LDL Exposure Disrupts Mitochondrial Function and Dynamics in a Hippocampal Neuronal Cell Line.

Hypercholesterolemia has been associated with cognitive dysfunction and neurodegenerative diseases. Moreover, this metabolic condition disrupts the blood-brain barrier, allowing low-density lipoprotein (LDL) to enter the central nervous system. Thus, we investigated the effects of LDL exposure on mitochondrial function in a mouse hippocampal neuronal cell line (HT-22). HT-22 cells were exposed to human LDL (50 and 300 µg/mL) for 24 h. After this, intracellular lipid droplet (LD) content, cell viability, cell death, and mitochondrial parameters were assessed. We found that the higher LDL concentration increases LD content compared with control. Both concentrations increased the number of Annexin V-positive cells, indicating apoptosis. Moreover, in mitochondrial parameters, the LDL exposure on hippocampal neuronal cell line leads to a decrease in mitochondrial complexes I and II activities in both concentrations tested and a reduction in Mitotracker™ Red fluorescence and Mitotracker™ Red and Mitotracker™ Green ratio in the higher concentration, indicating mitochondrial impairment. The LDL incubation induces mitochondrial superoxide production and decreases superoxide dismutase activity in the lower concentration in HT-22 cells. Finally, LDL exposure increases the expression of genes associated with mitochondrial fusion (OPA1 and mitofusin 2) in the lower concentration. In conclusion, our findings suggest that LDL exposure induces mitochondrial dysfunction and modulates mitochondrial dynamics in the hippocampal neuronal cells.

[Mechanism of ligustilide in attenuating OGD/R-induced HT22 cell injury based on FtMt inhibition of ferroptosis].

This study investigated the role and mechanism of ligustilide(LIG) in attenuating oxygen-glucose deprivation/reoxyge-nation(OGD/R)-induced damage to mouse hippocampal neuron cells(HT22) by inhibiting ferroptosis through mitochondrial ferritin(FtMt). An in vitro model of OGD/R-induced HT22 cell damage was established. HT22 cells were randomly divided into normal group, model group, LIG groups(5, 10, and 20 µmol·L~(-1)), and ferrostatin-1(Fer-1, 2 µmol·L~(-1)) group. Cell viability was mea-sured using the CCK-8 method, and lactate dehydrogenase(LDH) release was measured using an LDH assay kit. Cell morphology was observed under an inverted microscope, and mitochondrial ultrastructure was observed using transmission electron microscopy. Intracellular Fe~(2+) content was detected using a chemiluminescence method. To further investigate the mechanism of FtMt inhibition of ferroptosis, FtMt in HT22 cells was silenced and divided into normal group, model group, LIG group(20 µmol·L~(-1)), si-NC group, si-FtMt group, and si-FtMt+20 µmol·L~(-1) LIG group. Immunofluorescence and Western blot were used to detect FtMt expression. Chemiluminescence was used to measure the content of NADPH/NADP~+, GSH, MDA, and ATP in HT22 cells. The mtROS fluorescence intensity was observed by laser confocal microscopy, and intracellular Fe~(2+) content was measured by flow cytometry. The expression of ferroptosis-related proteins Ferrtin, GPX4, and ACSL4 was detected by Western blot. The results showed that compared with the model group, LIG significantly increased the survival rate of HT22 cells, improved the morphology of damaged HT22 cells and mitochondrial ultrastructure, decreased intracellular Fe~(2+) content, and reduced the expression of the pro-ferroptosis protein ACSL4 while increasing the expression of anti-ferroptosis proteins Ferrtin and GPX4. After silencing FtMt, LIG promoted FtMt expression. Compared with the si-FtMt group, LIG significantly increased the content of NADPH/NADP~+ and GSH, reduced mtROS fluorescence intensity and MDA content, increased ATP activity, decreased intracellular Fe~(2+) content, inhibited the expression of pro-ferroptosis protein ACSL4, and increased the expression of anti-ferroptosis proteins Ferrtin and GPX4. In summary, LIG improved mitochondrial function by upregula-ting FtMt expression to inhibit ferroptosis, thereby alleviating OGD/R-induced damage to HT22 cells.

