Toxicity In HT22 Cells Research Articles

MiR-153 promotes neural differentiation by activating the cell adhesion/Ca2+ signaling pathway and targeting ion channel activity in HT-22 cells by bioinformatic analysis

MicroRNAs have been studied extensively in neurodegenerative diseases. In a previous study, miR-153 promoted neural differentiation and projection formation in mouse hippocampal HT-22 cells. However, the pathways and molecular mechanism underlying miR-153-induced neural differentiation remain unclear. To explore the molecular mechanism of miR-153 on neural differentiation, we performed RNA sequencing on miR-153-overexpressed HT-22 cells. Based on RNA sequencing, differentially expressed genes (DEGs) and pathways in miR-153-overexpressed cells were identified. The Database for Annotation, Visualization and Integrated Discovery and Gene Set Enrichment Analysis were used to perform functional annotation and enrichment analysis of DEGs. Targetscan predicted the targets of miR-153. The Search Tool for the Retrieval of Interacting Genes and Cytoscape, were used to construct protein-protein interaction networks and identify hub genes. Q-PCR was used to detect mRNA expression of the identified genes. The expression profiles of the identified genes were compared between embryonic days 9.5 (E9.5) and E11.5 in the embryotic mouse brain of the GDS3442 dataset. Cell Counting Kit-8 assay was used to determine cell proliferation and cellular susceptibility to amyloid β-protein (Aβ) toxicity in miR-153-overexpressed cells. The results indicated that miR-153 increased cell adhesion/Ca2+ (Cdh5, Nrcam, and P2rx4) and Bdnf/Ntrk2 neurotrophic signaling pathway, and decreased ion channel activity (Kcnc3, Kcna4, Clcn5, and Scn5a). The changes in the expression of the identified genes in miR-153-overexpressed cells were consistent with the expression profile of GDS3442 during neural differentiation. In addition, miR-153 overexpression decreased cellular susceptibility to Aβ toxicity in HT-22 cells. In conclusion, miR-153 overexpression may promote neural differentiation by inducing cell adhesion and the Bdnf/Ntrk2 pathway, and regulating electrophysiological maturity by targeting ion channels. MiR-153 may play an important role in neural differentiation; the findings provide a useful therapeutic direction for neurodegenerative diseases.

Novel Carbon Dots Derived from Moutan Cortex Significantly Improve the Solubility and Bioavailability of Mangiferin.

Mangiferin (MA), a bioactive C-glucosyl xanthone with a wide range of interesting therapeutic properties, has recently attracted considerable attention. However, its application in biomedicine is limited by poor solubility and bioavailability. Carbon dots (CDs), novel nanomaterials, have immense promise as carriers for improving the biopharmaceutical properties of active components because of their outstanding characteristics. In this study, a novel water-soluble carbon dot (MC-CDs) was prepared for the first time from an aqueous extract of Moutan Cortex Carbonisata, and characterized by various spectroscopies, zeta potential and high-resolution transmission electron microscopy (HRTEM). The toxicity effect was investigated using the CCK-8 assay in vitro. In addition, the potential of MC-CDs as carriers for improving the pharmacokinetic parameters was evaluated in vivo. The results indicated that MC-CDs with a uniform spherical particle size of 1-5 nm were successfully prepared, which significantly increased the solubility of MA in water. The MC-CDs exhibited low toxicity in HT-22 cells. Most importantly, the MC-CDs effectively affected the pharmacokinetic parameters of MA in normal rats. UPLC-MS analysis indicated that the area under the maximum blood concentration of MA from mangiferin-MC-CDs (MA-MC-CDs) was 1.6-fold higher than that from the MA suspension liquid (MA control) after oral administration at a dose of 20 mg/kg. Moutan Cortex-derived novel CDs exhibited superior performance in improving the solubility and bioavailability of MA. This study not only opens new possibilities for the future clinical application of MA but also provides evidence for the development of green biological carbon dots as a drug delivery system to improve the biopharmaceutical properties of insoluble drugs.

