Cell Line HT22 Research Articles

Oxysophocarpine Prevents the Glutamate-Induced Apoptosis of HT-22 Cells via the Nrf2/HO-1 Signaling Pathway.

Oxysophocarpine (OSC), a quinolizidine alkaloid, shows neuroprotective potential, though its mechanisms are unclear. The aim of the present study was to investigate the neuroprotective effects of OSC through the nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) signaling pathway using the HT-22 cell line. Assessments of cell viability were conducted utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Assessments of oxidative stress (OS) were conducted through the quantification of reactive oxygen species (ROS). The integrity of the mitochondrial membrane potential (MMP) was scrutinized using fluorescent probe technology. Apoptosis levels were quantified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The trafficking of Nrf2 within the cell nucleus was examined through immunofluorescence analysis. Furthermore, Western blotting (WB) was applied to evaluate the expression levels of proteins implicated in apoptosis and the Nrf2/HO-1 pathway. To further probe the influence of OSC on the overexpression of antioxidant enzymes, cells were subjected to transfection with HO-1 siRNA. The results showed that OSC inhibited glutamate-induced OS, as evidenced by reduced cell viability and ROS levels. Furthermore, the apoptotic condition induced by glutamate in HT-22 cells was significantly reduced following OSC treatment. More interestingly, the Nrf2/HO-1 signaling pathway was upregulated following OSC treatment. These results suggest that OSC can exert neuroprotective effects by regulating the Nrf2/HO-1 pathway to inhibit neuronal cell apoptosis, potentially aiding in the treatment of neurodegenerative diseases.

Potential PDE4A inhibition-mediated neuroprotective effects of psoralidin.

This study aimed to investigate the effect of psoralidin, a natural phenolic coumarin compound, on MK-801-induced neurotoxicity that may cause Alzheimer's disease and to determine the phosphodiesterase(PDE)-related molecular mechanism of action. In this study, neurotoxicity was performed using the MK-801 in the HT-22 cell line. The effects of compounds on the proliferation of HT-22 cells were determined by Real-Time Cell Analysis (RTCA). After measuring the total protein concentration, the PDE4A protein level was determined using the Western blot method. Psoralidin (100, 200, 400 µM) has been shown to have a neuroprotective effect against MK-801-induced neurotoxicity, as indicated by Real-Time Cell Analysis. In HT-22 cells, the half maximal effective concentration (EC50) value of psoralidin was calculated to be 230.4 µM, IC50 value of MK-801 was calculated to be 62.4 µM at 24 hours. It has been determined that psoralidin (200, 400 µM) inhibits PDE4A by using the Western blot method. This research uncovers that psoralidin has neuroprotective effects in MK801-associated accumulation of the excitatory amino acid glutamate neurodegeneration and Alzheimer's disease.

Mechanisms of Differential Sensitivity to Ethanol-Induced Apoptosis in Mouse Spinal Cord at Different Developmental Stages-Akt/GSK Signaling and BAX.

The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages and the molecular mechanisms involved. A mouse ethanol intervention model was established on postnatal days 4, 7, and 12. Primary cells were derived from the spinal cord at postnatal day 4. Western blotting, immunofluorescence, and flow cytometry were used to detect apoptosis-related proteins in the spinal cord and primary cells. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes originating from the Gene Expression Omnibus dataset GSE184615 was conducted. Effects on Akt/GSK3β pathway proteins were investigated using the GSK3β inhibitor AR-A014418, and the Akt inhibitor DHA. Lentiviral knockdown and overexpression of intervening GSK3β were used in HT22 cell lines to investigate the effects of alcohol on GSK 3β and caspase proteins. J-aggregates, reactive oxygen species assays, and calcein-AM assays were used to investigate mitochondrial function and cell viability. Ethanol caused downregulation of Akt activity and upregulation of GSK3β activity and apoptosis. DHA, AR-A014418, and knockdown of GSK3β effectively counteracted ethanol-induced apoptosis, whereas overexpression of GSK3β enhanced the injury process. PI3K activity was unchanged during these processes. Fluorescence colocalization analysis indicated that BAX was translocated to mitochondria during the apoptotic process. BAX was downregulated as the spinal cord developed, consistent with a reduced susceptibility to ethanol-induced apoptosis. Akt/GSK3β signaling and BAX together determine the direction of alcohol-induced apoptosis and its susceptibility to change during developmental stages in the spinal cord.

