Mouse Hippocampal Cells Research Articles

A new ferroptosis inhibitor, isolated from Ajuga nipponensis, protects neuronal cells via activating NRF2-antioxidant response elements (AREs) pathway

Ferroptosis is a new form of cell death, and inhibition of ferroptosis is a promising strategy to treat neurological diseases. In this work, sixteen compounds were isolated from Ajuga nipponensis and assayed for anti-ferroptosis activity in HT22 mouse hippocampal neuronal cells. Ajudecunoid C (1, ADC), a new neoclerodane diterpenoid, showed significant inhibitory activity against erastin and RSL3-induced ferroptosis with EC50 values of 4.1 ± 1.0 and 3.6 ± 0.3 μM, respectively. Experimental results demonstrated that ADC effectively prevented ferroptosis through scavenging free radical and activating NRF2-antioxidant response elements (AREs) pathway. This study reveals that ADC, as a new ferroptosis inhibitor, is a promising lead compound for the development of drugs against ferroptosis-related neurological diseases.

Antioxidative and Anti-inflammatory Effects of Kojic Acid in Aβ-Induced Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a common cause of dementia that is clinically characterized by the loss of memory and cognitive functions. Currently, there is no specific cure for the management of AD, although natural compounds are showing promising therapeutic potentials because of their safety and easy availability. Herein, we evaluated the neuroprotective properties of kojic acid (KA) in an AD mouse model. Intracerebroventricular injection (i.c.v) of Aβ1-42 (5 μL/5min/mouse) into wild-type adult mice induced AD-like pathological changes in the mouse hippocampus by increasing oxidative stress and neuroinflammation, affecting memory and cognitive functions. Interestingly, oral treatment of kojic acid (50mg/kg/mouse for 3weeks) reversed the AD pathology by reducing the expression of amyloid-beta (Aβ) and beta-site amyloid precursor protein cleaving enzyme1 (BACE-1). Moreover, kojic acid reduced oxidative stress by enhancing the expression of nuclear factor erythroid-related factor 2 (Nrf2) and heme oxygenase 1 (HO1). Also, kojic acid reduced the lipid peroxidation and reactive oxygen species in the Aβ + kojic acid co-treated mice brains. Moreover, kojic acid decreased neuroinflammation by inhibiting Toll-like receptor 4, phosphorylated nuclear factor-κB, tumor necrosis factor-alpha, interleukin 1-beta (TLR-4, p-NFκB, TNFα, and IL-1β, respectively), and glial cells. Furthermore, kojic acid enhanced synaptic markers (SNAP-23, SYN, and PSD-95) and memory functions in AD model mice. Additionally, kojic acid treatment also decreased Aβ expression, oxidative stress, and neuroinflammation in vitro in HT-22 mouse hippocampal cells. To the best of our knowledge, this is the first study to show the neuroprotective effects of kojic acid against an AD mouse model. Our findings could serve as a favorable and alternative strategy for the discovery of novel drugs to treat AD-related neurodegenerative conditions.

Momordica charantia L. Extract Protects Hippocampal Neuronal Cells against PAHs-Induced Neurotoxicity: Possible Active Constituents Include Stigmasterol and Vitamin E.

Polycyclic aromatic hydrocarbons (PAHs) have been recognized to cause neurobehavioral dysfunctions and disorder of cognition and behavioral patterns in childhood. Momordica charantia L. (MC) has been widely known for its nutraceutical and health-promoting properties. To date, the effect of MC for the prevention and handling of PAHs-induced neurotoxicity has not been reported. In the current study, the neuroprotective effects of MC and its underlying mechanisms were investigated in mouse hippocampal neuronal cell line (HT22); moreover, in silico analysis was performed with the phytochemicals MC to decipher their potential function as neuroprotectants. MC was demonstrated to possess neuroprotective effect by reducing reactive oxygen species’ (ROS’) production and down-regulating cyclin D1, p53, and p38 mitogen-activated protein kinase (MAPK) protein expressions, resulting in the inhibition of cell apoptosis and the normalization of cell cycle progression. Additionally, 28 phytochemicals of MC and their competence on inhibiting cytochrome P450 (CYP: CYP1A1, CYP1A2, and CYP1B1) functions were resolved. In silico analysis of vitamin E and stigmasterol revealed that their binding to either CYP1A1 or CYP1A2 was more efficient than the binding of each positive control (alizarin or purpurin). Together, MC is potentially an interesting neuroprotectant including vitamin E and stigmasterol as probable active components for the prevention for PAHs-induced neurotoxicity.

