzheimer's Disease.

Natural xanthones have diversity pharmacological activities. Here, a series of xanthones isolated from the pericarps of Garcinia mangostana Linn, named α-Mangostin, 8-Deoxygartanin, Gartanin, Garciniafuran, Garcinone C, Garcinone D, and γ-Mangostin were investigated. Biological screening performed in vitro and in Escherichia coli cells indicated that most of the xanthones exhibited significant inhibition of self-induced β-amyloid (Aβ) aggregation and also β-site amyloid precursor protein-cleaving enzyme 1, acted as potential antioxidants and biometal chelators. Among these compounds, α-Mangostin, Gartanin, Garcinone C and γ-Mangostin showed better antioxidant properties to scavenge Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) free radical than Trolox, and potent neuroprotective effects against glutamate-induced HT22 cell death partly by up-regulating HO-1 protein level and then scavenging reactive oxygen species. Moreover, Gartanin, Garcinone C and γ-Mangostin could be able to penetrate the blood-brain barrier (BBB) in vitro. These findings suggest that the natural xanthones have multifunctional activities against Alzheimer's disease (AD) and could be promising compounds for the therapy of AD.

The 5-Lipoxygenase Inhibitor Zileuton Confers Neuroprotection against Glutamate Oxidative Damage by Inhibiting Ferroptosis.

5-Lipoxygenase (5-LOX) inhibitors have been shown to be protective in several neurodegenerative disease models; however, the underlying mechanisms remain unclear. We investigated whether 5-LOX inhibitor zileuton conferred direct neuroprotection against glutamate oxidative toxicity by inhibiting ferroptosis, a newly identified iron-dependent programmed cell death. Treatment of HT22 mouse neuronal cell line with glutamate resulted in significant cell death, which was inhibited by zileuton in a dose-dependent manner. Consistently, zileuton decreased glutamate-induced production of reactive oxygen species but did not restore glutamate-induced depletion of glutathione. Moreover, the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (ZVAD-fmk) neither prevented HT22 cell death induced by glutamate nor affected zileuton protection against glutamate oxidative toxicity, suggesting that zileuton did not confer neuroprotection by inhibiting caspase-dependent apoptosis. Interestingly, glutamate-induced HT22 cell death was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. Moreover, zileuton protected HT22 neuronal cells from erastin-induced ferroptosis. However, we did not observe synergic protective effects of zileuton and ferrostatin-1 on glutamate-induced cell death. These results suggested that both the 5-LOX inhibitor zileuton and the ferropotosis inhibitor ferrostatin-1 acted through the same cascade to protect against glutamate oxidative toxicity. In conclusion, our results suggested that zileuton protected neurons from glutamate-induced oxidative stress at least in part by inhibiting ferroptosis.

Polyozellin, a key constituent of the edible mushroom Polyozellus multiplex, attenuates glutamate-induced mouse hippocampal neuronal HT22 cell death.

Polyozellus multiplex (PM), a Korean edible mushroom, has biological activities such as chemoprevention of stomach cancer, inhibition of lipid peroxidation, and reduction of prolyl endopeptidase activity. However, there are little reports on the protective effects of PM or its constituents against glutamate-induced mouse hippocampal neuronal cell (HT22) death. In this study, polyozellin (PZ), a key constituent of PM, was applied to glutamate-treated HT22 cells to evaluate its neuroprotective mechanisms. PZ (25 μM) dramatically increased the HT22 cell viability when the cell death was induced by 5 mM glutamate for 12 h, which was mediated by inhibition of Ca(2+) influx, intracellular reactive oxygen species (ROS) production, and lipid peroxidation. PZ also regulated expression of Bid, Bcl-2, and apoptosis-inducing factor (AIF), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). These data suggest that PM and its constituent PZ might be useful for prevention and treatment of neurodegenerative disorders.

Isolation and identification of twelve metabolites of isocorynoxeine in rat urine and their neuroprotective activities in HT22 cell assay.

Isocorynoxeine, one of the major alkaloids from Uncaria Hook, shows the effects of lowering blood pressure, vasodilatation, and protection against ischemia-induced neuronal damage. In this paper, the metabolism of isocorynoxeine was investigated in rats. Twelve metabolites and the parent drug were isolated by using solvent extraction and repeated chromatographic methods, and determined by spectroscopic methods including UV, MS, NMR, and CD experiments. Seven new compounds were identified as 11-hydroxyisocorynoxeine, 5-oxoisocorynoxeinic acid-22-O-β-D-glucuronide, 10-hydroxyisocorynoxeine, 17-O-demethyl-16,17-dihydro-5-oxoisocorynoxeine, 5-oxoisocorynoxeinic acid, 21-hydroxy-5-oxoisocorynoxeine, and oxireno[18, 19]-5-oxoisocorynoxeine, together with six known compounds identified as isocorynoxeine, 18,19-dehydrocorynoxinic acid, 18,19-dehydrocorynoxinic acid B, corynoxeine, isocorynoxeine-N-oxide, and corynoxeine-N-oxide. Possible metabolic pathways of isocorynoxeine are proposed. Furthermore, the activity assay for the parent drug and some of its metabolites showed that isocorynoxeine exhibited a significant neuroprotective effect against glutamate-induced HT22 cell death at the maximum concentration. However, little or weak neuroprotective activities were observed for M-3, M-6, M-7, and M-10. Our present study is important to further understand their metabolic fate and disposition in humans.