Idebenone protects against ethanol toxicity in HT-22 cells through strengthening neuroimmune response

Idebenone, an analogue of coenzyme Q10, may function as a neuroprotective agent with its antioxidant and anti-inflammatory properties. The current report was designed to examine the beneficial effects of idebenone on ethanol-related neurotoxicity in hippocampal neuronal HT-22 cells in vitro and annotate the neuroprotective mechanism of idebenone. 75 mM ethanol was applied to the cells for 24h to develop ethanol toxicity. Then, different concentrations of idebenone (final concentration in the well to be 1, 2.5, and 5 μM) were applied to HT-22 cells for 24 h to explore the protective impact against ethanol-induced hippocampal damage. Cell viability was evaluated with MTT test. MDA, SOD, and GSH concentrations were examined to interpret oxidative damage. Moreover, the effects of idebenone on IL-1β, IL-6, and IL-23 neuroimmune-related genes expression levels were assigned by the RT-PCR analysis. In our study, 75 mM ethanol decreased neuronal cell viability by approximately 61%. All concentrations of idebenone were not toxic to neurons. In addition, idebenone increased cell viability by reducing the damage caused by alcohol. Idebenone reversed the reduction in antioxidant capacity caused by ethanol through decreasing MDA and increasing SOD and GSH levels. In addition, idebenone attenuated ethanol-induced impairment in neuroimmune and neuroinflammatory responses by reducing IL-1β, IL-6, and IL-23 mRNA expression levels. Treatment with idebenone increased antioxidant capacity and a significant improvement was achieved in neuroimmune and neuroinflammatory parameters. Possible mechanisms underlying these beneficial effects cover the down-regulation of IL-1β, IL-6, and IL-23 receptors, and antioxidant restoration of idebenone.

Neuroprotective Effects of Ethanol Extract of Polyscias fruticosa (EEPF) against Glutamate-Mediated Neuronal Toxicity in HT22 Cells.

In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.

Activated AMPK-mediated glucose uptake and mitochondrial dysfunction is critically involved in the glutamate-induced oxidative injury in HT22 cell

BackgroundAccumulation of glutamate damages neurons via the reactive oxygen species (ROS) injury, which was involved in the development of neurodegenerative diseases. However, the mechanism of neuronal oxidative stress damage caused by glutamate and the intervention targets still needs to be further studied. This study explored whether 5′ adenosine monophosphate-activated protein kinase (AMPK)-induced glucose metabolic and mitochondrial dysfunction were related to glutamate-dependent ROS injury of the neuron. MethodsNeuronal oxidative stress injury was induced by glutamate treatment in HT-22 cells. Western blotting was used to evaluate the phosphorylation of the AMPK. The XF24 Flux Analyzer was used to measure the effect of glutamate and Compound C (a well-known pharmacological inhibitor of AMPK phosphorylation) on the cellular oxygen consumption rate (OCR) of HT-22 cells. Glucose uptake, intracellular ROS, mitochondrial potential, apoptosis and cell viability were quantified using biochemical assays. ResultsGlutamate caused the phosphorylation of AMPK and subsequently promoted the glucose uptake. Furthermore, AMPK-mediated glucose uptake enhanced OCR and increased the intracellular ROS levels in neurons. The pharmacological inhibition of AMPK phosphorylation by Compound C attenuated glutamate-induced toxicity in HT22 cells by regulating the glucose uptake/mitochondrial respiration/ROS pathway. ConclusionsThe AMPK phosphorylation/glucose uptake/mitochondrial respiration/ROS pathway was involved in glutamate-induced excitotoxic injury in HT22 cells. The inhibition of AMPK phosphorylation may be a potential target for the development of therapeutic agents for treating the glutamate-induced neurotoxicity.

Neuroprotective effect of histamine H3 receptor blockade on methamphetamine-induced cognitive impairment in mice

