Hippocampal Neuronal Cell Line Research Articles

Anti-diabetes and neuroprotection potential and primary safety studies of Isatis tinctoria L. hydroalcoholic leaf extract

PurposeNatural plant raw materials, previously underestimated in therapeutics, are becoming the subject of research for new applications in medicine. In our research, the hydroalcoholic extract of Isatis tinctoria leaf, rich in flavonoid compounds such as vicenin-2 and quercetin, was examined as a potential antidiabetic and neuroprotective agent. MethodsThe effect of the extract and its main flavonoid compounds on protein glycation, alpha-glucosidase activity, and acetylcholinesterase activity was tested. In vitro, in the mouse hippocampal neuronal cell line and in vivo, using a mouse model, the safety of the extract was screened for. ResultsOur experiments demonstrated significant inhibition of protein glycation, alpha-glucosidase activity, acetylcholinesterase activity, and β-amyloid aggregation by the extract, in a concentration-dependent manner. The extract had a strong reducing effect and did not exhibit cytotoxicity up to a concentration of 25 mg/mL. Intraperitoneal administration of the extract to mice did not have negative effects on body mass, locomotor activity, coordination, and liver cell integrity. ConclusionsOur research sheds new light on this raw material and deepens knowledge of its activity. This may result in the recognition of its therapeutic effects and even in its introduction in the modern treatment of diseases characterized by pathological changes associated with hyperglycemia, oxidation, and inflammation.

Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulation.

Neurodegenerative diseases are often characterized by mitochondrial dysfunction. In Alzheimer's disease, abnormal tau phosphorylation disrupts mitophagy, a quality control process through which damaged organelles are selectively removed from the mitochondrial network. The precise mechanism through which this occurs remains unclear. Previously, we showed that tau which has been mutated at Thr-231 to glutamic acid to mimic an Alzheimer's-relevant phospho-epitope expressed early in disease selectively inhibits oxidative stress-induced mitophagy in C. elegans. Here, we use immortalized mouse hippocampal neuronal cell lines to extend that result into mammalian cells. Specifically, we show that phosphomimetic tau at Ser-396/404 (EC) or Thr-231/Ser-235 (EM) partly inhibits mitophagy induction by paraquat, a potent inducer of mitochondrial oxidative stress. Moreover, a combination of immunologic and biochemical approaches demonstrates that the levels of the mitophagy receptor FKBP8, significantly decrease in response to paraquat in cells expressing EC or EM tau mutants, but not in cells expressing wildtype tau. In contrast, paraquat treatment results in a decrease in the levels of the mitophagy receptors FUNDC1 and BNIP3 in the presence of both wildtype tau and the tau mutants. Interestingly, FKBP8 is normally trafficked to the endoplasmic reticulum during oxidative stress induced mitophagy, and our results support a model where this trafficking is impacted by disease-relevant tau, perhaps through a direct interaction. We provide new insights into the molecular mechanisms underlying tau pathology in Alzheimer's disease and highlight FKBP8 receptor as a potential target for mitigating mitochondrial dysfunction in neurodegenerative diseases.

Role of FMRP in AKT/mTOR pathway-mediated hippocampal autophagy in fragile X syndrome

Fragile X syndrome (FXS) is caused by epigenetic silencing of the Fmr1 gene, leading to the deletion of the coding protein FMRP. FXS induces abnormal hippocampal autophagy and mTOR overactivation. However, it remains unclear whether FMRP regulates hippocampal autophagy through the AKT/mTOR pathway, which influences the neural behavior of FXS. Our study revealed that FMRP deficiency increased the protein levels of p-ULK-1 and p62 and decreased LC3II/LC3I level in Fmr1 knockout (KO) mice. The mouse hippocampal neuronal cell line HT22 with knockdown of Fmr1 by lentivirus showed that the protein levels of p-ULK-1 and p62 were increased, whereas LC3II/LC3I was unchanged. Further observations revealed that FMRP deficiency obstructed autophagic flow in HT22 cells. Therefore, FMRP deficiency inhibited autophagy in the mouse hippocampus and HT22 cells. Moreover, FMRP deficiency increased reactive oxygen species (ROS) level, decreased the co-localization between the mitochondrial outer membrane proteins TOM20 and LC3 in HT22 cells, and caused a decrease in the mitochondrial autophagy protein PINK1 in HT22 cells and Fmr1 KO mice, indicating that FMRP deficiency caused mitochondrial autophagy disorder in HT22 cells and Fmr1 KO mice. To explore the mechanism by which FMRP deficiency inhibits autophagy, we examined the AKT/mTOR signaling pathway in the hippocampus of Fmr1 KO mice, found that FMRP deficiency caused overactivation of the AKT/mTOR pathway. Rapamycin-mediated mTOR inhibition activated and enhanced mitochondrial autophagy. Finally, we examined whether rapamycin affected the neurobehavior of Fmr1 KO mice. The Fmr1 KO mice exhibited stereotypical behavior, impaired social ability, and learning and memory impairment, while rapamycin treatment improved behavioral disorders in Fmr1 KO mice. Thus, our study revealed the molecular mechanism by which FMRP regulates autophagy function, clarifying the role of hippocampal neuron mitochondrial autophagy in the pathogenesis of FXS, and providing novel insights into potential therapeutic targets of FXS.