Daidzein ameliorates experimental traumatic brain injury-induced neurological symptoms by suppressing oxidative stress and apoptosis.

Traumatic brain injury (TBI) causes deficits in neurological function, induces pathological changes, and increases oxidative stress. The current investigation aimed to determine Daidzein's neuroprotective potential in experimental TBI. Initially, the HT-22 cell line exposed to H2O2 underwent in vitro examination, and the results showed that Daidzein had a neuroprotective effect evident from enhanced cell viability and decreased NO generation. Using three different Daidzein doses-1 mg/kg, 5 mg/kg, and 10 mg/kg-in the in vivo experiment, the potential of Daidzein was evaluated against TBI. The neurological severity score (NSS), kondziela's screen test, and elevated plus maze showed improvements after treatment with Daidzein manifested by decreased score, enhanced motor coordination, and anti-anxiety effects. Additionally, Daidzein improved mechanical allodynia and restored the breakdown of the blood-brain barrier. The FTIR spectral analysis showed restoration of the biochemical compositional changes. Furthermore, H & E and Toluidine blue staining revealed an improvement in the histopathological alterations. The RT-qPCR revealed an increase in mRNA expression level of Nrf2, HO-1, and Bcl-2 and the downregulation of Keap-1, Bax and Cleaved caspase-3 expressions. Thus, exhibiting its antioxidant and antiapoptotic potential. The RT-qPCR also manifested a decrease in mRNA expression of GFAP and Iba-1. Further immunohistochemistry results indicated Daidzein's antioxidant and antiapoptotic properties by upregulating Nrf2 and downregulating cleaved caspase-3. Daidzein also lowered the apoptosis index and improved neuronal survival evidenced by flow cytometric analysis. In addition to this, Daidzein notably increased the antioxidant enzyme levels and decreased the oxidative stress markers. The current study's findings point to the neuroprotective potential of the phytoestrogen Daidzein as it lessened neurological abnormalities, decreased oxidative stress, and lowered proapoptotic protein expression.

Bis-chalcones obtained via one-pot synthesis as the anti-neurodegenerative agents and their effect on the HT-22 cell line

In the area of research on neurodegenerative diseases, the current challenge is to search for appropriate research methods that would detect these diseases at the earliest possible stage, but also new active structures that would reduce the rate of the disease progression and minimize the intensity of their symptoms experienced by the patient. The chalcones are considered in the context of candidates for new drugs dedicated to the fight against neurodegenerative diseases. The synthesis of bis-chalcone derivatives (3a-3d), as aim molecules was performed. Their structures were established by applying 1H NMR, 13C NMR, MS, FT-IR and UV–Vis spectra. All bis-chalcones were synthesized from terephthalaldehyde and appropriate aromatic ketone as substrates in the Claisen-Schmidt condensation method and evaluated in the biological tests and in silico analysis. Compounds exerted antioxidant activity using the HORAC method (3a-3d) and decreased the activities of GPx, COX-2 (3b-3d), GR (3a-3c) and CAT (3a,3b). The high anti-neurodegenerative potential of all four bis-chalcones was observed by inhibition of acetyl- (AChE) and butyrylcholinesterase (BChE) and a positive effect on the mouse hippocampal neuronal HT-22 cell line (LDH release and PGC-1α, PPARγ and GAPDH protein expression). TD-DFT method (computing a number of descriptors associated with HOMO–LUMO electron transition: electronegativity, chemical hardness and potential, first ionization potential, electron affinity) was employed to study the spectroscopic properties. This method showed that the first excited state of compounds was consistent with their maximum absorption in the computed UV–Vis spectra, which showed good agreement with the experimental spectrum using PBE1PBE functional. Using in silico approach, interactions of bis-chalcones with selected targets (aryl hydrocarbon receptor (AhR) PAS-A Domain, ligand binding domain of human PPAR-γ, soman-aged human BChE-butyrylthiocholine complex, Torpedo californica AChE:N-piperidinopropyl-galanthamine complex and the COX-2-celecoxib complex) were characterized. Results obtained in in silico models were consistent with in vitro experiments.