Exploring molecular mechanisms of diazinon toxicity in HT22 hippocampal neurons through integrated miRNA and mRNA profiling.

Diazinon (DZN), a persistent organophosphate insecticide, has been associated with neurotoxic effects, particularly in the hippocampus. However, the specific molecular mechanisms of DZN-induced hippocampal toxicity remain unknown. In this study, we analyzed the mRNA and miRNA expression patterns of HT22 cells following exposure to DZN (125 µM), and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted subsequently. The integration of miRNA sequencing (miRNA-seq) and mRNA sequencing (mRNA-seq) data identified 33 differentially expressed miRNAs (DEMIs, 15 up-regulated and 18 down-regulated) and 271 differentially expressed mRNAs (DEMs, 69 up-regulated and 202 down-regulated) targeted by the DEMIs. Moreover, the 3 most central mRNAs (ITGAV, FN1, and EGFR) and 7 associated miRNAs (mmu-miR-700-5p, mmu-miR-26a-2-3p, mmu-miR-452-3p, mmu-miR-25-3p, mmu-miR-582-5p, mmu-miR-467a-5p, and mmu-miR-467b-5p) were screened and validated using quantitative real-time PCR. Furthermore, the GO analysis revealed that the identified DEMs were enriched in biological adhesion extracellular matrix, and growth factor binding, while the KEGG analysis suggested that the enriched DEMs were involved in ECM-receptor interaction, mTOR signaling pathway, MAPK signaling pathway, and AMPK signaling pathway. Our results may aid in elucidating the underlying mechanisms associated with DZN-induced hippocampal toxicity and provide valuable insights into the pathogenesis of neurotoxicity triggered by other organophosphorus pesticides.

Sulfiredoxin-1 accelerates erastin-induced ferroptosis in HT-22 hippocampal neurons by driving heme Oxygenase-1 activation.

Ferroptosis, a recently identified non-apoptotic form of cell death, is strongly associated with neurological diseases and has emerged as a potential therapeutic target. Nevertheless, the fundamental mechanisms are still predominantly unidentified. In the current investigation, sulfiredoxin-1 (SRXN1) has been identified as a crucial regulator that enhances the susceptibility to ferroptosis in HT-22 mouse hippocampal cells treated with erastin. Utilizing TMT-based proteomics, a significant increase in SRXN1 expression was observed in erastin-exposed HT-22 cells. Efficient amelioration of erastin-induced ferroptosis was achieved via the knockdown of SRXN1, which resulted in the reduction of intracellular Fe2+ levels and reactive oxygen species (ROS) in HT-22 cells. Notably, the activation of Heme Oxygenase-1 (HO-1) was found to be crucial for inducing SRXN1 expression in HT-22 cells upon treatment with erastin. SRXN1 increased intracellular ROS and Fe2+ levels by activating HO-1 expression, which promoted erastin-induced ferroptosis in HT-22 cells. Inhibiting SRXN1 or HO-1 alleviated erastin-induced autophagy in HT-22 cells. Additionally, upregulation of SRXN1 or HO-1 increased the susceptibility of HT-22 cells to ferroptosis, a process that was counteracted by the autophagy inhibitor 3-Methyladenine (3-MA). These results indicate that SRXN1 is a key regulator of ferroptosis, activating the HO-1 protein through cellular redox regulation, ferrous iron accumulation, and autophagy in HT-22 cells. These findings elucidate a novel molecular mechanism of erastin-induced ferroptosis sensitivity and suggest that SRXN1-HO-1-autophagy-dependent ferroptosis serves as a promising treatment approach for neurodegenerative diseases.

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.

Hippocampal Lactate-Infusion Enhances Spatial Memory Correlated with Monocarboxylate Transporter 2 and Lactylation.