Protective Effects of Gintonin on Reactive Oxygen Species-Induced HT22 Cell Damages: Involvement of LPA1 Receptor-BDNF-AKT Signaling Pathway.

Gintonin is a kind of ginseng-derived glycolipoprotein that acts as an exogenous LPA receptor ligand. Gintonin has in vitro and in vivo neuroprotective effects; however, little is known about the cellular mechanisms underlying the neuroprotection. In the present study, we aimed to clarify how gintonin attenuates iodoacetic acid (IAA)-induced oxidative stress. The mouse hippocampal cell line HT22 was used. Gintonin treatment significantly attenuated IAA-induced reactive oxygen species (ROS) overproduction, ATP depletion, and cell death. However, treatment with Ki16425, an LPA1/3 receptor antagonist, suppressed the neuroprotective effects of gintonin. Gintonin elicited [Ca2⁺]i transients in HT22 cells. Gintonin-mediated [Ca2⁺]i transients through the LPA1 receptor-PLC-IP3 signaling pathway were coupled to increase both the expression and release of BDNF. The released BDNF activated the TrkB receptor. Induction of TrkB phosphorylation was further linked to Akt activation. Phosphorylated Akt reduced IAA-induced oxidative stress and increased cell survival. Our results indicate that gintonin attenuated IAA-induced oxidative stress in neuronal cells by activating the LPA1 receptor-BDNF-TrkB-Akt signaling pathway. One of the gintonin-mediated neuroprotective effects may be achieved via anti-oxidative stress in nervous systems.

Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury.

Salidroside is the active ingredient extracted from Rhodiola rosea, and has been reported to show protective effects in cerebral ischemia, but the exact mechanisms of neuronal protective effects are still unrevealed. In this study, the protective effects of salidroside (1 µmol/L) in ameliorating neuronal injuries induced by oxygen-glucose deprivation (OGD), which is a classical model of cerebral ischemia, were clarified. The results showed that after 8 h of OGD, the mouse hippocampal neuronal cell line HT22 cells showed increased cell death, accompanied with mitochondrial fragmentation and augmented mitophagy. However, the cell viability of HT22 cells showed significant restoration after salidroside treatment. Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment. The protective effects of salidroside were further related to the prevention of mitochondrial over-fission. The results showed that mTOR could be recruited to the mitochondria after salidroside treatment, which might be responsible for inhibiting excessive mitophagy caused by OGD. Thus, salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function, which may provide evidence for further translational studies of salidroside in ischemic diseases.

Ischemia Injury induces mPTP opening by reducing Sirt3

Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Analysis of the role of Pur\u03b1 in the pathogenesis of Alzheimer's disease based on RNA-seq and ChIP-seq

Purine rich element binding protein A (Purα), encoded by the Purα gene, is an important transcriptional regulator that binds to DNA and RNA and is involved in processes such as DNA replication and RNA translation. Purα also plays an important role in the nervous system. To identify the function of Pura, we performed RNA sequence (RNA-seq) analysis of Purɑ-KO mouse hippocampal neuron cell line (HT22) to analyze the effect of Purα deletion on neuronal expression profiles. And combined with ChIP-seq analysis to explore the mechanism of Purα on gene regulation. In the end, totaly 656 differentially expressed genes between HT22 and Purα-KO HT22 cells have been found, which include 7 Alzheimer’s disease (AD)-related genes and 5 Aβ clearance related genes. 47 genes were regulated by Purα directly, the evidence based on CHIP-seq, which include Insr, Mapt, Vldlr, Jag1, etc. Our study provides the important informations of Purα in neuro-development. The possible regulative effects of Purα on AD-related genes consist inthe direct and indirect pathways of Purα in the pathogenesis of AD.

Selective Overexpression of Collybistin in Mouse Hippocampal Pyramidal Cells Enhances GABAergic Neurotransmission and Protects against PTZ-Induced Seizures.

Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABA type-A receptors (GABAARs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3–, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knock-out (KO) mice results in absence of gephyrin and gephyrin-dependent GABAARs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3– in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3– in hippocampal pyramidal cells or hippocampal interneurons. The results show that: (1) the accumulation of gephyrin and GABAARs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3– overexpression; (2) overexpression of CBSH3– in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission; and (3) these enhanced inhibitory synapses provide protection against pentylenetetrazole (PTZ)-induced seizures. The results indicate that this AAV vector carrying CBSH3– can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain.

Gossypitrin, A Naturally Occurring Flavonoid, Attenuates Iron-Induced Neuronal and Mitochondrial Damage.

The disruption of iron homeostasis is an important factor in the loss of mitochondrial function in neural cells, leading to neurodegeneration. Here, we assessed the protective action of gossypitrin (Gos), a naturally occurring flavonoid, on iron-induced neuronal cell damage using mouse hippocampal HT-22 cells and mitochondria isolated from rat brains. Gos was able to rescue HT22 cells from the damage induced by 100 µM Fe(II)-citrate (EC50 8.6 µM). This protection was linked to the prevention of both iron-induced mitochondrial membrane potential dissipation and ATP depletion. In isolated mitochondria, Gos (50 µM) elicited an almost complete protection against iron-induced mitochondrial swelling, the loss of mitochondrial transmembrane potential and ATP depletion. Gos also prevented Fe(II)-citrate-induced mitochondrial lipid peroxidation with an IC50 value (12.45 µM) that was about nine time lower than that for the tert-butylhydroperoxide-induced oxidation. Furthermore, the flavonoid was effective in inhibiting the degradation of both 15 and 1.5 mM 2-deoxyribose. It also decreased Fe(II) concentration with time, while increasing O2 consumption rate, and impairing the reduction of Fe(III) by ascorbate. Gos–Fe(II) complexes were detected by UV-VIS and IR spectroscopies, with an apparent Gos-iron stoichiometry of 2:1. Results suggest that Gos does not generally act as a classical antioxidant, but it directly affects iron, by maintaining it in its ferric form after stimulating Fe(II) oxidation. Metal ions would therefore be unable to participate in a Fenton-type reaction and the lipid peroxidation propagation phase. Hence, Gos could be used to treat neuronal diseases associated with iron-induced oxidative stress and mitochondrial damage.

Combination of endothelial progenitor cells and BB-94 significantly alleviates brain damage in a mouse model of diabetic ischemic stroke.

Ischemic stroke is a complication of chronic macrovascular disease in type 2 diabetes. However, the pathogenesis of diabetic ischemic stroke has not yet been fully clarified. The aim of the present study was to investigate the underlying effects of endothelial progenitor cells (EPCs) and the matrix metalloproteinase inhibitor BB-94 on diabetic stroke. In vitro experiments were performed using oxygen-glucose deprivation/reoxygenation (OGD/R) model cells, established using HT22 mouse hippocampal cells. MTT assays and flow cytometry revealed that BB-94 prominently induced the proliferation of the OGD/R model cells and prevented their apoptosis. When EPCs and BB-94 were applied to the OGD/R model cells in combination, proliferation was further accelerated and oxidative damage was attenuated. In vivo experiments were also performed using a middle cerebral artery occlusion (MCAO) mouse model. The results of modified neurological severity scoring and oxidative stress marker analysis demonstrated that EPCs and BB-94 prominently alleviated cerebral ischemia/reperfusion injury in the MCAO model mice. Furthermore, reverse transcription-quantitative PCR and western blot assays revealed that EPCs in combination with BB-94 significantly downregulated the expression of matrix metalloproteinases (MMPs) and upregulated the expression of tissue inhibitor of metalloproteinases 1 in OGD/R cells and MCAO model mice. The results suggest that EPCs were successfully isolated and identified, and the OGD/R cell and MCAO mouse models were successfully established. They also indicate that EPCs alone or in combination with BB-94 may exert protective effects against ischemic stroke via the reduction of MMP expression.

Isorhynchophylline Relieves Ferroptosis-Induced Nerve Damage after Intracerebral Hemorrhage Via miR-122-5p/TP53/SLC7A11 Pathway.