Bromodomain-containing protein 4 knockdown promotes neuronal ferroptosis in a mouse model of subarachnoid hemorrhage.

Neuronal cell death is a common outcome of multiple pathophysiological processes and a key factor in neurological dysfunction after subarachnoid hemorrhage. Neuronal ferroptosis in particular plays an important role in early brain injury. Bromodomain-containing protein 4, a member of the bromo and extraterminal domain family of proteins, participated in multiple cell death pathways, but the mechanisms by which it regulates ferroptosis remain unclear. The primary aim of this study was to investigate how bromodomain-containing protein 4 affects neuronal ferroptosis following subarachnoid hemorrhage in vivo and in vitro. Our findings revealed that endogenous bromodomain-containing protein 4 co-localized with neurons, and its expression was decreased 48 hours after subarachnoid hemorrhage of the cerebral cortex in vivo. In addition, ferroptosis-related pathways were activated in vivo and in vitro after subarachnoid hemorrhage. Targeted inhibition of bromodomain-containing protein 4 in neurons increased lipid peroxidation and intracellular ferrous iron accumulation via ferritinophagy and ultimately led to neuronal ferroptosis. Using cleavage under targets and tagmentation analysis, we found that bromodomain-containing protein 4 enrichment in the Raf-1 promoter region decreased following oxyhemoglobin stimulation in vitro. Furthermore, treating bromodomain-containing protein 4-knockdown HT-22 cell lines with GW5074, a Raf-1 inhibitor, exacerbated neuronal ferroptosis by suppressing the Raf-1/ERK1/2 signaling pathway. Moreover, targeted inhibition of neuronal bromodomain-containing protein 4 exacerbated early and long-term neurological function deficits after subarachnoid hemorrhage. Our findings suggest that bromodomain-containing protein 4 may have neuroprotective effects after subarachnoid hemorrhage, and that inhibiting ferroptosis could help treat subarachnoid hemorrhage.

Neuroprotective Activity of a Non-Covalent Imatinib+TP10 Conjugate in HT-22 Neuronal Cells In Vitro.

This study evaluated the probable relevance of a non-covalent conjugate of imatinib with TP10 in the context of a neuroprotective effect in Parkinson's disease. Through the inhibition of c-Abl, which is a non-receptor tyrosine kinase and an indicator of oxidative stress, imatinib has shown promise in preclinical animal models of this disease. The poor distribution of imatinib within the brain tissue triggered experiments in which a conjugate was obtained by mixing the drug with TP10, which is known for exhibiting high translocation activity across the cell membrane. The conjugate was tested on the HT-22 cell line with respect to its impact on MPP+-induced oxidative stress, apoptosis, necrosis, cytotoxicity, and mortality. Additionally, it was checked whether the conjugate activated the ABCB1 protein. The experiments indicated that imatinib+PEG4+TP10 reduced the post-MPP+ oxidative stress, apoptosis, and mortality, and these effects were more prominent than those obtained after the exposition of the HT-22 cells to imatinib alone. Its cytotoxicity was similar to that of imatinib itself. In contrast to imatinib, the conjugate did not activate the ABCB1 protein. These favorable qualities of imatinib+PEG4+TP10 make it a potential candidate for further in vivo research, which would confirm its neuroprotective action in PD-affected brains.