Lactate has emerged as a key player in regulating neural functions and cognitive processes. Beyond its function as an energy substrate and signal molecule, recent research has revealed lactate to serve as an epigenetic regulator in the brain. However, the molecular mechanisms by which lactate regulates spatial memory and its role in the prevention of cognitive disorders remain unclear. Herein, we injected L-lactate (10 µmol/kg/d for 6 d) into the mouse's hippocampus, followed by the Morris water maze (MWM) test and molecular analyses. Improved spatial memory performances were observed in mice injected with lactate. Besides, lactate upregulated the expression of synaptic proteins post-synaptic density 95 (PSD95), synaptophysin (SYP), and growth associated protein 43 (GAP43) in hippocampal tissues and HT22 cells, suggesting a potential role in synaptic transmission and memory formation. The facilitative role of monocarboxylate transporter 2 (MCT2), a neuron-specific lactate transporter, in this process was confirmed, as MCT2 antagonists attenuated the lactate-induced upregulation of synaptic proteins. Moreover, lactate induced protein lactylation, a post-translational modification, which could be suppressed by MCT2 inhibition. RNA sequencing of lactated-injected hippocampal tissues revealed a comprehensive gene expression profile influenced by lactate, with significant changes in genes associated with transcriptional progress. These data demonstrate that hippocampal lactate injection enhances spatial memory in mice, potentially through the upregulation of synaptic proteins and induction of protein lactylation, with MCT2 playing a crucial role in these processes. Our findings shed light on the multi-faceted role of lactate in neural function and memory regulation, opening new avenues for therapeutic interventions targeting cognitive disorders.

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.

The Neuroprotective Effect of Sophocarpine against Glutamate-Induced HT22 Cell Cytotoxicity.

Neuronal cell death and dysfunction of the central nervous system can be caused by oxidative stress, which is associated with the development of neurodegenerative diseases. Sophocarpine, an alkaloid compound derived from Sophora moorcroftiana (Benth.) Baker seeds, has a wide range of medicinal value. This study sought to determine how sophocarpine exerts neuroprotective effects by inhibited oxidative stress and apoptosis in mouse hippocampus neuronal (HT22) cells. 20mM glutamate-induced HT22 cells were used to develop an in vitro model of oxidative stress damage. The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. According to the instructions on the kits to detect reactive oxygen species (ROS) levels and oxidative stress indicators. HT22 cells were examined using immunofluorescence and Western Blotting to detect Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) expression. The expression of proteins and messenger RNA (mRNA) for heme oxygenase-1 (HO-1) was examined by Western Blotting and Quantitative real time polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential (MMP) and Cell apoptosis were used by 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC- 1) kit and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit, respectively. Finally, the expression of pro-apoptotic proteins was detected by Western Blotting. The result demonstrated that sophocarpine (1.25 µM-10 µM) can significantly inhibit glutamate-induced cytotoxicity and ROS generation, improve the activity of antioxidant enzymes. Sophocarpine increased the expression of HO-1 protein and mRNA and the nuclear translocation of Nrf2 to play a cytoprotective role; however, cells were transfected with small interfering RNA targeting HO-1 (si-HO-1) reversed the above effects of sophocarpine. In addition, sophocarpine significantly inhibited glutamate induced mitochondrial depolarization and further inhibited cell apoptosis by reducing the expression level of caspase-related proteins.

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.

AMPK Activation by Cimicifuga racemosa Extract Ze 450 Is Associated with Metabolic Effects and Cellular Resilience against Age-Related Pathologies in Different Tissue Cell Types.