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.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Cadmium exposure induced neuronal ferroptosis and cognitive deficits via the mtROS-ferritinophagy pathway

Exposure to environmental cadmium (Cd) is known to cause neuronal death and cognitive decline in humans. Ferroptosis, a novel iron-dependent type of regulated cell death, is involved in various neurological disorders. In the present study, Cd exposure triggered ferroptosis in the mouse hippocampus and in the HT22 murine hippocampal neuronal cell line, as indicated by significant increases in ferroptotic marker expression, intracellular iron levels, and lipid peroxidation. Interestingly, ferroptosis of hippocampal neurons in response to Cd exposure relied on the induction of autophagy since the suppression of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) substantially ameliorated Cd-induced ferroptosis. Furthermore, nuclear receptor coactivator 4 (NCOA4)-mediated degradation of ferritin was required for the Cd-induced ferroptosis of hippocampal neurons, demonstrating that NCOA4 knockdown decreased intracellular iron levels and lipid peroxidation and increased cell survival, following Cd exposure. Moreover, Cd-induced mitochondrial reactive oxygen species (mtROS) generation was essential for the ferritinophagy-mediated ferroptosis of hippocampal neurons. Importantly, pretreatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated Cd-induced hippocampal neuronal death and cognitive impairment in mice. Taken together, these findings indicate that ferroptosis is a novel mechanism underlying Cd-induced neurotoxicity and cognitive impairment and that the mtROS-ferritinophagy axis modulates Cd-induced neuronal ferroptosis.

Itaconate inhibits corticosterone-induced necroptosis and neuroinflammation via up-regulating menin in HT22 cells.

Corticosterone (CORT) damages hippocampal neurons as well as induces neuroinflammation. The tricarboxylic acid cycle metabolite itaconate has an anti-inflammatory role. Necroptosis is a form of programmed cell death, also known as inflammatory cell death. Menin is a multifunctional scaffold protein, which deficiency aggravates neuroinflammation. In this study, we explored whether itaconate inhibits CORT-induced neuroinflammation as well as necroptosis and further investigated the mediatory role of Menin in this protective effect of itaconate by using an exposure of CORT to HT22 cells (a hippocampal neuronal cell line). The viability of HT22 cells was examined by the cell counting kit 8 (CCK-8). The morphology of HT22 cells was observed by transmission electron microscope (TEM). The expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) were evaluated by western blotting. The contents of inflammatory factors were detected by an enzyme-linked immunosorbent assay (ELISA) kit. Our results showed that CORT increases the contents of pro-inflammatory factors (IL-1β, TNF-α) as well as decreases the contents of anti-inflammatory factors (IL-4, IL-10) in HT22 cells. We also found that CORT increases the expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) and decreases the cell viability in HT22 cells, indicating that CORT induces necroptosis in HT22 cells. Itaconate improves CORT-induced neuroinflammation and necroptosis. Furthermore, itaconate upregulates the expression of Menin in CORT-exposed HT22 cells. Importantly, silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced necroptosis and neuroinflammation. In brief, these results indicated that itaconate protects HT22 cells against CORT-induced neuroinflammation and necroptosis via upregulating Menin.

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.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons.