Cytosolic Escape of Mitochondrial DNA Triggers cGAS-STING Pathway-Dependent Neuronal PANoptosis in Response to Intermittent Hypoxia.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), characterized by neuronal cell death and neurocognitive impairment. We focus on the accumulated mitochondrial DNA (mtDNA) in the cytosol, which acts as a damage-associated molecular pattern (DAMP) and activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a known trigger for immune responses and neuronal death in degenerative diseases. However, the specific role and mechanism of the mtDNA-cGAS-STING axis in IH-induced neural damage remain largely unexplored. Here, we investigated the involvement of PANoptosis, a novel type of programmed cell death linked to cytosolic mtDNA accumulation and the cGAS-STING pathway activation, in neuronal cell death induced by IH. Our study found that PANoptosis occurred in primary cultures of hippocampal neurons and HT22 cell lines exposed to IH. In addition, we discovered that during IH, mtDNA released into the cytoplasm via the mitochondrial permeability transition pore (mPTP) activates the cGAS-STING pathway, exacerbating PANoptosis-associated neuronal death. Pharmacologically inhibiting mPTP opening or depleting mtDNA significantly reduced cGAS-STING pathway activation and PANoptosis in HT22 cells under IH. Moreover, our findings indicated that the cGAS-STING pathway primarily promotes PANoptosis by modulating endoplasmic reticulum (ER) stress. Inhibiting or silencing the cGAS-STING pathway substantially reduced ER stress-mediated neuronal death and PANoptosis, while lentivirus-mediated STING overexpression exacerbated these effects. In summary, our study elucidates that cytosolic escape of mtDNA triggers cGAS-STING pathway-dependent neuronal PANoptosis in response to IH, mainly through regulating ER stress. The discovery of the novel mechanism provides theoretical support for the prevention and treatment of neuronal damage and cognitive impairment in patients with OSA.

Synthesis, structural characterization and cytoprotective activity of a new mixed oxidovanadium(IV) compound with nitrilotriacetate and imidazole ligands

Herein, we have shown that the water molecule in [VO(nta)(H2O)]- can be replaced by an imidazole moiety which is a versatile binding site in various biological systems. These findings offer a new approach for synthesizing mixed ligand complexes with desired physicochemical and biological properties. In this study, a new hetero-ligand oxidovanadium(IV) monohydrate salt was successfully synthesized and structurally characterized. The compound consists of a mononuclear [VO(nta)(im)]- entity, where nitrilotriacetato–(nta) and imidazole (im) ligands are incorporated, along with an imidazolinium counterion, [im(H)][VO(nta)(im)]H2O. To characterize the complex in a solid state, various techniques were employed including elemental analysis, thermogravimetric analysis (TG-FTIR), single-crystal X-ray diffraction method and IR spectroscopy. In addition, the investigation included a study of the speciation of the complex ion [VO(nta)(im)]- in an aqueous solution. Furthermore, the influence of imidazole as an auxiliary ligand (im) in the coordination sphere of V(IV) is investigated in terms of its effects on ABTS+• radical scavenging activity and cytoprotective properties against oxidative damage in the hippocampal neuronal HT22 cell line. Finally, this study compares and discusses the physicochemical properties and biological activities of the newly synthesised compound and those containing [VO(nta)(H2O)]- complex anions.

Targeting cathepsin S promotes activation of OLF1-BDNF/TrkB axis to enhance cognitive function

BackgroundCathepsin S (CTSS) is a cysteine protease that played diverse roles in immunity, tumor metastasis, aging and other pathological alterations. At the cellular level, increased CTSS levels have been associated with the secretion of pro-inflammatory cytokines and disrupted the homeostasis of Ca2+ flux. Once CTSS was suppressed, elevated levels of anti-inflammatory cytokines and changes of Ca2+ influx were observed. These findings have inspired us to explore the potential role of CTSS on cognitive functions.MethodsWe conducted classic Y-maze and Barnes Maze tests to assess the spatial and working memory of Ctss−/− mice, Ctss+/+ mice and Ctss+/+ mice injected with the CTSS inhibitor (RJW-58). Ex vivo analyses including long-term potentiation (LTP), Golgi staining, immunofluorescence staining of sectioned whole brain tissues obtained from experimental animals were conducted. Furthermore, molecular studies were carried out using cultured HT-22 cell line and primary cortical neurons that treated with RJW-58 to comprehensively assess the gene and protein expressions.ResultsOur findings reported that targeting cathepsin S (CTSS) yields improvements in cognitive function, enhancing both working and spatial memory in behavior models. Ex vivo studies showed elevated levels of long-term potentiation levels and increased synaptic complexity. Microarray analysis demonstrated that brain-derived neurotrophic factor (BDNF) was upregulated when CTSS was knocked down by using siRNA. Moreover, the pharmacological blockade of the CTSS enzymatic activity promoted BDNF expression in a dose- and time-dependent manner. Notably, the inhibition of CTSS was associated with increased neurogenesis in the murine dentate gyrus. These results suggested a promising role of CTSS modulation in cognitive enhancement and neurogenesis.ConclusionOur findings suggest a critical role of CTSS in the regulation of cognitive function by modulating the Ca2+ influx, leading to enhanced activation of the BDNF/TrkB axis. Our study may provide a novel strategy for improving cognitive function by targeting CTSS.