ObjectiveMethamphetamine (METH) exposure is commonly believed to result in cognitive impairment. Histamine H3 receptor (H3R) antagonists reportedly have potential applications for treating cognitive impairment accompanied by various neuropsychiatric disorders. The present study aimed to investigate the effect of H3R blockade by Thioperamide (THIO) on METH-induced cognitive impairment and the underlying mechanism. MethodsIn Experiment 1, C57BL/6 mice received daily injections of saline or 5 mg/kg METH for 5 consecutive days. The Novel Object Recognition (NOR) and Morris water maze (MWM) tasks were used to assess cognitive functions of mice. H3R protein expression and apoptosis were subsequently measured in the hippocampus. In Experiment 2, HT22 cells were first treated with ddH2O or 3 mM METH. The cell survival rate and H3R protein level were subsequently assessed. In Experiment 3, the animals were first treated with saline or 20 mg/kg THIO for 7 days, followed by co-administration of either saline or 5 mg/kg METH for an additional 5 days. The remaining experiments were carried out in the same manner as Experiment 1. In Experiment 4, HT22 cells were pretreated with either ddH2O or 5 mM THIO for 2 h, followed by ddH2O or 3 mM METH treatment for an additional 12 h. The remaining experiments were carried out in the same manner as Experiment 2. In Experiment 5, the changes in MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 protein levels were examined in the hippocampus of all mice from Experiment 3 and HT22 cells from Experiment 4. ResultsMETH-treated mice showed significantly worsened NOR and MWM performance, along with markably hippocampal apoptosis. A significantly lower cell survival rate was observed in METH-treated HT22 cells. Increased levels of H3R protein were found in both METH-treated mice and HT22 cells. THIO significantly improved METH-induced cognitive impairment in mice and toxicity in HT22 cells. METH significantly increased the level of p-MEK1/2 and p-ERK1/2 proteins in the hippocampus of mice and HT22 cells, which was reversed by THIO pretreatment. ConclusionOur findings reveal that H3R blockade by THIO yields a neuroprotective effect against METH-induced cognitive impairment in mice and toxicity in HT22 cells via the raf-MEK-ERK signaling pathway.

Screening of the active Ingredients in Huanglian Jiedu decoction through amide bond-Immobilized magnetic nanoparticle-assisted cell membrane chromatography.

Introduction: The Huanglian Jiedu decoction (HLJDD) is a Chinese herbal formula that exerts neuroprotective effects by alleviating oxidative stress injuries and may potentially be prescribed for treating Alzheimer's disease; however, its active ingredients have not yet been identified. Cell membrane chromatography is a high-throughput method for screening active ingredients, but traditional cell membrane chromatography requires multiple centrifugation steps, which affects its separation efficiency. Magnetic nanoparticles are unparalleled in solid-liquid separation and can overcome the shortcomings of traditional cell membrane chromatography. Methods: In this study, the neuroprotective effects of the components of HLJDD were screened through a novel magnetic nanoparticle-assisted cell membrane chromatography method. Magnetic nanoparticles and cell membranes were stably immobilized by amide bonds. Magnetic bead (MB)-immobilized cell membranes of HT-22 cells were incubated with the HLJDD extract to isolate specific binding components. The specific binding components were then identified by ultraperformance liquid chromatography (UPLC)-Orbitrap Fusion Tribrid MS after solid-phase extraction. The bioactivity of these components was analyzed in an HT-22 cellular model of glutamate-induced injury. Results and Discussion: The preparation method of the composite of cell membrane and MBs has the advantages of simple preparation and no introduction of toxic organic reagents. MBs not only provide support for cell membranes, but also greatly improve the separation efficiency compared with traditional cell membrane chromatography. Fifteen of these components were found to specifically bind to the cell membranes, and seven of them were confirmed to reduce varying degrees of glutamate-induced toxicity in HT-22 cells. In conclusion, our findings suggest that the amide bond-based immobilization of magnetic nanoparticles on cell membranes, along with solid-phase extraction and UPLC, is an effective method for isolating and discovering the bioactive components of traditional Chinese medicines.

Rhinacanthin-C but Not -D Extracted from Rhinacanthus nasutus (L.) Kurz Offers Neuroprotection via ERK, CHOP, and LC3B Pathways.

Neurodegenerative diseases present an increasing problem as the world’s population ages; thus, the discovery of new drugs that prevent diseases such as Alzheimer’s, and Parkinson’s diseases are vital. In this study, Rhinacanthin-C and -D were isolated from Rhinacanthus nasustus, using ethyl acetate, followed by chromatography to isolate Rhinacanthin-C and -D. Both compounds were confirmed using NMR and ultra-performance-LCMS. Using glutamate toxicity in HT-22 cells, we measured cell viability and apoptosis, ROS build-up, and investigated signaling pathways. We show that Rhinacanthin-C and 2-hydroxy-1,4-naphthoquinone have neuroprotective effects against glutamate-induced apoptosis in HT-22 cells. Furthermore, we see that Rhinacanthin-C resulted in autophagy inhibition and increased ER stress. In contrast, low concentrations of Rhinacanthin-C and 2-hydroxy-1,4-naphthoquinone prevented ER stress and CHOP expression. All concentrations of Rhinacanthin-C prevented ROS production and ERK1/2 phosphorylation. We conclude that, while autophagy is present in HT-22 cells subjected to glutamate toxicity, its inhibition is not necessary for cryoprotection.