Isorhynchophylline (IRN), a component of traditional Chinese herb Uncaria rhynchophylla, possesses strong antioxidant activity. Ferroptosis induced by iron overload causes cell oxidative stress after intracerebral hemorrhage (ICH). Therefore, this study aims to explore the effects of IRN on the ferroptosis following ICH. In this study, mouse hippocampal HT-22 cells were treated with ferric ammonium citrate (FAC) alone or together with IRN, and we found IRN reduced the FAC-induced cell damage. Then, cells were treated with IRN following treatment with FAC after transfection with miR-122-5p inhibitor, and the results showed IRN reduced the FAC-induced decrease of miR-122-5p levels and relieved the ferroptosis by detecting ferroptotic marker proteins, iron ion concentration and oxidative stress level; after transfection with miR-122-5p inhibitor, the protective effects of IRN against FAC-induced ferroptosis in these cells were weakened. TP53 (also known as p53) was verified as a target of miR-122-5p by using dual luciferase reporter assay, and restoration of TP53 attenuated the effects of miR-122-5p on ferroptotic marker proteins expression, iron ion concentration and lipid ROS levels, as well as solute carrier family seven member 11 (SLC7A11) mRNA expression. SLC7A11 siRNA reversed the inhibitory effects of IRN on FAC-induced ferroptosis and oxidative stress levels. Subsequently, IRN increased the mNSS score, and decreased brain water content and EB content in ICH model. Moreover, IRN decreased ferroptosis and lipid ROS level, upregulated the expression of miR-122-5p and SLC7A11 mRNA, and inhibited TP53 expression. Our findings reveal that IRN protects neurocyte from ICH-induced ferroptosis via miR-122-5p/TP53/SLC7A11 pathway, which may provide a potential therapeutic mechanism for ICH.

Synergistic Neuroprotective Effects of Mature Silkworm and Angelica gigas Against Scopolamine-Induced Mild Cognitive Impairment in Mice and H2O2-Induced Cell Death in HT22 Mouse Hippocampal Neuronal Cells.

We previously reported that mature Bombyx mori silkworm (SW) ameliorated scopolamine (Sco)-induced amnesia, and Angelica gigas (AG) prevented cognitive impairment. SW is known for its gastroprotective effects such as improving liver function and alleviating the effects of Parkinson's disease. AG is known for its neuroprotective effects and for lowering the effects of low-density lipoprotein cholesterol. However, the neuroprotective effect of combined SW and AG (SWA-1) treatment and the underlying molecular mechanism by which SWA-1 regulates neurodegenerative diseases remains unclear. We evaluated the neuroprotective effect of SWA-1 against Sco-induced mild cognitive impairment in mice and H2O2-induced cell death in HT22 mouse hippocampal neuronal cells and elucidated the underlying molecular mechanism. Morris water maze and Y-maze tests were performed to examine the learning and memory abilities of mice. The underlying molecular mechanism was investigated by using western blotting. We demonstrated that SWA-1 significantly protects against H2O2-induced cell death in HT22 mouse hippocampal neuronal cells. SWA-1 also significantly reversed Sco-induced spatial learning and memory impairment. Specifically, SWA-1 upregulates the protein levels of phosphorylated extracellular signal-related kinase (Erk1/2) and phosphorylated p38 MAP kinase (p38). SWA-1 remarkably decreased the apoptotic index Bax/Bcl2 expression in the hippocampus of Sco-treated mice. Our results suggest that SWA-1 may be administered as alternative therapy for cognitive impairment and neurodegenerative diseases and should be studied further in human trials.