Human YTH Domain Family 2 (YTHDF2)-Dependent N6-Methyladenosine Mediates Cerebral Ischemia/Reperfusion Injury via Oxidative Stress

Our study aimed to explore whether YT521-B homology domain family protein 2 (YTHDF2)-dependent m6A is involved in oxidative stress induced by I/R in vitro. We established a cell model of I/R by oxygen-glucose deprivation/re-oxygenation (OGD/R) in HT22 cell line. The shRNAs were used to silence YTHDF2 and Nrf2. The expression of YTHDF2 and Nrf2, levels of m6A, and the indicators related to oxidative stress (GSH, SOD and MDA) was detected in different cell groups. CCK8, flow cytometry, and ki67 fluorescence staining was used to evaluate the cell viability and apoptosis. The levels of YTHDF2, m6A and MDA were increased in cells, while the levels of GSH and SOD were reduced by OGD/R. Also, the apoptosis in cells was increased after OGD/R, and with decreased cell viability. The knockdown of YTHDF2 could reduce the level of m6A, increase the expression of Nrf2. Moreover, the levels of GSH and SOD were increased after exposure to YTHDF2-shRNA, while the level of MDA was decreased, and the cell viability was increased. Our study showed that YTHDF2-Dependent N6A mediates cerebral I/R injury via oxidative stress in vitro, which may constitute a new target for stroke.

Crocus Officinalis (L.) Extract on Human Colerectal Cancer Cell Line (HT-22): Investigation in Vitro

Crocus sativus L. has been used as spice, food colouring and medicinal plant for thousands of years. In this study, antioxidant properties and cytotoxic effect of saffron extract were evaluated in HT-22 cell lines. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of saffron extract in different solvents (water, ethanol, ethyl-acetate, n-hexane). Cell viability was determined by MTT assay. Antioxidant parameters such as DPPH, FRAP, total polyphenol and flavonoid amounts were measured. The highest DPPH value was found as 15.90±0.12 µg/mL in ethyl acetate solvent. Total polyphenols were 89.06±0.02 mg GAE/g extract, flavonoids were 76.98±2.08 µg QE/g extract and FRAP was 67.93±0.98 mg/g extract in ethanol solvent. In colon cancer, IC50 value 94±1.37 µg/mL was found to be the highest value. This is thought to be due to the high antioxidant capacity in ethanol solvent. While antioxidant capacity was not found to be high, antiproliferation effect was found to be high. Saffron may also be considered as a promising chemotherapeutic agent in cancer treatment in the future.

4-Hydroxysesamin, a Modified Natural Compound, Attenuates Neuronal Apoptosis After Ischemic Stroke via Inhibiting MAPK Pathway.