Cimicifuga racemosa extracts (CREs) have gained well-established use for the treatment of menopausal symptoms such as hot flushes and excessive sweating, and weight gain. While the clinical effects of CREs have been well documented, the mechanisms underlying these effects are largely unknown. More recently, the metabolic effects of the CRE Ze 450 were demonstrated in cultured cells in vitro and in mouse models of obesity in vivo. At the molecular level, metabolic regulation, enhanced insulin sensitivity, and increased glucose uptake were linked to the activation of AMP-activated protein kinase (AMPK). Therefore, we tested the effects of Ze 450 on AMPK phosphorylation and thus activation in cells from different tissues, i.e., murine C2C12 myoblast cells, human HEPG2 liver cells, mouse HT22 neuronal cells, and in murine 3T3L1 adipocytes. Using a FRET-based HTRF-assay, we found that Ze 450 induced AMPK phosphorylation and the activation of this key enzyme of metabolic regulation in cells from various different tissues including C2C12 (muscle), HEPG2 (liver), HT22 (hippocampal), and 3T3-L1 (adipocyte) cells. In C2C12 muscle cells, enhanced AMPK activation was accompanied by reduced mitochondrial respiration and enhanced glucose uptake. Further, Ze 450 enhanced the resilience of the cells against oxidative death induced by ferroptosis inducers erastin or RSL3. Our findings suggest a general effect of Cimicifuga racemosa on AMPK activation in different tissues and across species. This may have a significant impact on expanded therapeutic applications of Ze 450, since AMPK activation and the related metabolic effects have been previously associated with anti-aging effects and the prevention of the metabolic syndrome.

The role of DRP1 mediated mitophagy in HT22 cells apoptosis induced by silica nanoparticles.

Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca2+) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca2+, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.

Fatsia japonica extract exerts antioxidant and anti-neuroinflammatory effects on neuronal cells and a zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Fatsia japonica is a traditional medicine used to treat various diseases, including inflammation-related disorders. However, its antineuroinflammatory and neuroprotective effects remain unclear. AIM OF THE STUDY: We aimed to evaluate the anti-neuroinflammatory and neuroprotective effects of F. japonica extract to identify the underlying mechanisms. MATERIALS AND METHODS: The components of F. japonica extract were profiled using ultra-high-performance liquid chromatography-mass spectrometry. The effects of F. japonica extract were investigated in BV2 microglia and HT22 hippocampal cells. Furthermore, in vivo effects of F. japonica extract were assessed using zebrafish models treated with H2O2 and LPS to evaluate the effects of in vivo. RESULTS: We identified 27 compounds in the F. japonica extract. F. japonica extract demonstrated anti-inflammatory properties by suppressing LPS-induced inflammatory responses in both BV2 cells and zebrafish, along with inhibiting the activation of the nuclear factor (NF)-κB (p65) pathway. The protective effects of this extract were also observed on glutamate-treated HT22 cells and in H2O2-induced zebrafish. Furthermore, F. japonica extract upregulated nuclear factor E2-related (Nrf) 2/heme oxygenase (HO)-1 expression in BV2 and HT22 cells. CONCLUSIONS: F. japonica extract exerted anti-neuroinflammatory and neuroprotective effects through Nrf2/HO-1 and the NF-κB pathway.

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.

Deferoxamine Mitigates Ferroptosis and Inflammation in Hippocampal Neurons After Subarachnoid Hemorrhage by Activating the Nrf2/TXNRD1 Axis.

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood-brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis.

Inhibitory effect of gingerone on apoptosis of HT22 cells by alleviation oxidative stress damage after OGD/R through activating Nrf2/HO-1 signaling pathway / 吉林大学学报(医学版)