Neural Cellular Response Upon Exposure to Brominated Flame Retardants

Brominated Flame Retardants (BFRs) are ubiquitously utilized to reduce flammability in a wide range of household products including carpets, upholstery, and paints. While useful chemicals, BFRs also migrate from their products into the environment. This has resulted in continuous, population-level exposure which has been correlated to impaired learning and memory. To determine the effects of multiple BFRs on different stages of neuronal development, human Neural Stem Cells (NSCs) and mouse hippocampal HT-22 cells were exposed to Tetrabromobisphenol-A (TBBPA), Hexabromocyclododecane (HBCD), or 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) (current, phasing out, and phased out BFRs, respectively). Cell viability analysis was assessed by MTT staining after 48 hr of exposure, and demonstrated that HBCD was more toxic to both NSCs (3 μM IC50) and HT-22 cells (15 μM IC50) than TBBPA (NSC 20 μM, HT-22 50 μM) and BDE-47 (NSC 9 μM, HT-22 60 μM). HT-22 cellular and nuclear morphology demonstrated evidence of apoptosis and altered morphology at the IC50 for each BFR after both 24 and 48 hr exposure. Flow cytometry analysis provided further support for a time and concentration-dependent increase in apoptosis as indicated by an increase in annexin V staining in cells. Both 24 and 48 hr HBCD (50 or 100 μM) or BDE-47 (100 μM) exposure induced significant increases in apoptosis, although 100 μM TBBPA only induced apoptosis and necrosis after 48 hr. Interestingly, these chemicals also induced cell cycle alterations with increasing exposure time. Upon 24 hr exposure, HBCD (50 or 100 μM) induced significant S-phase arrest which was maintained upon 48 hr exposure. However, only upon 48 hr exposure did BDE-47 (50 and 100 μM) and TBBPA (100 μM) induce significant S-phase arrest. These data demonstrate that BFRs can induce chemical-dependent toxicity in neural cells, possibly by multiple mechanisms. Further study is needed to determine if BFR-induced neural cell death would adversely affect learning and memory in vivo.

Gene Expression of Mouse Hippocampal Stem Cells Grown in a Galactose-Derived Molecular Gel Compared to In Vivo and Neurospheres

Background: N-heptyl-D-galactonamide (GalC7) is a small synthetic carbohydrate derivative that forms a biocompatible supramolecular hydrogel. In this study, the objective was to analyze more in-depth how neural cells differentiate in contact with GalC7. Method: Direct (ex vivo) cells of the fresh hippocampus and culture (In vitro) of the primary cells were investigated. In vitro, investigation performed under three conditions: on culture in neurospheres for 19 days, on culture in GalC7 gel for 7 days, and on culture in both neurospheres and GalC7 gel. Total RNA was isolated with TRIzol from each group, Sox8, Sox9, Sox10, Dcx, and Neurod1 expression levels were measured by qPCR. Result: Sox8 and Sox10, oligodendrocyte markers, and Sox9, an astrocyte marker, were expressed at a much higher level after 7 days of culture in GalC7 hydrogel compared to all other conditions. Dcx, a marker of neurogenesis, and Neurod1, a marker of neuronal differentiation, were expressed at better levels in the GalC7 gel culture compared to the neurosphere. Conclusions: These results show that the GalC7 hydrogel brings different and interesting conditions for inducing the differentiation and maturation of neural progenitor cells compared with polymer-based scaffolds or cell-only conditions. The differences observed open new perspectives in tissue engineering, induction, and transcript analysis.

S100B/RAGE/Ceramide signaling pathway is involved in sepsis-associated encephalopathy

AimsSepsis-associated encephalopathy (SAE) is one of the most common complications of sepsis, and it might lead to long-term cognitive dysfunction and disability. This study aimed to explore the role of S100 calcium binding protein B (S100B)/RAGE/ceramide signaling pathway in SAE. Main methodsFPS-ZM1 (an inhibitor of RAGE), myriocin and GW4869 (an inhibitor of ceramide) were used to explore the role of S100B/RAGE/ceramide in acute brain injury and long-term cognitive impairment in sepsis. In addition, Mdivi-1 (inhibitor of Drp1) and Drp1 siRNA were utilized to assess the effects of C2-ceramide on neuronal mitochondria, and to explore the specific underlying mechanism in C2 ceramide-induced death of HT22 mouse hippocampal neuronal cells. Key findingsWestern blot analysis showed that sepsis significantly up-regulated S100B and RAGE. Nissl staining and Morris water maze (MWM) test revealed that inhibition of RAGE with FPS-ZM1 markedly attenuated cecal ligation and puncture (CLP)-induced brain damage and cognitive dysfunction. Furthermore, FPS-ZM1 relieved sepsis-induced C2-ceramide accumulation and abnormal mitochondrial dynamics. Moreover, inhibition of ceramide also showed similar protective effects both in vivo and in vitro. Furthermore, Mdivi-1 and Drp1 siRNA significantly reduced C2-ceramide-induced neuronal mitochondrial fragmentation and cell apoptosis in vitro. SignificanceThis study confirmed that S100B regulates mitochondrial dynamics through RAGE/ceramide pathway, in addition to the role of this pathway in acute brain injury and long-term cognitive impairment during sepsis.

Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves.

Dendropanax morbifera 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, Ca2+ 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.

Kaempferia parviflora Rhizome Extract Inhibits Glutamate-Induced Toxicity in HT-22 Mouse Hippocampal Neuronal Cells and Extends Longevity in Caenorhabditis elegans.

Simple SummaryIn aging societies, age-associated diseases are recognized to diminish people’s quality of life. Kaempferia parviflora Wall. ex Baker or “Kra-chai-dam” is a medicinal plant that is known to exhibit numerous pharmacological effects such as anti-inflammation, antimicrobial, and sexual-enhancing activity, and has been used as a nerve tonic. Due to its known neuropharmacological activities, we hypothesize that its rhizome extract might possess both neuroprotective and longevity-enhancing properties. We found that the extract protected glutamate-induced cytotoxicity of mouse hippocampal HT-22 neuronal cells, thus reducing apoptotic cell death. Concomitantly, cells had lower levels of intracellular reactive oxygen species, and changes in the expression of related proteins were found to support the molecular neuroprotection. Furthermore, we found the extract could extend the lifespan of Caenorhabditis elegans. Overall, our study proved that the K. parviflora rhizome extract possessed neuroprotective and longevity-enhancing properties.Kaempferia parviflora Wall. ex Baker (KP) or “Kra-chai-dam” has been shown to exhibit several pharmacological effects including anti-inflammation, antimicrobial, and sexual-enhancing activity. The objectives of this study included an investigation of the effect of KP rhizome extract against glutamate-induced toxicity in mouse hippocampal HT-22 neuronal cells, determination of the underlying mechanism of neuroprotection, and an evaluation of the effect of KP extract on the longevity of Caenorhabditis elegans. HT-22 cells were co-treated with glutamate (5 mM) and KP extract (25, 50, and 75 μg/mL) for 14 h. Cell viability, intracellular reactive oxygen species (ROS) assay, fluorescence-activated cell sorting (FACS) analysis, and Western blotting were performed. The longevity effect of KP extract on C. elegans was studied by lifespan measurement. In HT-22 cells, co-treatment of glutamate with KP extract significantly inhibited glutamate-mediated cytotoxicity and decreased intracellular ROS production. Additionally, the glutamate-induced apoptosis and apoptotic-inducing factor (AIF) translocation were blocked by KP extract co-treatment. Western blot analysis also demonstrated that KP extract significantly diminished extracellular signal-regulated kinase (ERK) phosphorylation induced by glutamate, and brain-derived neurotrophic factor (BDNF) was recovered to the control. Moreover, this KP extract treatment prolonged the lifespan of C. elegans. Altogether, this study suggested that KP extract possesses both neuroprotective and longevity-inducing properties, thus serving as a promising candidate for development of innovative health products.

Functional genomics study of protein inhibitor of activated STAT1 in mouse hippocampal neuronal cells revealed by RNA sequencing.

Protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) E3 ligase, was considered to be an inhibitor of STAT1 by inhibiting the DNA-binding activity of STAT1 and blocking STAT1-mediated gene transcription in response to cytokine stimulation. PIAS1 has been determined to be involved in modulating several biological processes such as cell proliferation, DNA damage responses, and inflammatory responses, both in vivo and in vitro. However, the role played by PIAS1 in regulating neurodegenerative diseases, including Alzheimer’s disease (AD), has not been determined. In our study, significantly different expression levels of PIAS1 between normal controls and AD patients were detected in four regions of the human brain. Based on a functional analysis of Pias1 in undifferentiated mouse hippocampal neuronal HT-22 cells, we observed that the expression levels of several AD marker genes could be inhibited by Pias1 overexpression. Moreover, the proliferation ability of HT-22 cells could be promoted by the overexpression of Pias1. Furthermore, we performed RNA sequencing (RNA-seq) to evaluate and quantify the gene expression profiles in response to Pias1 overexpression in HT-22 cells. As a result, 285 significantly dysregulated genes, including 79 upregulated genes and 206 downregulated genes, were identified by the comparison of Pias1/+ cells with WT cells. Among these genes, five overlapping genes, including early growth response 1 (Egr1), early growth response 2 (Egr2), early growth response 3 (Egr3), FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1), were identified by comparison of the transcription factor binding site (TFBS) prediction results for STAT1, whose expression was evaluated by qPCR. Three cell cycle inhibitors, p53, p18 and p21, were significantly downregulated with the overexpression of Pias1. Analysis of functional enrichment and expression levels showed that basic region leucine zipper domain-containing transcription factors including zinc finger C2H2 (zf-C2H2), homeobox and basic/helix-loop-helix (bHLH) in several signaling pathways were significantly involved in PIAS1 regulation in HT-22 cells. A reconstructed regulatory network under PIAS1 overexpression demonstrated that there were 43 related proteins, notably Nr3c2, that directly interacted with PIAS1.