The 4-hydroxysesamin (4-HS, a di-tetrahydrofuran lignin) is a modified sesamin that was prepared in the laboratory. This preclinical study was designed to preliminarily investigate the neuroprotective properties of 4-HS. In vitro, neuronal injury and inflammation were simulated by oxygen-glucose deprivation and lipopolysaccharide (LPS) exposure in mouse hippocampal neuronal HT22 cell line, and treated with 4-HS and/or metformin (MET, MAPK pathway activator for exploring mechanism). CCK-8, flow cytometry, and enzyme-linked immunosorbent assay were performed to evaluate cell viability, apoptosis, and inflammation. Apoptosis- and pathway-related proteins were detected by Western blotting. Middle cerebral artery occlusion (MCAO) was constructed as a stroke model and treated with 4-HS for in vivo confirmation. Histological staining was used for in vivo evaluation of 4-HS properties. The 4-HS showed similar anti-inflammatory activity to sesamin but did not affect the cell viability of HT22 cells. In vitro, 4-HS improved the cell viability, ameliorated neuronal apoptosis, along with the reversion of apoptotic proteins (Bax, cleaved-caspase 3/9, Bcl-2) expression and inflammatory cytokines (IL-6, TNF-α, IL-10) in LPS-treated HT22 cells. The 4-HS suppressed the phosphorylation of ERK, JNK, and p38 but the addition of MET reversed 4-HS-induced changes of phenotype and protein expression in LPS-treated cells. In vivo, 4-HS showed apparent improvement in cerebral infarction, brain tissue morphology, neuronal architecture, apoptosis, and inflammation of MCAO mice, and also showed inhibiting effects on the phosphorylation of ERK, JNK, and p38, confirming in vivo results. In this first pre-clinical study on 4-HS, we preliminarily demonstrated the neuroprotective properties of 4-HS both in cell and animal models, and proposed that the underlying mechanism might be associated with the MAPK pathway.

Glutamate Provides Cytoprotective Effect for Astrocytes Against Ischemic Insult and Promotes Astrogliosis.

Glutamate-mediated excitotoxicity has been extensively explored as a therapeutic target for the development of potential treatments of neurological disorders including stroke. However, the effect of glutamate on astrocytes under pathological conditions has been less studied. Using primary astrocyte culture, we determined the effect of glutamate on astrocytes against ischemic insult. Glutamate provided a cytoprotective effect and acted as an alternative substrate for ATP production in primary astrocytes against oxygen glucose deprivation reoxygenation insult, which was blocked by glutamate uptake inhibition. The cytoprotective effect of glutamate appears to be astrocyte-specific, as glutamate dose-dependently induces cytotoxic action in murine hippocampal HT-22 cell line. Interestingly, the cytoprotective effect of glutamate against glucose deprivation was short-last, as no protection was observed after 3-day glucose deprivation. We determined the metabolic phenotype of primary astrocyte cultured in glucose or glutamate. Primary astrocytes cultured in glutamate displayed a different metabolic phenotype when compared to those cultured in glucose, evidenced by higher basal and maximal oxygen consumption rate (OCR), higher ATP production and proton leak-coupled OCR, as well as lower glycolysis. Furthermore, glutamate exposure resulted in astrocyte activation, evidenced by an increase in astrocyte size and GFAP expression. Our study demonstrated that glutamate exerts a dual effect on astrocytes under ischemic condition. Glutamate provides an alternative substrate for energy metabolism in the absence of glucose, thereby protecting astrocytes against ischemic insults. On the other hand, glutamate exposure induces astrogliosis. Modulation of glutamate uptake and metabolism in astrocytes may provide novel targets for alleviating ischemic injury and improving function recovery after ischemic stroke.

Investigation of the Protective Role of Quercetin on Oxidative Stress and Endoplasmic Stress Pathway in 4-aminopyridine-induced Neuronal Damage