Objective:To discuss the protective effect of gingerone on the hippocampal neuron HT22 cells after oxygen-glucose deprivation/reoxygenation(OGD/R),and to clarify the related mechanism.Methods:The HT22 cells were cultured,and the OGD/R cell injury model was established by setting the gradient of OGD/R time.The HT22 cells were divided into control group,OGD/R group,OGD/R+ 1 μmol·L-1 gingerone group,OGD/R + 10 μmol·L-1 gingerone group,OGD/R+100 μmol·L-1 gingerone group,and OGD/R+0.2%dimethyl sulfoxide(DMSO)group.The viability of the cells in various groups was detected by CCK-8 assay;the survival rates of the cells in various groups were calculated to determine the optimal drug concentration of gingerone.The cells were divided into control,OGD/R group,OGD/R+ gingerone,and OGD/R+gingerone+nuclear factor erythroid-2-related factor 2(Nrf2)inhibitor(ML385)groups.The cells in OGD/R + gingerone group were treated with gingerone for 4 h before OGD treatment for 8 h followed by reoxygenation for 8 h,and the cells in OGD/R+gingerone+ ML385 group were treated with 10 μmol·L-1 ML385 for 6 h before gingerone treatment.The viability of the cells in various groups was detected by CCK-8 assay;the expression levels of Nrf2,heme oxygenase-1(HO-1),B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)proteins in the cells in various groups were detected by Western blotting method;the activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)in the cell culture supernatant in various groups were detected by enzyme-linked immunosorbent assay(ELISA)method.Results:Compared with control group,the survival rate of the HT22 cells was below 50%after treated with OGD for 8 h and reoxygenation for 8 h,so the HT22 cell OGD/R model was established by treated with OGD for 8 h and reoxygenation for 8 h.Compared with OGD/R group,the survival rates of the cells in OGD/R+different doses of gingerone groups were increased to various extents,and the survival rate of the cells in OGD/R+ 100 μmol·L-1 gingerone group was significantly increased(P<0.01);so 100 μmol·L-1 gingerone was used for the subsequent experiment.Compared with control group,the viability of the cells in OGD/R group was significantly decreased(P<0.01),and the expression levels of Nrf2,HO-1,and Bax proteins in the cells were significantly increased(P<0.01),while the expression level of Bcl-2 protein in the cells was significantly decreased(P<0.05),and the SOD activity in the cell culture supernatant was significantly decreased(P<0.01),and the level of MDA was significantly increased(P<0.01);compared with OGD/R group,the viability of the cells in OGD/R + gingerone group was significantly increased(P<0.01),and the expression levels of Nrf2,HO-1,and Bcl-2 proteins in the cells were significantly increased(P<0.05 or P<0.01),while the expression level of Bax protein in the cells was decreased(P<0.05),the SOD activity in the cell culture supernatant was significantly increased(P<0.01),and the level of MDA was significantly decreased(P<0.01);compared with OGD/R + gingerone group,the viability of the cells in OGD/R + gingerone + ML385 group was significantly decreased(P<0.01),and the expression levels of Nrf2,HO-1,and Bcl-2 proteins were significantly decreased(P<0.01),while the expression level of Bax protein in the cells was significantly increased(P<0.01),the SOD activity in the cell culture supernatant was significantly decreased(P<0.01),and the level of MDA was significantly increased(P<0.05).Conclusion:Gingerone alleviates the oxidative stress damage,and thereby plays an inhibiory effect on the apoptosis of the HT22 neurons by activating the Nrf2/HO-1 signaling pathway after OGD/R.

Construction and effect of a high glucose induced hippocampal neuron metabolic memory cell model in HT-22 mice / 天津医药