A Novel Diselenide-Probucol-Analogue Protects Against Methylmercury-Induced Toxicity in HT22 Cells by Upregulating Peroxide Detoxification Systems: a Comparison with Diphenyl Diselenide.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant whose mechanisms of action involve oxidation of endogenous nucleophilic groups (mainly thiols and selenols), depletion of antioxidant defenses, and disruption of neurotransmitter homeostasis. Diphenyl diselenide-(PhSe)2-a model diaryl diselenide, has been reported to display significant protective effects against MeHg-induced neurotoxicity under both in vitro and in vivo experimental conditions. In this study, we compared the protective effects of (PhSe)2 with those of RC513 (4,4'-diselanediylbis(2,6-di-tert-butylphenol), a novel diselenide-probucol-analog) against MeHg-induced toxicity in the neuronal (hippocampal) cell line HT22. Although both (PhSe)2 and RC513 significantly mitigated MeHg- and tert-butylhydroperoxide (t-BuOOH)-cytotoxicity, the probucol analog exhibited superior protective effects, which were observed earlier and at lower concentrations compared to (PhSe)2. RC513 treatment (at either 0.5µM or 2µM) significantly increased glutathione peroxidase (GPx) activity, which has been reported to counteract MeHg-toxicity. (PhSe)2 was also able to increase GPx activity, but only at 2µM. Although both compounds increased the Gpx1 transcripts at 6h after treatments, only RC513 was able to increase mRNA levels of Prx2, Prx3, Prx5, and Txn2, which are also involved in peroxide detoxification. RC513 (at 2µM) significantly increased GPx-1 protein expression in HT22 cells, although (PhSe)2 displayed a minor (nonsignificant) effect in this parameter. In agreement, RC513 induced a faster and superior capability to cope with exogenously-added peroxide (t-BuOOH). In summary, when compared to the prototypical organic diaryl diselenide [(PhSe)2], RC513 displayed superior protective properties against MeHg-toxicity in vitro; this was paralleled by a more pronounced upregulation of defenses related to detoxification of peroxides, which are well-known MeHg-derived intermediate oxidant species.

Hibiscus sabdariffa extract protects HT-22 cells from glutamate-induced neurodegeneration by upregulating glutamate transporters and exerts lifespan extension in C. elegans via DAF-16 mediated pathway

BACKGROUND: Glutamate toxicity is involved in several neurodegenerative conditions, including Alzheimer’s disease. OBJECTIVE: The study aims to investigate the neuroprotective efficacy of ethanol extract of Hibiscus sabdariffa calyces (HS) against glutamate-induced toxicity in HT-22 cells and anti-aging property in Caenorhabditis elegans. METHODS: HT-22 cells were pre-treated with HS followed by glutamate and evaluated for the neuroprotective effect using cell viability assay, confocal microscopic analysis, qPCR, Western blot, and docking analysis. Induction of anti-aging property in C. elegans with HS extract was analyzed through physiological assays and qPCR analysis. RESULTS: GC-MS analysis of the HS extract showed the presence of 19 compounds with antioxidant properties including oleamide, 2-(diethoxymethyl)furan and 5-methylfurfural. In vitro studies reveal that glutamate exerted toxicity in HT-22 cells by inducing oxidative stress, depleting glutathione, downregulating glutamate transporters, antioxidant genes, inducing autophagy (Beclin-1, Atg-5, Atg-7, LC3-II) by the activation of MAPK (p38, JNK) pathway, and causing apoptosis. However, pre-treatment with HS extract (5, 10μg/ml) reversed the effect and offered neuroprotection. In silico studies showed that the compounds of HS extract can bind effectively and inhibit the activity of NMDAR, calpain-1 and GSK-3β. In C. elegans, HS extended lifespan, reduced the accumulation of lipofuscin, modulated healthspan-related genes and downregulated the expression of daf-2. CONCLUSION: Our results indicate that HS with its bioactive components exhibits neuroprotective activity by upregulating glutamate transporters, inhibiting autophagy and exerts anti-aging property through DAF-16 dependent mechanism.