Quercetin (QU) is a flavonoid found in different fruits and vegetables. Studies report that QU may have positive effects on neurological diseases. However, the effect of QU on 4-aminopyridine (4-AP)-induced neurodegeneration in neuronal cells is still not fully elucidated. In this study, the effects of QU on 4-AP-induced hippocampal neuron damage in vitro and the possible role of oxidative stress and endoplasmic reticulum stress in this effect were investigated. The study was carried out using the HT-22 hippocampal neuronal cell line. The effect of pre-treatment with QU on cell viability after 4-AP-induced neuronal damage was determined by the XTT test. Cells were evaluated histopathologically for apoptotic nuclear change (ANC) using DAPI staining. The effects of QU on oxidative stress (total oxidant state (TOS) and total antioxidant status (TAS)) occurring after neuronal damage were evaluated with colorimetric commercial kits and endoplasmic reticulum stress markers (activating transcription factor 4 (ATF-4) and C/EBP homologous protein). (CHOP) was measured with the ELISA kits. While the cell viability rate decreased in the cells treated with 4-AP, it was determined that pre-treatment with QU reversed this situation. In terms of histopathology, treatment with 4-AP increased the number of ANC, while QU pre-treatment reduced it. In addition, in terms of biochemical evaluations, TOS, ATF-4, and CHOP increased in neuronal cells after 4-AP, and QU was determined to suppress this increase. In addition, QU normalized the decreased TAS levels following the 4-AP application. As a result, in the HT-22 cell line, it was found that QU treatment had a neuroprotective effect by suppressing oxidative stress and endoplasmic reticulum stress in 4-AP-induced neuronal damage.

Investigation of the Effect of Pumpkin (Cucurbita pepo L.) Seed Oil on Pentylenetetrazole-induced Neuronal Damage in HT-22 Cell Line

Recent studies have shown the positive effects of Cucurbita pepo L. (pumpkin) seed oil (PSO) in different disease models. However, the effect of PSO on neurological diseases has not been clarified yet. Therefore, this study aims to elucidate the effects of BBS on pentylenetetrazole (PTZ)-induced neuronal damage and the possible roles of oxidative and nitrosative stress in this effect in vitro. The HT-22 hippocampal neuronal cell line was used in the study. Cell survival after PTZ-induced neuronal damage was evaluated with the XTT test in the groups. While the effects of BBS on total antioxidant status (TAS) and total oxidant status (TOS) after PTZ were measured with colorimetric commercial kits, its effects on neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) levels were also determined by ELISA kits. In light of the data obtained, it was found that pre-treatment with PSO prevented the decrease in cell survival after exposure to PTZ. In addition, it has been found that PSO normalizes the increase in TOS, nNOS, and NO in neuronal cells after PTZ. As a result, it was determined that the treatment of neuronal cells with PSO prevented neuronal damage caused by PTZ and showed neuroprotective properties. It is thought that PSO may achieve these effects through oxidative and nitrosative systems. Enrichment of a daily diet with PSO might be beneficial in reducing the risks of neurological diseases.

Study of ATF4/CHOP axis-mediated mitochondrial unfolded protein response in neuronal apoptosis induced by methylmercury

Methylmercury (MeHg) is a widely distributed environmental pollutant that can easily cross the blood-brain barrier and accumulate in the brain, thereby damaging the central nervous system. Studies have shown that MeHg-induced mitochondrial damage and apoptosis play a crucial role in its neurotoxic effects. Mitochondrial unfolded protein response (UPRmt) is indispensable to maintain mitochondrial protein homeostasis and ensure mitochondrial function, and the ATF4/CHOP axis is one of the signaling pathways to activate UPRmt. In this study, the role of the ATF4/CHOP axis-mediated UPRmt in the neurotoxicity of MeHg has been investigated by C57BL/6 mice and the HT22 cell line. We discovered that mice exposed to MeHg had abnormal neurobehavioral patterns. The pathological section showed a significant decrease in the number of neurons. MeHg also resulted in a reduction in mtDNA copy number and mitochondrial membrane potential (MMP). Additionally, the ATF4/CHOP axis and UPRmt were found to be significantly activated. Subsequently, we used siRNA to knock down ATF4 or CHOP and observed that the expression of UPRmt-related proteins and the apoptosis rate were significantly reduced. Our research showed that exposure to MeHg can over-activate the UPRmt through the ATF4/CHOP axis, leading to mitochondrial damage and ultimately inducing neuronal apoptosis.

