Procyanidin C1 Activates the Nrf2/HO-1 Signaling Pathway to Prevent Glutamate-Induced Apoptotic HT22 Cell Death.
ABSTRACT: 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.
Project description:In the course of searching for bioactive secondary metabolites from marine fungi, TMC-256C1 was isolated from an ethyl acetate extract of the marine-derived fungus Aspergillus sp. SF6354. TMC-256C1 displayed anti-neuroinflammatory effect in BV2 microglial cells induced by lipopolysaccharides (LPS) as well as neuroprotective effect against glutamate-stimulated neurotoxicity in mouse hippocampal HT22 cells. TMC-256C1 was shown to develop a cellular resistance to oxidative damage caused by glutamate-induced cytotoxicity and reactive oxygen species (ROS) generation in HT22 cells, and suppress the inflammation process in LPS-stimulated BV2 cells. Furthermore, the neuroprotective and anti-neuroinflammatory activities of TMC-256C1 were associated with upregulated expression of heme oxygenase (HO)-1 and nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) in HT22 and BV2 cells. We also found that TMC-256C1 activated p38 mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in HT22 and BV2 cells. These results demonstrated that TMC-256C1 activates HO-1 protein expression, probably by increasing nuclear Nrf2 levels via the activation of the p38 MAPK and PI3K/Akt pathways.
Project description:OBJECTIVES:Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death. MATERIALS AND METHODS:Nitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS:Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function. CONCLUSIONS:The 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.
Project description:In this study, we examined the neuroprotective effects of MeOH extract and bioactive compounds obtained from <i>Reynoutria elliptica</i> 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 <i>R. elliptica</i> seeds strongly protected HT22 cells from glutamate toxicity. To clarify the responsible compound for the neuroprotective effects, we took an interest in procyanidins of <i>R. elliptica</i> 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 <i>R. elliptica</i> seeds using the LC/MS-guided isolation approach was applied, and procyanidin B2 3?-<i>O</i>-gallate (<b>1</b>) was successfully isolated. The structure of <b>1</b> was elucidated by analyzing the nuclear magnetic resonance spectroscopic data and LC/MS analysis. The neuroprotective activities of <b>1</b> were thoroughly examined using HT22 cells. Compound <b>1</b> exhibited a strong antioxidant efficacy and blocked glutamate-mediated increase in the reactive oxygen species (ROS) accumulation. Furthermore, compound <b>1</b> 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 <i>R. elliptica</i> 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.
Project description:Oxidative cell damage contributes to neuronal degeneration in many central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, and ischemia. Nrf2 signaling-mediated heme oxygenase (HO)-1 expression acts against oxidants that are thought to play a key role in the pathogenesis of neuronal diseases. Cudraflavone B is a prenylated flavone isolated from C. tricuspidata which has shown anti-proliferative activity, mouse brain monoamine oxidase (MAO) inhibitory effects, apoptotic actions in human gastric carcinoma cells and mouse melanoma cells, and hepatoprotective activity. In this study, cudraflavone B showed neuroprotective effects and reactive oxygen species (ROS) inhibition against glutamate-induced neurotoxicity by inducing the expression of HO-1 in mouse hippocampal HT22 cells. Furthermore, cudraflavone B caused the nuclear accumulation of nuclear factor-E2-related factor 2 (Nrf2) and increased the promoter activity of antioxidant response elements (ARE) in mouse hippocampal HT22 cells. In addition, we found that the Nrf2-midiated HO-1 expression by cudraflavone B is involved in the cell protective response and ROS reductions, and cudraflavone B-induced expression of HO-1 was mediated through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in HT22 cells. Our results demonstrated the potential application of naturally occurring cudraflavone B as a therapeutic agent from neurodegenerative disease.
Project description:<i>Dendropanax morbifera</i> leaves (DML) have long been used as traditional medicine to treat diverse symptoms in Korea. Ethyl acetate-soluble extracts of DML (DMLE) rescued HT22 mouse hippocampal neuronal cells from glutamate (Glu)-induced oxidative cell death; however, the protective compounds and mechanisms remain unknown. Here, we aimed to identify the neuroprotective ingredients and mechanisms of DMLE in the Glu-HT22 cell model. Five antioxidant compounds were isolated from DMLE and characterized as chlorogenic acid, hyperoside, isoquercitrin, quercetin, and rutin by spectroscopic methods. Isoquercitrin and quercetin significantly inhibited Glu-induced oxidative cell death by restoring intracellular reactive oxygen species (ROS) levels and mitochondrial superoxide generation, Ca<sup>2+</sup> dysregulation, mitochondrial dysfunction, and nuclear translocation of apoptosis-inducing factor. These two compounds significantly increased the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the presence or absence of Glu treatment. Combinatorial treatment of the five compounds based on the equivalent concentrations in DMLE showed that significant protection was found only in the cells cotreated with isoquercitrin and quercetin, both of whom showed prominent synergism, as assessed by drug-drug interaction analysis. These findings suggest that isoquercitrin and quercetin are the active principles representing the protective effects of DMLE, and these effects were mediated by the Nrf2/HO-1 pathway.