Chrysanthemum indicum Prevents Hydrogen Peroxide-Induced Neurotoxicity by Activating the TrkB/Akt Signaling Pathway in Hippocampal Neuronal Cells.

Oxidative stress-mediated neuronal damage is associated with the pathogenesis and development of neurodegenerative diseases. Chrysanthemum indicum has antioxidant properties. However, the neuroprotective effects and the cellular mechanism of C. indicum ethanol extract (CIE) against oxidative damage in hippocampal neuronal cells have not been clearly elucidated. Therefore, this study investigated whether CIE has protective effects against hydrogen peroxide (H2O2)-induced oxidative toxicity in HT22 cells. CIE pretreatment significantly improved neuronal cell viability. Moreover, the formation of intracellular reactive oxygen species and apoptotic bodies, and mitochondrial depolarization were significantly reduced in HT22 cells with H2O2-induced oxidative toxicity. Furthermore, CIE increased the phosphorylation of tropomyosin-related kinase receptor B (TrkB), protein kinase B (Akt), cAMP response element-binding protein, the expression of brain-derived neurotrophic factor, antioxidant enzymes, and the nuclear translocation of nuclear factor erythroid 2-related factor 2 by activating the TrkB/Akt signaling pathway. In contrast, the addition of K252a, a TrkB inhibitor, or MK-2206, an Akt-selective inhibitor, reduced the neuroprotective and antioxidant effects of CIE. Taken together; CIE exhibits neuroprotective and antioxidant effects against oxidative damage. Therefore, it can be a potential agent for treating oxidative stress-related neurodegenerative diseases.

Cissus verticillata Extract Decreases Neuronal Damage Induced by Oxidative Stress in HT22 Cells and Ischemia in Gerbils by Reducing the Inflammation and Phosphorylation of MAPKs.

In the present study, we examined the effects of Cissus verticillata leaf extracts (CVE) against hydrogen peroxide (H2O2)- and ischemia-induced neuronal damage in HT22 cells and gerbil hippocampus. Incubation with CVE produced concentration-dependent toxicity in HT22 cells. Significant cellular toxicity was observed with >75 μg/mL CVE. CVE treatment at 50 μg/mL ameliorated H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell death in HT22 cells. In addition, incubation with CVE significantly mitigated the increase in Bax and decrease in Bcl-2 induced by H2O2 treatment in HT22 cells. In an in vivo study, the administration of CVE to gerbils significantly decreased ischemia-induced motor activity 1 d after ischemia, as well as neuronal death and microglial activation 4 d after ischemia, respectively. CVE treatment reduced the release of interleukin-1β, interleukin-6, and tumor necrosis factor-α 6 h after ischemia. Furthermore, CVE treatment significantly ameliorated ischemia-induced phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2, and p38. These results suggest that CVE has the potential to reduce the neuronal damage induced by oxidative and ischemic stress by reducing the inflammatory responses and phosphorylation of MAPKs, suggesting that CVE could be a functional food to prevent neuronal damage induced by ischemia.

Germinated Brown Rice Attenuates Cell Death in Vascular Cognitive Impaired Mice and Glutamate-Induced Toxicity In HT22 Cells.

Germinated brown rice (GBR) with unpolishing, soaking, and germinating processes can improve the texture, flavor, and nutritional value, including GABA and phenolic contents. The effect of GBR was first investigated in vascular cognitive impaired mice and glutamate-induced toxicity in HT22 cells with respect to standard pure GABA. Feeding mice with GBR for 5 weeks showed neuroprotection. In this study, the modified bilateral common carotid artery occlusion mice model was mild but a significant difference in cognitive impairment was still shown. Like pure GABA, GBR decreased cognitive deficits in memory behavioral tests and significantly attenuated hippocampal neuronal cell death at P < 0.001. Similarly to 0.125 μM of GABA, 100 μg/mL of GBR increased HT22 cell viability after glutamate toxicity. GBR affected less apoptotic cell death and less blocking by the GABAA antangonist bicuculline in comparison to GABA. When the results are taken together, the underlying mechanism of GBR protection may mediate though the GABAA receptor and its phenolic contents.

Flavonoids from Chionanthus retusus (Oleaceae) Flowers and Their Protective Effects against Glutamate-Induced Cell Toxicity in HT22 Cells.