Neuronal MeCP2 in the dentate gyrus regulates mossy fiber sprouting of mice with temporal lobe epilepsy

Sprouting of mossy fibers, one of the most consistent findings in tissue from patients with mesial temporal lobe epilepsy, exhibits several uncommon axonal growth features and has been considered a paradigmatic example of circuit plasticity that occurs in the adult brain. Clarifying the mechanisms responsible may provide new insight into epileptogenesis as well as axon misguidance in the central nervous system. Methyl-CpG-binding protein 2 (MeCP2) binds to methylated genomic DNA to regulate a range of physiological functions implicated in neuronal development and adult synaptic plasticity. However, exploring the potential role of MeCP2 in the documented misguidance of axons in the dentate gyrus has not yet been attempted. In this study, a status epilepticus-induced decrease of neuronal MeCP2 was observed in the dentate gyrus (DG). An essential regulatory role of MeCP2 in the development of functional mossy fiber sprouting (MFS) was confirmed through stereotaxic injection of a recombinant adeno-associated virus (AAV) to up- or down-regulate MeCP2 in the dentate neurons. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to identify the binding profile of native MeCP2 using micro-dissected dentate tissues. In both dentate tissues and HT22 cell lines, we demonstrated that MeCP2 could act as a transcription repressor on miR-682 with the involvement of the DNA methylation mechanism. Further, we found that miR-682 could bind to mRNA of phosphatase and tensin homolog (PTEN) in a sequence specific manner, thus leading to the suppression of PTEN and excessive activation of mTOR. This study therefore presents a novel epigenetic mechanism by identifying MeCP2/miR-682/PTEN/mTOR as an essential signal pathway in regulating the formation of MFS in the temporal lobe epileptic (TLE) mice. Significance statementUnderstanding the mechanisms that regulate axon guidance is important for a better comprehension of neural disorders. Sprouting of mossy fibers, one of the most consistent findings in patients with mesial temporal lobe epilepsy, has been considered a paradigmatic example of circuit plasticity in the adult brain. Although abnormal regulation of DNA methylation has been observed in both experimental rodents and humans with epilepsy, the potential role of DNA methylation in this well-documented example of sprouting of dentate axon remains elusive. This study demonstrates an essential role of methyl-CpG-binding protein 2 in the formation of mossy fiber sprouting. The underlying signal pathway has been also identified. The data hence provide new insight into epileptogenesis as well as axon misguidance in the central nervous system.

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage

BackgroundEarly brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. MethodsThe rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. ResultsWe observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. ConclusionThis study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.

Supraphysiologic doses of 17β-estradiol aggravate depression-like behaviors in ovariectomized mice possibly via regulating microglial responses and brain glycerophospholipid metabolism

Background17β-Estradiol (E2) is generally considered neuroprotective in humans. However, the current clinical use of estrogen replacement therapy (ERT) is based on the physiological dose of E2 to treat menopausal syndrome and has limited therapeutic efficacy. The efficacy and potential toxicity of superphysiological doses of ERT for menopausal neurodegeneration are unknown.MethodsIn this study, we investigated the effect of E2 with a supraphysiologic dose (0.5 mg/kg, sE2) on the treatment of menopausal mouse models established by ovariectomy. We performed the open field, Y-maze spontaneous alternation, forced swim tests, and sucrose preference test to investigate behavioral alterations. Subsequently, the status of microglia and neurons was detected by immunohistochemistry, HE staining, and Nissl staining, respectively. Real-time PCR was used to detect neuroinflammatory cytokines in the hippocampus and cerebral cortex. Using mass spectrometry proteomics platform and LC–MS/ MS-based metabolomics platform, proteins and metabolites in brain tissues were extracted and analyzed. BV2 and HT22 cell lines and primary neurons and microglia were used to explore the underlying molecular mechanisms in vitro.ResultssE2 aggravated depression-like behavior in ovariectomized mice, caused microglia response, and increased proinflammatory cytokines in the cerebral cortex and hippocampus, as well as neuronal damage and glycerophospholipid metabolism imbalance. Subsequently, we demonstrated that sE2 induced the pro-inflammatory phenotype of microglia through ERα/NF-κB signaling pathway and downregulated the expression of cannabinoid receptor 1 in neuronal cells, which were important in the pathogenesis of depression.ConclusionThese data suggest that sE2 may be nonhelpful or even detrimental to menopause-related depression, at least partly, by regulating microglial responses and glycerophospholipid metabolism.