Unveiling the agonistic properties of Preyssler-type Polyoxotungstates on purinergic P2 receptors.

The Preyssler-type polyoxotungstate ({P5W30}) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P5W30} on the cytosolic Ca2+ concentration. Changes in cytosolic Ca2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca2+ indicator Fluo3. 31P-Nuclear magnetic resonance measurements of {P5W30} indicate its stability in the medium used for cytosolic Ca2+ measurements for over 30 min. The findings reveal that addition of {P5W30} to the extracellular medium induces a sustained increase of the cytosolic Ca2+ concentration within minutes. This Ca2+ increase is triggered by extracellular Ca2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 µM {P5W30}. In addition, after the {P5W30}-induced cytosolic Ca2+ increase, the transient Ca2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 µM {P5W30}. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P5W30} in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P5W30} could serve as a novel agonist of purinergic P2 receptors.

CB1 receptor mediates anesthetic drug ketamine­induced neuroprotection against glutamate in HT22 cells.

The anesthetic drug, ketamine (KTM) has been shown to induce therapeutic effects against major depressive disorder (MDD), however the related underlying mechanisms remain unclear. In the present study, HT22 neuronal cells were treated with glutamate to imitate oxidative stress injury in MDD, and it was hypothesized that the cannabinoid type 1 (CB1) receptor mediates KTM-induced neuroprotection via ameliorating mitochondrial function in glutamate-treated neuronal cells. Compared with the control, glutamate decreased cell viability and intracellular antioxidants, including glutathione (GSH), catalase and superoxide dismutase 2 levels, and inhibited mitochondrial function simultaneously. Moreover, glutamate increased lactate dehydrogenase release, cellular apoptosis level, cleaved caspase-3 expression and intracellular oxidants, such as reactive oxygen species, oxidized GSH and mitochondrial superoxide in the cells. The presence of KTM, however, significantly decreased the glutamate-induced oxidative stress injury, ameliorated the antioxidant/oxidant levels in the cells, enhanced mitochondrial function and upregulated CB1 receptor expression (P<0.05). Co-administration of the CB1 receptor antagonist AM251 markedly abolished the KTM-induced cytoprotective effects and ameliorations of antioxidant/oxidant levels and mitochondrial function, and also reversed CB1 upregulation (P<0.05). These observations indicated that KTM decreases the oxidative stress injury caused by glutamate in HT22 neuronal cells, and the neuroprotective effects may be mediated by the CB1 receptor.

γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells.

The excessive activation of glutamate in the brain is a factor in the development of vascular dementia. γ-Oryzanol is a natural compound that has been shown to enhance brain function, but more research is needed to determine its potential as a treatment for vascular dementia. This study investigated if γ-oryzanol can delay or improve glutamate neurotoxicity in an in vitro model of differentiated HT-22 cells and explored its neuroprotective mechanisms. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h then given γ-oryzanol at appropriate concentrations or memantine (10 µmol/L) for another 24 h. Glutamate produced reactive oxygen species and depleted glutathione in the cells, which reduced their viability. Mitochondrial dysfunction was also observed, including the inhibition of mitochondrial respiratory chain complex I activity, the collapse of mitochondrial transmembrane potential, and the reduction of intracellular ATP levels in the HT-22 cells. Calcium influx triggered by glutamate subsequently activated type II calcium/calmodulin-dependent protein kinase (CaMKII) in the HT-22 cells. The activation of CaMKII-ASK1-JNK MAP kinase cascade, decreased Bcl-2/Bax ratio, and increased Apaf-1-dependent caspase-9 activation were also observed due to glutamate induction, which were associated with increased DNA fragmentation. These events were attenuated when the cells were treated with γ-oryzanol (0.4 mmol/L) or the N-methyl-D-aspartate receptor antagonist memantine. The results suggest that γ-oryzanol has potent neuroprotective properties against glutamate excitotoxicity in differentiated HT-22 cells. Therefore, γ-oryzanol could be a promising candidate for the development of therapies for glutamate excitotoxicity-associated neurodegenerative diseases, including vascular dementia.

Arecoline induces neurotoxicity in HT22 cells via the promotion of endoplasmic reticulum stress and downregulation of the Nrf2/HO-1 pathway.