The dried flowers of Chionanthus retusus were extracted with 80% MeOH, and the concentrate was divided into EtOAc, n-BuOH, and H2O fractions. Repeated SiO2, octadecyl SiO2 (ODS), and Sephadex LH-20 column chromatography of the EtOAc fraction led to the isolation of four flavonols (1–4), three flavones (5–7), four flavanonols (8–11), and one flavanone (12), which were identified based on extensive analysis of various spectroscopic data. Flavonoids 4–6 and 8–11 were isolated from the flowers of C. retusus for the first time in this study. Flavonoids 1, 2, 5, 6, 8, and 10–12 significantly inhibited NO production in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) and glutamate-induced cell toxicity and effectively increased HO-1 protein expression in mouse hippocampal HT22 cells. Flavonoids with significant neuroprotective activity were also found to recover oxidative-stress-induced cell damage by increasing HO-1 protein expression. This article demonstrates that flavonoids from C. retusus flowers have significant potential as therapeutic materials in inflammation and neurodisease.

Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate

BackgroundAcanthus ebracteatus (AE), an herb native to Asia, has been recognized in traditional folk medicine not only for its antioxidant properties and various pharmacological activities but also as an ingredient of longevity formulas. However, its anti-neurodegenerative potential is not yet clearly known. This work aimed to evaluate the protective effect of AE leaf extract against glutamate-induced oxidative damage in mouse hippocampal HT22 cells, a neurodegenerative model system due to a reduction in glutathione levels and an increase in reactive oxygen species (ROS).MethodsCell viability, apoptosis, and ROS assays were performed to assess the protective effect of AE leaf extract against glutamate-induced oxidative toxicity in HT22 cells. The antioxidant capacity of AE was evaluated using in vitro radical scavenging assays. The subcellular localization of apoptosis-inducing factor (AIF) and the mRNA and protein levels of genes associated with the nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidant system were determined to elucidate the mechanisms underlying the neuroprotective effect of AE leaf extract.ResultsWe demonstrated that AE leaf extract is capable of attenuating the intracellular ROS generation and HT22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with the extract significantly reduced apoptotic cell death via inhibition of AIF nuclear translocation. The increases in Nrf2 levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with the extract.ConclusionsThe results suggested that AE leaf extract possesses neuroprotective activity against glutamate-induced oxidative injury and may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.

Laccase Fermentation of Clove Extract Increases Content of Dehydrodieugenol, Which Has Neuroprotective Activity against Glutamate Toxicity in HT22 Cells.

Enzyme fermentation is a type of food processing technique generally used to improve the biological activities of food and herbal medicines. In this study, a Syzygii Flos (clove) extract was fermented using laccase derived from Trametes versicolor (LTV). The fermented clove extract showed greater neuroprotective effects against glutamate toxicity on HT22 than the non-fermented extract did. HPLC analysis revealed that the eugenol (1) and dehydrodieugenol (2) contents had decreased and increased, respectively, after fermentation. The content of 2 peaked at 1 h after fermentation to 103.50 ± 8.20 mg/gex (not detected at zero time), while that of 1 decreased to 79.54 ± 4.77 mg/gex (185.41 ± 10.16 mg/gex at zero time). Compound 2 demonstrated promising HT22 neuroprotective properties with inhibition of Ca2+ influx, the overproduction of intracellular reactive oxygen species, and lipid peroxidation. In addition, LTV showed the best fermentation efficacy compared with laccases derived from Pleurotus ostreatus and Rhus vernicifera.

Nature-based molecules combined with rivastigmine: A symbiotic approach for the synthesis of new agents against Alzheimer's disease

Starting from nature as original source, new potential agents with pleiotropic activities have been synthesized and evaluated as neuroprotective agents. In this work, novel nature-based hybrids, combining antioxidant motifs with rivastigmine, have been designed and synthesized. The biological results revealed that the new compounds inhibit both AChE and BuChE. In particular, lipoic acid hybrids LA1, LA2, LA3 resulted to be the most potent inhibitors of BuChE showing IC50 values ranging from 340 to 378 nM. Analogously, all the compounds were able to inhibit the self β-amyloid1-42 aggregation. The gallic acid hybrid GA2 as well as the 2-chromonecarboxylic acid hybrids CA1 and CA2 prevented the self-mediated Aβ aggregation with percentages of inhibition ranging from 53% to 59%. Finally, some of them also show potent neuroprotective effects against glutamate-induced cell death and low toxicity in HT22 cells.