JOURNAL/nrgr/04.03/01300535-202412000-00026/figure1/v/2024-04-08T165401Z/r/image-tiff Gamma-aminobutyric acid (GABA)ergic neurons, the most abundant inhibitory neurons in the human brain, have been found to be reduced in many neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia. Our previous study identified the upregulation of microRNA-502-3p (miR-502-3p) and downregulation of GABA type A receptor subunit α-1 in Alzheimer's disease synapses. This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function. In vitro studies were performed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs. In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunit α-1 mRNA. Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunit α-1 gene and suppresses the luciferase activity. Furthermore, quantitative reverse transcription-polymerase chain reaction, miRNA in situ hybridization, immunoblotting, and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunit α-1 level, while suppression of miR-502-3p increased the level of GABA type A receptor subunit α-1 protein. Notably, as a result of the overexpression of miR-502-3p, cell viability was found to be reduced, and the population of necrotic cells was found to be increased. The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney (HEK) recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function, suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function. Additionally, the levels of proteins associated with Alzheimer's disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression. The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p. We propose that micro-RNA, in particular miR-502-3p, could be a potential therapeutic target to modulate GABAergic synapse function in neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia.

VPS13D affects epileptic seizures by regulating mitochondrial fission and autophagy in epileptic rats

Abnormal mitochondrial dynamics can lead to seizures, and improved mitochondrial dynamics can alleviate seizures. Vacuolar protein sorting 13D (VPS13D) is closely associated with regulating mitochondrial homeostasis and autophagy. However, further investigation is required to determine whether VPS13D affects seizures by influencing mitochondrial dynamics and autophagy. We aimed to investigate the influence of VPS13D on behavior in a rat model of acute epileptic seizures. Hence, we established an acute epileptic seizure rat model and employed the CRISPR/CAS9 technology to construct a lentivirus to silence the Vps13d gene. Furthermore, we used the HT22 mouse hippocampal neuron cell line to establish a stable strain with suppressed expression of Vps13d in vitro. Then, we performed quantitative proteomic and bioinformatics analyses to confirm the mechanism by which VPS13D influences mitochondrial dynamics and autophagy, both in vitro and in vivo using the experimental acute epileptic seizure model. We found that knockdown of Vps13d resulted in reduced seizure latency and increased seizure frequency in the experimental rats. Immunofluorescence staining and western blot analysis revealed a significant increase in mitochondrial dynamin-related protein 1 expression following Vps13d knockdown. Moreover, we observed a significant reduction in LC3II protein expression levels and the LC3II/LC3I ratio (indicators for autophagy) accompanied by a significant increase in P62 expression (an autophagy adaptor protein). The proteomic analysis confirmed the up-regulation of P62 protein expression. Therefore, we propose that VPS13D plays a role in modulating seizures by influencing mitochondrial dynamics and autophagy.

Genetic screen identified PRMT5 as a neuroprotection target against cerebral ischemia.

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remain largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. PRMT5 was identified as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. PRMT5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that PRMT5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, mice were treated with middle cerebral artery occlusion with the PRMT5 inhibitor EPZ015666 and found that PRMT5 inhibition sustains protection against neuronal death in vivo. Together, these findings revealed a novel epigenetic mechanism of PRMT5 in cerebral ischemia and uncovered a potential target for neuroprotection.

Genetic screen identified PRMT5 as a neuroprotection target against cerebral ischemia

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remain largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. PRMT5 was identified as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. PRMT5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that PRMT5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, mice were treated with middle cerebral artery occlusion with the PRMT5 inhibitor EPZ015666 and found that PRMT5 inhibition sustains protection against neuronal death in vivo. Together, these findings revealed a novel epigenetic mechanism of PRMT5 in cerebral ischemia and uncovered a potential target for neuroprotection.

Notoginsenoside R1 attenuates bupivacaine induced neurotoxicity by activating Jak1/Stat3/Mcl1 pathway

Bupivacaine, a common amide local anesthetic, can provide effective analgesia or pain relief but can also cause neurotoxicity, which remains a mounting concern in clinic and animal care. However, the precise underlying mechanisms have not been fully elucidated. A natural compound, notoginsenoside R1 (NG-R1) has been reported to exhibit a neuroprotective role in stress conditions. In this study, we explored the function and mechanism of NG-R1 in alleviating bupivacaine-induced neurotoxicity in mouse hippocampal neuronal (HT-22) and mouse neuroblastoma (Neuro-2a) cell lines. Our results exhibited that NG-R1 treatment can significantly rescue the decline of cell survival induced by bupivacaine. Tunel staining and western blotting showed that NG-R1 could attenuate BPV‑induced cell apoptosis. Besides, we focused on Mcl1 as a potential target as it showed opposite expression tendency in response to NG-R1 and bupivacaine exposure. Mcl1 knockdown blocked the inhibitory effect of NG-R1 on cell apoptosis against bupivacaine treatment. Intriguingly, we found that NG-R1 can upregulate Mcl1 transcription by activating Stat3 and promote its nuclear translocation. In addition, NG-R1 can also promote Jak1 phosphorylation and docking analysis provide a predicted model for interaction between NG-R1 and phosphorylated Jak1. Taken together, our results demonstrated that NG-R1 can attenuate bupivacaine induced neurotoxicity by activating Jak1/Stat3/Mcl1 pathway