Arecoline, the predominant bioactive substance extracted from areca nut (AN), is the world's fourth most frequently used psychoactive material. Research has revealed that chewing AN can affect the central nervous system (CNS) and may lead to neurocognitive deficits that are possibly linked to the action of arecoline. However, the mechanism behind the neurotoxicity caused by arecoline remains unclear. This study aimed to investigate the neurotoxic effects of arecoline and its underlying mechanism. The results showed that arecoline caused cytotoxicity against HT22 cells in a dose-dependent manner and induced apoptosis by upregulating the expression of pro-apoptotic caspase and Bcl-2 family proteins. Furthermore, arecoline escalated intracellular reactive oxygen species (ROS) levels and Ca2+ concentration with increasing doses, thereby motivating endoplasmic reticulum stress (ERS) and ERS-associated apoptotic protein expression. Additionally, the study found that arecoline attenuates intracellular antioxidant defense by inhibiting the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus and decreasing downstream Heme oxygenase-1 (HO-1) levels. The specific inhibitor Sodium 4-phenylbutyrate (4-PBA) can dramatically attenuate arecoline-mediated cell apoptosis and ERS-associated apoptotic pathway expression by blocking ERS. The antioxidant N-Acetylcysteine (NAC) also effectively reverses the arecoline-mediated increase of ERS-related apoptotic pathway protein levels by scavenging intracellular ROS accumulation. In conclusion, this study suggests that arecoline induces neurotoxicity in HT22 cells via ERS mediated by oxidative stress- and Ca2+ disturbance, as well as by downregulation of the Nrf2/HO-1 pathway.

Silver nanoparticles induced synaptic degeneration via Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells.

Silver nanoparticles (AgNPs) have been widely used in biomedicine and cosmetics, increasing their potential risks in neurotoxicity. But the involved molecular mechanism remains unclear. This study aims to explore molecular events related to AgNPs-induced neuronal damage by RNA-seq, and elucidate the role of Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells synaptic degeneration induced by AgNPs. This study found that cell viabilities were decreased by AgNPs in a dose/time-dependent manner. AgNPs also increased protein expression of PINK1, Parkin, synaptophysin, and inhibited PGC-1α, MAP2 and APP protein expression, indicating AgNPs-induced synaptic degeneration involved in disturbance of mitophagy and mitochondrial biogenesis in HT22 cells. Moreover, inhibition of AgNPs-induced Ca2+/CaMKII activation and Drp1/ROS rescued mitophagy disturbance and synaptic degeneration in HT22 cells by reserving aforementioned protein express changes except for PGC-1α and APP protein. Thus, AgNPs-induced synaptic degeneration was mediated by Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells, and mitophagy is the sensitive to the mechanism. Our study will provide in-depth molecular mechanism data for neurotoxic evaluation and biomedical application of AgNPs.

Alcohol extract of Rubia yunnanensis: Metabolic alterations and preventive effects against OGD/R­induced oxidative damage in HT22 cells.

The present study investigated the inhibitory and neuroprotective effects of Rubia yunnanensis alcohol extract (RY-A) on oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells. In vitro cultured HT22 cells were randomly divided into control, OGD/R, OGD/R + 100 µmol/l edaravone and OGD/R + 10, 20 and 40 µg/ml RY-A groups. Oxygen-sugar deprivation was performed with 10 mmol/l sodium dithionite combined with sugar-free DMEM medium for 2 h, followed by re-glycolization and reoxygenation for 2 h to establish an in vitro OGD/R model. Cell morphology was observed under a phase contrast microscope. Cell survival rate was detected by thiazolyl blue and lactate dehydrogenase and oxidative stress-related indexes were detected by commercial kits. The effects and metabolic alterations of RY-A treatment after OGD/R were evaluated using ultra-high performance liquid chromatography and mass spectrometry. Protein levels were further examined by western blotting. The results showed that cells in the OGD/R group were swollen and lacked protrusions, had significantly reduced viability and had significantly elevated oxidative stress-related indexes of reactive oxygen species, nitric oxide levels and malondialdehyde content and significantly reduced activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, compared with controls. Compared with the OGD/R group, the RY-A group had significantly improved cell morphology and significantly increased cell viability and in terms of oxidative stress, exhibited significantly reduced reactive oxygen species, nitric oxide levels and malondialdehyde content, as well as significantly increased superoxide dismutase and glutathione peroxidase activities. Metabolomic analysis identified changes in 20 metabolites, including L-tryptophan, ornithine, eicosapentaenoic acid-d5, isosafrole and xanthine. Metabolomics analysis showed that the pathways affected included those related to phenylalanine, tyrosine and tryptophan biosynthesis, the prolactin signaling pathway and amphetamine addiction. These results suggested that RY-A had significant preventive effects on an in vitro model of cerebral ischemia-reperfusion injury simulated by OGD/R and the mechanism may be related to increased tryptophan content, activation of indoleamine 2,3-dioxygenase enzymes and inhibition of oxidative stress.