Objective To construct an in vitro"metabolic memory"cell model of HT-22 mouse hippocampal neurons induced by high glucose,and to investigate the effect of"metabolic memory"on apoptosis and histone acetylation in HT-22 cells.Methods HT-22 cells were cultured in high glucose medium(glucose concentration was 55 mmol/L)and conventional glucose medium(glucose concentration was 25 mmol/L),and cells were divided into the control group(NG 4,6 and 8 groups,25 mmol/L glucose was cultured for 4,6 and 8 days,respectively),the high glucose group(HG 4,6 and 8 groups,respectively)and the metabolic memory group(HG2NG2,HG2NG4,HG2NG6,HG4NG2 and HG4NG4 groups,high glucose culture for 2 days to 25 mmol/L glucose culture for 2,4 or 6 days,high glucose culture for 4 days to 25 mmol/L glucose culture for 2 or 4 days).Cell viability was detected by CCK-8 method.The release of lactate dehydrogenase(LDH)in cell culture supernatant was detected,and the optimal time to establish a"metabolic memory"model was selected.Subsequently,cells were divided into the NG4 group,the NG8 group,the HG4 group,the HG4NG4 group and the HG8 group,and the cell morphology of each group was observed by optical microscope.The apoptosis rate was detected by flow cytometry.The activities of deacetylase(HDAC)and histone acetyltransferase(HAT)were detected by enzyme-linked immunosorbent assay(ELISA).Western blot assay was used to detect expression levels of histone deacetylase 4(HDAC4),B lymphocyte tumor 2(Bcl-2),Bcl-2 related X protein(Bax)and Caspase-3 protein.Results The HG4NG4 group was the ideal cell model with high glucose metabolic memory.Cells of the NG4 group and the NG8 group were interwoven into a dense network,growing well,with spindle shaped cells and distinct synaptic structures.However,in the HG4 group and the HG8 group,the cell body became round,synaptic structure disappeared and growth was inhibited.In the HG4NG4 group,the number of cells increased but their morphology was damaged.Results of flow cytometry showed that compared with the NG8 group,the apoptosis rates were significantly increased in the HG8 group and the HG4NG4 group(P＜0.05).ELISA results showed that compared with the NG8 group,the expression levels of HDAC4,Bax,and Caspase-3 proteins increased in the HG8 group and the HG4NG4 group,while the expression level of Bcl-2 protein significantly decreased(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels of HAT and HDAC in the HG4NG4 group.Western blot reslts showed that compared with the NG8 group,the levels of HDAC4,Bax and Caspase-3 protein increased in the HG8 group and the HG4NG4 group(P＜0.05).Compared with the HG8 group,there were no significant differences in protein expression levels in the HG4NG4 group.Conclusion HT-22 mouse hippocampal neurons cultured with 55mmol/L high glucose for 4 days,and then cultured with 25 mmol/L glucose for 4 days are the ideal"metabolic memory"cell model.The mechanism may be related to the increased activity of HDAC,HAT and HDAC4 expression in the hyperglycemic model.

Effect of Xiaoxuming decoction on OGD/R-induced synaptic plasticity in HT22 cells / 中国病理生理杂志

AIM:To explore the protective effect of Xiaoxuming decoction(XXMD)on synaptic plasticity in the context of cerebral ischemia-reperfusion injury following ischemic stroke.METHODS:An oxygen-glucose depriva-tion/reoxygenation(OGD/R)model was employed in vitro using mouse hippocampal neurons(HT22 cells)to simulate ischemia-reperfusion injury.Cell viability was assessed using a CCK-8 assay to determine the optimal XXMD concentra-tion.The HT22 cells were divided into two groups:control and model(OGD/R).Cellular morphological changes were ob-served using an inverted microscope.The levels of IL-1β,IL-6 and TNF-α in the supernatant were quantified by ELISA.Ultrastructural changes were examined by transmission electron microscopy.Immunofluorescence staining was used to de-tect neuron markers NeuN and synaptic proteins NF200 and MAP2.The protein levels of NF200 and MAP2 were analyzed by Western blot.RESULTS:The highest cell survival rate occurred at an XXMD concentration of 100 mg/L(P<0.05).Compared with control group,the cells in model group exhibited round shape and shrinkage,mitochondrial swelling or vacuolization,and a marked decrease in survival rate.There were significant increases in IL-1β,IL-6 and TNF-α levels(P<0.05).Immunofluorescence intensity and protein levels of NeuN,NF200 and MAP2 were notably reduced(P<0.05).Treatment with XXMD improved cell morphology,ultrastructure and survival rate(P<0.05),and decreased in-flammatory factor levels(P<0.05).Compared with model group,the cells in OGD/R+XXMD group showed significantly increased immunofluorescence intensity and protein levels of NeuN,NF200 and MAP2(P<0.05).CONCLUSION:Xiaoxuming decoction may mitigate OGD/R-induced injury,potentially by inhibiting inflammatory responses and enhanc-ing synaptic plasticity.