Neuroprotective effect of Aster yomena ethanolic extract in HT-22 and SK-N-MC cells based on antioxidant activity

The antioxidant potentials of ethanolic extracts derived from Aster yomena (A. yomena) were evaluated by assessing their total phenolic and flavonoid contents and radical scavenging activities. Our findings revealed that the 60% ethanolic extract of A. yomena exhibited the most robust antioxidant properties among all extracts tested. Specifically, the IC50 values for the 2,2′-azino-bis (3-ethyl benzothiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activities of the 60% ethanolic extract from A. yomena were determined to be 1,640.30 μg/mL and 2,655.10 μg/mL, respectively. Moreover, the inhibitory effect on malondialdehyde increased with the 60% ethanolic extract from A. yomena. To assess the neuroprotective effects, we examined the impact of the 60% ethanolic extract from A. yomena against H2O2-induced cytotoxicity in HT-22 (mouse hippocampal neuronal cell line) and SK-N-MC (human neuroblastoma cell line) cells. The results demonstrated a significant improvement in cell viability and reduced intracellular oxidative stress. Furthermore, the major bioactive compounds present in the 60% ethanolic extract from A. yomena were identified as chlorogenic acid and rutin through high-performance liquid chromatography (HPLC) analysis.

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.

IL-17A promotes the progression of Alzheimer’s disease in APP/PS1 mice

BackgroundAlzheimer’s disease (AD), which is the most common cause of dementia in elderly individuals, is a progressive neurodegenerative disorder. Neuroinflammation, which is an immune response that is activated by glial cells in the central nervous system, plays an important role in neurodegenerative diseases. Many studies have shown that interleukin-17A (IL-17A) plays an important role in AD, but research on the pathological effects of IL-17A on AD is limited.MethodsWe report the effect of IL-17A on AD progression in APPswe/PS1dE9 (APP/PS1) mice, which are the most widely used AD model mice. The BV2 cell line, which is a microglial cell line derived from C57/BL6 mice, was used to establish a cell model to verify the role of IL-17A in neuroinflammation at the cellular level. The HT22 hippocampal neuronal cell line was used to investigate the relationship between IL-17A and Aβ deposition.ResultsIn this research, we found that IL-17A promotes the progression of AD in the APP/PS1 mouse model. The role of IL-17A in neuroinflammation is related to tumour necrosis factor (TNF)-α. Circulating IL-17A stimulates the secretion of TNF-α by microglia through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signalling pathway, thus exacerbating neuroinflammation. In addition, intraperitoneal injection of IL-17A antibody (IL17Ab) significantly improved the cognitive function of APP/PS1 mice.ConclusionsIL-17A increased TNF-α levels in the brain and exacerbated neuroinflammation through the TLR4/NF-κB signalling pathway and microglial activation in APP/PS1 mice. Moreover, IL-17A promoted the progression of AD by enhancing neuroinflammation, inhibiting microglial phagocytosis, and promoting the deposition of β-amyloid 42 in AD model mice.

Oxymatrine Improves Oxidative Stress-Induced Senescence in HT22 Cells and Mice via the Activation of AMP-Activated Protein Kinase.