ABCA1-Labeled Exosomes in Serum Contain Higher MicroRNA-193b Levels in Alzheimer's Disease.

Objective We aimed to establish a method to determine whether microRNA-193b (miR-193b) levels in ABCA1-labeled serum exosomes might serve as a marker for the diagnosis of Alzheimer's disease. Methods We used immunocapture methods to determine the levels of ABCA1-labeled exosomal miR-193b in cultures of white blood cells (WBCs), red blood cells (RBCs), mouse hippocampal neuron HT-22 cells, and primary mouse neuronal cells. ABCA1-labeled exosomal miR-193b levels were also evaluated in the cerebrospinal fluid (CSF) and serum of APP/PS1 double-transgenic mice, as well as control subjects (n = 60) and study participants with subjective cognitive decline (SCD, n = 89), stage and mild cognitive impairment (MCI, n = 92), and dementia of the Alzheimer type (DAT, n = 92). Results ABCA1 levels of exosomes harvested from the medium of HT-22 cells and neurons were significantly higher than those of RBCs and WBCs (P < 0.05). Exosomal ABCA1 from the CSF of APP/PS1 mice were transmitted to the serum of wild-type mice after injection, and high miR-193b levels were observed in both the serum and CSF after injection. The ABCA1-labeled exosomal miR-193b levels were higher in the CSF of MCI and DAT patients compared with the CSF of the control group (P < 0.05). The ABCA1-labeled exosomal miR-193b were also slightly higher (P > 0.05) in the serum of SCD patients and significantly higher in the serum of MCI and DAT patients compared with the serum of the control group (P < 0.05). Conclusion This study provides a method to capture specific exosomes. Detection of serum exosomes labeled with ABCA1 may facilitate the early diagnosis of AD.

Neuroprotective Effect of Gallocatechin Gallate on Glutamate-Induced Oxidative Stress in Hippocampal HT22 Cells.

Oxidative stress leads to protein degeneration or mitochondrial dysfunction, causing neuronal cell death. Glutamate is a neurotransmitter that nerve cells use to send signals. However, the excess accumulation of glutamate can cause excitotoxicity in the central nervous system. In this study, we deciphered the molecular mechanism of catechin-mediated neuroprotective effect on glutamate-induced oxidative stress in mouse hippocampal neuronal HT22 cells. Cellular antioxidant activity was determined using the 1,1-diphenyl-picryl hydrazyl (DPPH) assay and 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining. Furthermore, the levels of intracellular calcium (Ca2+) as well as nuclear condensation and protein expression related to neuronal damage were assessed. All five catechins (epigallocatechin gallate, gallocatechin gallate (GCG), gallocatechin, epicatechin gallate, and epicatechin) showed strong antioxidant effects. Among them, GCG exhibited the highest neuroprotective effect against glutamate excitotoxicity and was used for further mechanistic studies. The glutamate-induced increase in intracellular Ca2+ was reduced after GCG treatment. Moreover, GCG reduced nuclear condensation and the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK) involved in cell death. The neuroprotective effect of GCG against glutamate-induced oxidative stress in HT22 cells was attributed to the reduction in intracellular free radicals and Ca2+ influx and also the inhibition of phosphorylation of ERK and JNK. Furthermore, the antioxidant effect of GCG was found to be likely due to the inhibition of phosphorylation of ERK and JNK that led to the effective suppression of neurocytotoxicity caused by glutamate in HT22 cells.