Reducing polypyrimidine tract­binding protein 1 fails to promote neuronal transdifferentiation on HT22 and mouse astrocyte cells under physiological conditions.

In contrast to prior findings that have illustrated the conversion of non-neuronal cells into functional neurons through the specific targeting of polypyrimidine tract-binding protein 1 (PTBP1), accumulated evidence suggests the impracticality of inducing neuronal transdifferentiation through suppressing PTBP1 expression in pathological circumstances. Therefore, the present study explored the effect of knocking down PTBP1 under physiological conditions on the transdifferentiation of mouse hippocampal neuron HT22 cells and mouse astrocyte (MA) cells. A total of 20 µM negative control small interfering (si)RNA and siRNA targeting PTBP1 were transfected into HT22 and MA cells using Lipo8000™ for 3 and 5 days, respectively. The expression of early neuronal marker ßIII-Tubulin and mature neuronal markers NeuN and microtubule-associated protein 2 (MAP2) were detected using western blotting. In addition, ßIII-tubulin, NeuN and MAP2 were labeled with immunofluorescence staining to evaluate neuronal cell differentiation in response to PTBP1 downregulation. Under physiological conditions, no significant changes in the expression of ßIII-Tubulin, NeuN and MAP2 were found after 3 and 5 days of knockdown of PTBP1 protein in both HT22 and MA cells. In addition, the immunofluorescence staining results showed no apparent transdifferentiation in maker levels and morphology. The results suggested that the knockdown of PTBP1 failed to induce neuronal differentiation under physiological conditions.

Adenosine A2A receptor antagonist KW6002 protects against A53T mutant alpha-synuclein-induced brain damage and neuronal apoptosis in Parkinson's disease mice by restoring autophagic flux.

BACKGROUND: Protein misfolding and inclusion body aggregation caused by α-Syn mutations in the brain often cause neurodegeneration and cognitive impairment, among which the A53T point mutation is more common. Inhibition of adenosine A2A receptor (A2AR) can alleviate the pathological symptoms of brain dysfunction caused by A53T-α-Syn protofibrils, but the mechanism of action is still unclear. AIM: This studies aimed to investigate the potential therapeutic role of the A2AR inhibitor KW6002 in a mouse model of brain synucleinopathy. METHODS: A53T-α-Syn fibre precursor cell nuclear protein was injected into the bilateral prefrontal cortex of mice to establish a synucleinopathy animal model, and the A2AR inhibitor KW6002 (5 mg/kg) was injected intraperitoneally to intervene. RESULT: The intracerebral injection of A53T-α-Syn protofibrils triggers the formation of inclusion bodies in the brain, leading to astrocyte activation, an increased number of apoptotic cells, and suppression of autophagic flux. The administration of KW6002 significantly reversed these phenomena. In vitro experiments revealed that A53T-α-Syn protofibrils inhibited HT-22 autophagy in mouse hippocampal neuronal cells, whereas KW6002 increased cellular autophagic flux, upregulated the expression of LAMP2A and Hsc70 proteins and inhibited the expression of SQSTM1 protein. The present study suggests that KW6002 reduces the level of α-Syn phosphorylation by inhibiting A2AR protein, at the same time, enhances the autophagic flux of neuronal cells, resulting in the degradation of A53T-α-Syn protofibrils and thus reducing the neuronal toxicity and apoptosis induced by A53T-α-Syn protofibrils. CONCLUSION: KW6002 has a significant protective effect on neuronal injury induced by A53T-α-Syn.

The Interplay Between Cholesterol and Amyloid-ß on HT22 Cell Viability, Morphology, and Receptor Tyrosine Kinase Signaling.