Neuroprotective Effects of Kinetin Against Glutamate-Induced Oxidative Cytotoxicity in HT22 Cells: Involvement of Nrf2 and Heme Oxygenase-1.

Oxidative stress is considered as one of key factors related to Alzheimer's disease (AD), while kinetin (KT) has been reported to exert anti-oxidative activities as well as neuroprotective effects both in vivo and in vitro. Thus, in this study, the neuroprotective effects of KT against glutamate-induced oxidative toxicity in HT22 cells were investigated. To evaluate the anti-oxidative capabilities of KT itself, several anti-oxidative assays in vitro were conducted. To evaluate the neuroprotective effects of KT, the levels of intracellular reactive oxygen species (ROS) and calcium influx, mitochondrial membrane potential (MMP), and cell death were measured by flow cytometry. Nuclear translocation of apoptosis inducing factor (AIF) and content of intracellular ATP were also determined. In addition, the phosphorylation levels of apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (p38) were evaluated as well. Besides, nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) were also examined to reveal underlying mechanisms. Results showed that KT rescued cell death, and suppressed the accumulation of intracellular ROS and the increase of intracellular calcium influx. In addition, KT maintained normal function of mitochondria and inhibited the phosphorylation of ASK-1, JNK, and p38. KT also promoted nuclear translocation of Nrf2 and enhanced the expression of HO-1 both at protein and mRNA level. Importantly, blockage of Nrf2 almost completely abolished the neuroprotective effects of KT, while blockage of HO-1 expression partly neutralized its neuroprotective effects. Our results indicated that KT can protect HT22 cells from glutamate-induced cell death by activating Nrf2 pathway and inducing expression of HO-1, suggesting KT might be a drug candidate for treatment of AD and other neurodegenerative disorders related to oxidative stress.

Neuroprotective compounds from Reynoutria sachalinensis.

Glutamate is a neurotransmitter in central nervous system. Overexpression of glutamate leads to oxidative stress, resulting in several neurodegenerative disorders that include Alzheimer's disease. The n-hexane fraction of stems and ethyl acetate (EtOAc) fraction of flowers of Reynoutria sachalinensis provide neuroprotection against glutamate-induced oxidative toxicity in HT22 cells. In this study, 1-decanol (1), β-amyrin (2), dammaran-3β-ol (3), campesterol (4), daucosterol (5), ergosterol peroxide (6), emodin 8-O-β-D-glucopyranoside (7), quercetin (8) and isoquercitrin (9) were isolated from n-hexane fractions of stems and EtOAc fractions of flowers of R. sachalinensis. Their neuroprotective activity was evaluated by MTT assay. 1-Decanol, campesterol, ergosterol peroxide, quercetin and isoquercitrin exhibited neuroprotective activity. These compounds decreased reactive oxygen species level, showed anti-oxidant activity with DPPH radical and in a H2O2 scavenging assay. Therefore, the neuroprotective activity of 1-decanol, campesterol, ergosterol peroxide, quercetin and isoquercitrin are associated with antioxidant activity.

Metabolic Alterations and the Protective Effect of Punicalagin Against Glutamate-Induced Oxidative Toxicity in HT22 Cells.

Oxidative stress is involved in many neurological diseases, including Alzheimer's disease. Punicalagin (PC) is a hydrolysable polyphenol derived from Punica granatum and a potent antioxidant. In this study, the neuroprotective effect of PC on glutamate-induced oxidative stress was evaluated in the mouse hippocampal cell line, HT22. PC treatment protected HT22 cells from glutamate-induced cell death in a concentration-dependent manner, potentially attenuated glutamate-induced intracellular reactive oxygen species (ROS) and restored the mitochondrial membrane depolarization. Metabolic alterations after glutamate-induced oxidative stress and the protective effect of PC were evaluated with HPLC and GC-MS profiling methods with multivariate statistical analyses. Alterations in ten metabolites were identified, including amino acids, aspartic acid, asparagine, threonine, anserine, cysteine, tryptophan, lysine, as well as fatty acids palmitic acid, stearic acid, and palmitoleic acid. Metabolic pathway analysis revealed the involvement of multiple affected pathways, such as cysteine and methionine metabolism, tryptophan metabolism, alanine, aspartate, and glutamate and fatty acid oxidation. These results clearly demonstrate that PC is a promising therapeutic agent for oxidative stress-associated diseases.