The accumulation of oxidative stress is one of the important factors causing cellular senescence. Oxymatrine (OM) is a natural quinolizidine alkaloid compound known for its antioxidant effects. This study aimed to investigate the anti-senescence potential of OM through oxidative stress-induced in vitro and in vivo models. By treating 600 μM of H2O2 to the HT22 mouse hippocampal neuronal cell line and by administering 150 mg/kg D-galactose to mice, we generated oxidative stress-induced senescence models. After providing 1, 2, and 4 μg/mL of OM to the HT22 mouse cell line and by administering 50 mg/kg OM to mice, we evaluated the enhancing effects. We evaluated different senescence markers, AMPK activity, and autophagy, along with DCFH-DA detection reaction and behavioral tests. In HT22 cells, OM showed a protective effect. OM, by reducing ROS and increasing p-AMPK expression, could potentially reduce oxidative stress-induced senescence. In the D-Gal-induced senescence mouse model, both the brain and heart tissues recovered AMPK activity, resulting in reduced levels of senescence. In neural tissue, to assess neurological recovery, including anxiety symptoms and exploration, we used a behavioral test. We also found that OM decreased the expression level of receptors for advanced glycation end products (RAGE). In heart tissue, we could observe the restoration of AMPK activity, which also increased the activity of autophagy. The results of our study suggest that OM ameliorates oxidative stress-induced senescence through its antioxidant action by restoring AMPK activity.

Circular RNA DLGAP4 alleviates sevoflurane-induced neurotoxicity by regulating miR-9-5p/Sirt1/BDNF pathway

BackgroundSevoflurane is a widely used anesthetic in infants. However, long and repeated exposure to this drug can cause developmental neurotoxicity. This study aimed to investigate the role and mechanism of circular RNA DLGAP4 (circDLGAP4) in sevoflurane-induced neurotoxicity. MethodsNeonatal mice and mouse hippocampal neuronal cell line HT22 were used to construct sevoflurane-induced nerve injury models. The role of circDLGAP4 in sevoflurane-induced neurotoxicity was evaluated by gain-and/or loss-of-function methods. Pathological alterations in hippocampus were analyzed by hematoxylin-eosin and Tunel staining. Cell injury was assessed by cell viability and apoptosis, which was detected by CCK-8 and flow cytometry. The expression of circDLGAP4 and miR-9-5p was determined by real-time PCR. Sirt1 and BDNF levels were measured by Western blot. Productions of TNF-α and IL-6 were examined by ELISA. Dual-luciferase reporter assay and/or RNA pull-down assay were used to confirm the direct binding among circDLGAP4, miR-9-5p, and Sirt1. Rescue experiments were used to further verify the mechanism of circDLGAP4. ResultsCircDLGAP4 expression was decreased by sevoflurane both in vivo and in vitro. Overexpression of circDLGAP4 elevated cell viability, reduced apoptosis and levels of TNF-α and IL-6, while circDLGAP4 knockdown showed the opposite effects in sevoflurane-induced HT22 cells. Mechanically, circDLGAP4 functioned via directly binding to and regulating miR-9-5p, followed by targeting the Sirt1/BDNF pathway. Additionally, circDLGAP4 upregulation relieved sevoflurane-induced nerve injury, reduced levels of TNF-α, IL-6 and miR-9-5p, but increased the expression of Sirt1 and BDNF in hippocampus. ConclusionsOur studies found that circDLGAP4 relieved sevoflurane-induced neurotoxicity by sponging miR-9-5p to regulate Sirt1/BDNF pathway.

Seipin overexpression attenuates cerebral ischemia-reperfusion injury via preventing apoptosis and autophagy.

Ischemic cerebrovascular disease (ICVD) is one of three fatal diseases in humans, along with heart disease and malignant tumors. Cerebral ischemia/reperfusion injury (CI/RI) is the primary cause of ICVD. Recently, seipin was reported to be crucial for lipid droplet formation, hepatic steatosis, and axonal atrophy. However, the function and mechanism of seipin in CI/RI has not been explicitly stated. Oxygen-glucose deprivation/reoxygenation (OGD/R) hippocampal neuron cell line (HT-22) and middle cerebral artery occlusion (MCAO) in rats were established. The levels of apoptosis- and autophagy-related proteins and seipin were confirmed by western blot. Cell proliferation was assessed with CCK-8, and ischemic infarction and pathological structure were monitored by TTC and H&E staining, and tissue apoptosis was assessed through TUNEL assay. The proliferative activity was decreased, and apoptosis and autophagy pathways could also be induced in the OGD/R HT-22 cells. Seipin overexpression accelerated viability and inhibited apoptosis and autophagy in the OGD/R HT-22 cells. Moreover, the data revealed that seipin overexpression could also attenuate cerebral infarction, apoptosis. Autophagy pathways could be repressed by seipin in the MCAO rats. Seipin has a protective role against CI/RI in rats, and its mechanism might be relevant to the suppression of apoptosis and autophagy, suggesting that seipin might be a latent target for CI/RI.