Project description:This data article describes the influence of <i>Cimicifuga racemosa</i> extract Ze 450 on neuronal cells in models of glutamate-induced excitotoxicity and cell death induced by oxidative stress. Effects of Ze 450 on glutamate-mediated excitotoxicity were assessed in primary cortical rat and mouse neurons and, further, glutamate-mediated oxidative stress was analyzed in HT22 cells lacking ionotropic glutamate receptors. This study especially focusses on mitochondrial parameters like mitochondrial ROS formation, mitochondrial membrane potential, ATP production and mitochondrial integrity. Further the effects of Ze 450 on lipid-peroxidation, metabolic activity, cell proliferation and cell death were assessed under control conditions and oxidative challenge evoked by millimolar concentrations of glutamate in HT22 cells. These data support the findings in HT22, mHypo and HepG2 liver cells (Rabenau et al., 2018) .
Project description:Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.
Project description:The aim of the present study was to examine the protective effect of ?-mangostin, a component of the mangosteen shell, against oxidative damage to nerve cells induced by excessive glutamate, a known excitatory neurotransmitter. To investigate the effect of ?-mangostin on apoptosis, 5 mM of glutamate was used to induce apoptotic cell death in mouse hippocampal HT22 cells. In this study, ?-mangostin was found to exert a stronger protection than N-acetyl cysteine against glutamate-induced cell damage. ?-Mangostin showed prevented glutamate-induced apoptosis in HT22 cells by reducing the production of reactive oxygen species and stimulating the expression of heme oxygenase-1 protein. In addition, glutamate significantly induced the accumulation of intracellular calcium ions, whereas treatment with ?-mangostin markedly reduced it. Hoechst 33342 staining showed an improvement in glutamate-induced nuclear condensation following ?-mangostin treatment. Furthermore, the number of annexin V-positive cells was significantly reduced following treatment with ?-mangostin. Western blot analysis showed the inhibition of glutamate-induced mitogen-activated protein kinase phosphorylation by ?-mangostin. ?-mangostin also inhibited the regulation of the intrinsic mitochondrial apoptotic pathway. Thus, the results of this study suggest that ?-mangostin is an active ingredient of mangosteen and exerts neuroprotective activities in HT22 cells.
Project description:The structure of a new procyanidin tetramer, which we call a crown procyanidin tetramer, with an unprecedented macrocyclic structure has been characterized for the first time. Its comprehensive spectroscopic analysis revealed that it is a symmetric procyanidin tetramer composed of four (-)-epicatechin sub-units linked alternatively via 4??8 or 4??6 B-type interflavanyl linkages to form the macrocyclic structure. This NMR-characterized carbon skeleton has never been reported before for procyanidins in grape or in wine, neither in the plant kingdom. Surprisingly, the crown procyanidin tetramer appeared to be specifically localized in grape skin, contrasting with the oligomeric and polymeric procyanidins present in seed, skin, and bunch stem. Moreover, this crown procyanidin tetramer showed promising protective effects against amyloid-? induced toxicity.
Project description:Glutamate induced excitotoxicity is common in diverse neurological disorders. RNF146 as an E3 ubiquitin ligase protects neurons against excitotoxicity via interfering with Poly (ADP-ribose) (PAR) polymer-induced cell death (parthanatos). However, the neuroprotective role of RNF146 has not been fully understood. We aimed to investigate the role of RNF146 in modulating autophagy in HT22 cells under glutamate excitotoxicity injury. Here we found that induction of RNF146 decreased the cellular damage and excitotoxicity induced by glutamate. RNF146 also suppressed the excessive autophagy, which is detrimental to HT22 cells survival, induced by glutamate or rapamycin treatment. In addition, we find that Wnt/?-catenin was a negative regulation factor for autophagy in glutamate excitotoxicity. Over-expression of RNF146 promoted Wnt/?-catenin signaling, which was related to destabilization of ?-catenin destruction complex. These results indicated that RNF146 acted as a neuroprotective agent against glutamate-induced excitatory damage, and this neuroprotection might be at least partly dependent on the inhibition of excessive autophagy by regulating Wnt/?-catenin signaling.