BACKGROUND: There is a lack of understanding in the molecular and cellular mechanisms of Alzheimer's disease that has hindered progress on therapeutic development. The focus has been on targeting toxic amyloid-ß (Aß) pathology, but these therapeutics have generally failed in clinical trials. Aß is an aggregation-prone protein that has been shown to disrupt cell membrane structure in molecular biophysics studies and interfere with membrane receptor signaling in cell and animal studies. Whether the lipid membrane or specific receptors are the primary target of attack has not been determined. OBJECTIVE: This work elucidates some of the interplay between membrane cholesterol and Aß42 on HT22 neuronal cell viability, morphology, and platelet-derived growth factor (PDGF) signaling pathways. METHODS: The effects of cholesterol depletion by methyl-ß-cyclodextrin followed by treatment with Aß and/or PDGF-AA were assessed by MTT cell viability assays, western blot, optical and AFM microscopy. RESULTS: Cell viability studies show that cholesterol depletion was mildly protective against Aß toxicity. Together cholesterol reduction and Aß42 treatment compounded the disruption of the PDGFα receptor activation. Phase contrast optical microscopy and live cell atomic force microscopy imaging revealed that cytotoxic levels of Aß42 caused morphological changes including cell membrane damage, cytoskeletal disruption, and impaired cell adhesion; cell damage was ameliorated by cellular cholesterol depletion. CONCLUSIONS: Cholesterol depletion impacted the effects of Aß42 on HT22 cell viability, morphology, and receptor tyrosine kinase signaling.

Sevoflurane alleviates oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells by activating the Keap1/Nrf2/ARE pathway to inhibit oxidative stress.

Objective: This study aimed to investigate the impact of sevoflurane on oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells and its associated mechanisms. Methods: HT22 cells were treated with sevoflurane, and an oxygen-glucose deprivation/reoxygenation injury model was established. The HT22 cells were randomly divided into the control group, oxygen-glucose deprivation/reoxygenation group, sevoflurane low-dose group, sevoflurane medium-dose group, and sevoflurane high-dose group. The proliferation of HT22 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis rate and mitochondrial membrane potential of HT22 cells were determined by flow cytometry. Protein expression levels of B-cell lymphoma-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) in HT22 cells were examined using Western blot. Reactive oxygen species (ROS) levels were measured with 2',7'-dichlorofluorescin diacetate (DCFH-DA). Malondialdehyde (MDA), glutathione peroxidase (GSH-Px) levels, and superoxide dismutase (SOD) enzyme activity in HT22 cells were determined using assay kits. Results: Compared to controls, OGD/R group had reduced cell viability, mitochondrial potential, Bcl-2, nuclear Nrf2, HO-1, GSH-Px levels, and SOD enzyme activity (p < 0.05), with increased apoptosis, Bax, cytoplasmic Nrf2, ROS, and MDA levels. Sevoflurane groups showed opposite trends (p < 0.05). Conclusion: Sevoflurane can mitigate oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells, and its mechanism may be related to the activation of the Keap1/Nrf2/ARE pathway to inhibit oxidative stress.

BANF1 promotes glutamate-induced apoptosis of HT-22 hippocampal neurons.

BACKGROUND: Glutamate exposure was fatal to HT-22 neuronal cells that derived from mouse hippocampus. This is often used as a model for hippocampus neurodegeneration in vitro. The targets relevant to glutamate-induced neuronal toxicity is not fully understood. In this study, we aimed to identify crucial factors associated with glutamate-induced cytotoxicity in HT-22 cells. METHODS: HT-22 cells were treated with 7.5 mM glutamate for 24 h and isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis conducted to identify the differentially expressed proteins. Differential proteins were subjected to Gene Ontology analyses. Upregulation of barrier to autointegration factor (BANF1/BANF1) protein was confirmed by RT-qPCR and western blotting. Cell viability was measured by CKK-8 and MTT assays. Cell apoptosis rates and intracellular reactive oxygen species (ROS) levels were detected using flow cytometry. RESULTS: A total of 5811 proteins were quantified by iTRAQ, 50 of which were recognized as significantly differential proteins (fold change ≥ 1.5 and P ≤ 0.05); 26 proteins were up-regulated and 24 were down-regulated after exposure to glutamate. GO enrichment analysis showed that the apoptotic signaling pathway was involved in cell death induced by glutamate. BANF1 expression level was markedly increased in HT-22 cells after glutamate treatment. Further, knockdown of BANF1 alleviated glutamate-mediated cell death with lower ROS levels. CONCLUSIONS: In conclusion, we successfully filtered out differential proteins relevant to glutamate-mediated cytotoxicity. BANF1 upregulation promoted glutamate-induced apoptosis of HT-22 cells by enhancing ROS generation.

Electro-conductive chitosan/graphene bio-nanocomposite scaffold for tissue engineering of the central nervous system.

Degenerative central nervous system (CNS) disorders and traumatic brain injuries are common nowadays. These may induce the loss of neuronal cells and delicate connections essential for optimal CNS function. The CNS tissue has restricted regeneration ability, hindering the development of effective therapies. Developing cell and tissue instructive materials may bring up new treatment possibilities. In this study, chitosan-graphene nano platelets (GNPs) composite films were developed to regenerate brain cells. This study evaluates the effects of GNP concentration (0.5, 1 and 2 wt%) and their alignment on mechanical, electrical, surface, protein adsorption and biological properties of the regenerative scaffolds. Incorporating and aligning GNPs into chitosan matrix improved all the physical and biological properties. On reinforced scaffolds, HT22 cell morphology mimics pyramidal brain cells, which are responsible for the brain's highly branched neural network. Additionally, the reinforced scaffolds supported Mesenchymal Stem like Cells growth and were biocompatible in vivo. The alignment of GNPs in the chitosan matrix offered the appropriate physicochemical and biological properties to promote adhesion, proliferation and shape morphogenesis of hippocampal HT22 neuronal cells. Overall, this study delineates the enormous potential offered by the GNP-reinforced scaffolds for regeneration of central nervous system, especially the brain.

Mitochondrial transplantation rescues neuronal cells from ferroptosis.

Ferroptosis is a type of oxidative cell death that can occur in neurodegenerative diseases and involves damage to mitochondria. Previous studies demonstrated that preventing mitochondrial dysfunction can rescue cells from ferroptotic cell death. However, the complexity of mitochondrial dysfunction and the timing of therapeutic interventions make it difficult to develop an effective treatment strategy against ferroptosis in neurodegeneration conditions. In this study, we explored the use of mitochondrial transplantation as a novel therapeutic approach for preventing ferroptotic neuronal cell death. Our data showed that isolated exogenous mitochondria were incorporated into both healthy and ferroptotic immortalized hippocampal HT-22 cells and primary cortical neurons (PCN). The mitochondrial incorporation was accompanied by increased metabolic activity and cell survival through attenuating lipid peroxidation and mitochondrial superoxide production. Further, the function of mitochondrial complexes I, III and V activities contributed to the neuroprotective activity of exogenous mitochondria. Similarly, we have also showed the internalization of exogenous mitochondria in mouse PCN; these internalized mitochondria were found to effectively preserve the neuronal networks when challenged with ferroptotic stimuli. The administration of exogenous mitochondria into the axonal compartment of a two-compartment microfluidic device induced mitochondrial transportation to the cell body, which prevented fragmentation of the neuronal network in ferroptotic PCN. These findings suggest that mitochondria transplantation may be a promising therapeutic approach for protecting neuronal cells from ferroptotic cell death.

Tat-CCT2 Protects the Neurons from Ischemic Damage by Reducing Oxidative Stress and Activating Autophagic Removal of Damaged Protein in the Gerbil Hippocampus.

CCT2 is a eukaryotic chaperonin TCP-1 ring complex subunit that mediates protein folding, autophagosome incorporation, and protein aggregation. In this study, we investigated the effects of CCT on oxidative and ischemic damage using in vitro and in vivo experimental models. The Tat-CCT2 fusion protein was efficiently delivered into HT22 cells in a concentration- and time-dependent manner, and the delivered protein was gradually degraded in HT22 cells. Incubation with Tat-CCT2 significantly ameliorated the 200 µM hydrogen peroxide (H2O2)-induced reduction in cell viability in a concentration-dependent manner, and 8 µM Tat-CCT2 treatment significantly alleviated H2O2-induced DNA fragmentation and reactive oxygen species formation in HT22 cells. In gerbils, CCT2 protein was efficiently delivered into pyramidal cells in CA1 region by intraperitoneally injecting 0.5 mg/kg Tat-CCT2, as opposed to control CCT2. In addition, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 mitigated ischemia-induced hyperlocomotive activity 1 d after ischemia and confirmed the neuroprotective effects by NeuN immunohistochemistry in the hippocampal CA1 region 4 d after ischemia. Tat-CCT2 treatment significantly reduced the ischemia-induced activation of astrocytes and microglia in the hippocampal CA1 region 4 d after ischemia. Furthermore, treatment with 0.2 or 0.5 mg/kg Tat-CCT2 facilitated ischemia-induced autophagic activity and ameliorated ischemia-induced autophagic initiation in the hippocampus 1 d after ischemia based on western blotting for LC3B and Beclin-1, respectively. Levels of p62, an autophagic substrate, significantly increased in the hippocampus following treatment with Tat-CCT2. These results suggested that Tat-CCT2 exerts neuroprotective effects against oxidative stress and ischemic damage by promoting the autophagic removal of damaged proteins or organelles.

Exosomes derived from M2-type microglia ameliorate oxygen-glucose deprivation/reoxygenation-induced HT22 cell injury by regulating miR-124-3p/NCOA4-mediated ferroptosis.

Background: Although it has been reported that miRNA carried by M2 microglial exosomes protects neurons from ischemia-reperfusion brain injury, the mechanism of action remains poorly understood. This study aimed to explore the miRNA signaling pathway by which M2-type microglia-derived exosomes (M2-exosomes) ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity in HT22 cells. Methods: BV2 microglia were induced by M2 polarization. Then, M2-exosomes were identified via transmission electron microscopy and special biomarker detection and co-cultured with HT22 cells. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Intracellular concentrations of reactive oxygen species (ROS), Fe2+, glutathione (GSH), and malondialdehyde (MDA) were determined using dichlorofluorescein fluorescence and biochemical determination. miR-124-3p levels were determined using qRT-PCR, and protein expressions were examined via western blotting. Results: OGD/R suppressed the proliferation and induced the accumulation of Fe2+, ROS, and MDA and reduction of GSH in mouse HT22 cells, suggesting ferroptosis of HT22 cells. OGD/R-induced changes in the above mentioned indexes was ameliorated by M2-exosomes but restored by the exosome inhibitor GW4869. M2-exosomes with (mimic-exo) or without miR-124-3p (inhibitor-exo) promoted and suppressed proliferation and ferroptosis-associated indexes of HT22 cells, respectively. Moreover, mimic-exo and inhibitor-exo inhibited and enhanced NCOA4 expression in HT22 cells, respectively. NCOA4 overexpression reversed the protective effects of miR-124-3p mimic-exo in OGD/R-conditioned cells. NCOA4 was targeted and regulated by miR-124-3p. Conclusions: M2-exosome protects HT22 cells against OGD/R-induced ferroptosis injury by transferring miR-124-3p and NCOA4 into HT22 cells, with the latter being a target gene for miR-124-3p.

Involvement of CXCL10 in Neuronal Damage under the Condition of Spinal Cord Injury and the Potential Therapeutic Effect of Nrg1.

OBJECTIVE: Few studies have reported the direct effect of C-X-C motif chemokine ligand 10 (CXCL10) and Neuregulin 1 (Nrg1) on neurons after spinal cord injury (SCI). This study reports the role of CXCL10 in the regulation of neuronal damage after SCI and the potential therapeutic effect of Nrg1. METHODS: The expression level of CXCL10 and Nrg1 in SCI mice was analyzed in the Gene Expression Omnibus DataSets, followed by immunohistochemical confirmation using a mouse SCI model. HT22 cells and NSC34 cells were treated with CXCL10 and Nrg1, individually or in combination, and then assayed for cell viability. The percentage of wound closure was determined through the cell scratch injury model using HT22 and NSC34 cells. Potential molecular mechanisms were also tested in response to either the individual administration of CXCL10 and Nrg1 or a mixture of both molecules. RESULTS: CXCL10 expression was significantly increased in both young and old mice subjected to SCI, while Nrg1 expression was significantly decreased. CXCL10 induced a decrease in cell viability, which was partially reversed by Nrg1. CXCL10 failed to inhibit scratch healing in HT22 and NSC34 cells, while Nrg1 promoted scratch healing. At the molecular level, CXCL10-activated cleaved caspase 9 and cleaved caspase 3 were both inhibited by Nrg1 through pERK1/2 signaling in HT22 and NSC34 cells. CONCLUSIONS: CXCL10 is upregulated in SCI. Despite the negative effect on cell viability, CXCL10 failed to inhibit the scratch healing of HT22 and NSC34 cells. Nrg1 may protect neurons by partially antagonizing the effect of CXCL10.

Mechanism of ligusticum cycloprolactam against neuroinflammation based on network pharmacology and experimental verification.

Ligustilide, a natural phthalide mainly derived from chuanxiong rhizomes and Angelica Sinensis roots, possesses anti-inflammatory activity, particularly in the context of the nervous system. However, its application is limited because of its unstable chemical properties. To overcome this limitation, ligusticum cycloprolactam (LIGc) was synthesized through structural modification of ligustilide. In this study, we combined network pharmacological methods with experimental verification to investigate the anti-neuroinflammatory effects and mechanisms of ligustilide and LIGc. Based on our network pharmacology analysis, we identified four key targets of ligustilide involved in exerting an anti-inflammatory effect, with the nuclear factor (NF)-κB signal pathway suggested as the main signalling pathway. To verify these results, we examined the expression of inflammatory cytokines and inflammation-related proteins, analysed the phosphorylation level of NF-κB, inhibitor of κBα (IκBα) and inhibitor of κB kinase α and ß (IKKα+ß), and evaluated the effect of BV2 cell-conditioned medium on HT22 cells in vitro. Our results, demonstrate for the first time that LIGc can downregulate the activation of the NF-κB signal pathway in BV2 cells induced by lipopolysaccharide, suppress the production of inflammatory cytokines and reduce nerve injury in HT22 cells mediated by BV2 cells. These findings suggest that LIGc inhibits the neuroinflammatory response mediated by BV2 cells, providing strong scientific support for the development of anti-inflammatory drugs based on natural ligustilide or its derivatives. However, there are some limitations to our current study. In the future, further experiments using in vivo models may provide additional evidence to support our findings.

The Role of LincRNA-EPS/Sirt1/Autophagy Pathway in the Neuroprotection Process by Hydrogen against OGD/R-Induced Hippocampal HT22 Cells Injury.

Cerebral ischemia/reperfusion (CI/R) injury causes high disability and mortality. Hydrogen (H2) enhances tolerance to an announced ischemic event; however, the therapeutic targets for the effective treatment of CI/R injury remain uncertain. Long non-coding RNA lincRNA-erythroid prosurvival (EPS) (lincRNA-EPS) regulate various biological processes, but their involvement in the effects of H2 and their associated underlying mechanisms still needs clarification. Herein, we examine the function of the lincRNA-EPS/Sirt1/autophagy pathway in the neuroprotection of H2 against CI/R injury. HT22 cells and an oxygen-glucose deprivation/reoxygenation (OGD/R) model were used to mimic CI/R injury in vitro. H2, 3-MA (an autophagy inhibitor), and RAPA (an autophagy agonist) were then administered, respectively. Autophagy, neuro-proinflammation, and apoptosis were evaluated by Western blot, enzyme-linked immunosorbent assay, immunofluorescence staining, real-time PCR, and flow cytometry. The results demonstrated that H2 attenuated HT22 cell injury, which would be confirmed by the improved cell survival rate and decreased levels of lactate dehydrogenase. Furthermore, H2 remarkably improved cell injury after OGD/R insult via decreasing pro-inflammatory factors, as well as suppressing apoptosis. Intriguingly, the protection of H2 against neuronal OGD/R injury was abolished by rapamycin. Importantly, the ability of H2 to promote lincRNA-EPS and Sirt1 expression and inhibit autophagy were abrogated by the siRNA-lincRNA-EPS. Taken together, the findings proved that neuronal cell injury caused by OGD/R is efficiently prevented by H2 via modulating lincRNA-EPS/Sirt1/autophagy-dependent pathway. It was hinted that lincRNA-EPS might be a potential target for the H2 treatment of CI/R injury.

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.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry.

Aconitine, a common and main toxic component of Aconitum, is toxic to the central nervous system. However, the mechanism of aconitine neurotoxicity is not yet clear. In this work, we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine. HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system. Meanwhile, to confirm the metabolic mechanism of aconitine toxicity on HT22 cells, the levels of lactate dehydrogenase, intracellular Ca2+, reactive oxygen species, glutathione and superoxide dismutase, and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology. Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid, which was followed by the accumulation of lactic acid and reduction of glucose. The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca2+ overload and oxidative stress, and eventually result in cell apoptosis. In general, we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.