H19-7 Cells Research Articles

Integration of metabolomics and transcriptomics to reveal metabolic characteristics and key targets associated with lncRNA Vof-16 in H19-7 cells

Cognitive disorders represent one of the most common chronic complications of diabetes. Our previous study has demonstrated that long non-coding RNA (lncRNA) Vof-16 is upregulated in the hippocampal tissue of streptozotocin (STZ)-induced diabetic rats. Despite this finding, the specific roles and underlying mechanisms of lncRNA Vof-16 in diabetes-related cognitive dysfunction remain largely unexplored. To elucidate the mechanism involved, lncRNA Vof-16 was overexpressed in rat hippocampal cell line H19-7 through lentivirus transfection. We integrated metabolomics and transcriptomics approaches to identify potential targets and metabolic pathways influenced by lncRNA Vof-16. Key proteins and pathways were subsequently validated using western blotting and immunofluorescence staining. Transcriptomics indicated that lncRNA Vof-16 overexpression may modulate autophagic activity in H19-7 cells. Metabolomic profiling revealed that the primary differential metabolic pathways included trehalose degradation, tryptophan metabolism, vitamin B6 metabolism, glycolysis, pterine biosynthesis, and the pentose phosphate pathway. Ingenuity Pathway Analysis (IPA) of gene-metabolite networks demonstrated that the high lncRNA Vof-16 expression group exhibited a significantly higher association with autophagy compared to the low lncRNA Vof-16 expression group. Western blot results confirmed that lncRNA Vof-16 overexpression led to decreased protein expression levels of ATG3 and ATG12. Specifically, lncRNA Vof-16 reduces autophagy in hippocampal neurons by targeting the elevated levels of phospho-p70S6K, a downstream effector of mTORC1, potentially contributing to the pathogenesis of diabetic cognitive impairment. The construction of gene-metabolite networks may offer promising new strategies for addressing the growing issue of diabetic cognitive impairment.

Mechanism of cAMP Response Element-binding Protein 1/Death-associated Protein Kinase 1 Axis-mediated Hippocampal Neuron Apoptosis in Rat Brain Injury After Cardiopulmonary Resuscitation

Brain injury represents a leading cause of deaths following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). This study explores the role of CREB1 (cAMP responsive element binding protein 1)/DAPK1 (death associated protein kinase 1) axis in brain injury after CPR. CA was induced by asphyxia in rats, followed by CPR. After CREB1 over-expression, the survival rate and neurological function score of rats were measured. Nissl and TUNEL staining evaluated the pathological condition of hippocampus and apoptosis of hippocampal neurons respectively. H19-7 cells were subjected to OGD/R and infected with oe-CREB1. CCK-8 assay and flow cytometry measured the cell viability and apoptosis. CREB1, DAPK1, and cleaved Caspase-3 expressions were examined using Western blot. The binding between CREB1 and DAPK1 was determined using ChIP and dual-luciferase reporter assays. CREB1 was poorly expressed while DAPK1 was highly expressed in rat hippocampus after CPR. CREB1 overexpression improved rat neurological function, repressed neuron apoptosis, and reduced cleaved Caspase-3 expression. CREB1 was enriched on the DAPK1 promoter and suppressed DAPK1 expression. DAPK1 overexpression reversed the inhibition of OGD/R-insulted apoptosis by CREB1 overexpression. To conclude, CREB1 suppresses hippocampal neuron apoptosis and mitigates brain injury after CPR by inhibiting DAPK1 expression.

Knocking down Trim47 ameliorated sevoflurane-induced neuronal cell injury and cognitive impairment in rats.

Sevoflurane (SEV), usually causing neuronal damage and cognitive dysfunction, is one of the most commonly used anesthetics in clinical practice. However, the function of Trim47 in SEV-induced neuronal impairment remains elusive. The aim of this study was to study the effect of knocking down Trim47 on the nerve injury induced by SEV. Nerve injury was induced in rats by 3% SEV, and H19-7 was used to establish a pathological model, and sh-Trim47 was transfected into H19-7 to study the function of Trim47. The effects of SEV on the expression of Trim47 in the hippocampus and cognitive function of rats were studied by neurological function score and Moris water maze (MWM). The mRNA and protein expression of TNF-α, IL-1β and IL-6 in the cells, along with the neuronal apoptosis in the hippocampus of rats in each group were studied by TUNEL or WB. Flow cytometry was used to study the effect of knockdown of Trim47 on cell apoptosis. CCK-8 was used to detect cell viability of H19-7 cells. Finally, the potential signaling pathway affected by knockdown of Trim47 after abrogation of SEV induction was investigated by WB. The results showed that, knockdown of Trim47 ameliorated SEV-induced neurological damage and cognitive deficits, inflammation and neuronal cell apoptosis in rats, and promoted hippocampal neuronal activity. Knockdown of Trim47 can inhibit the NF-κB signaling pathway and improve neuronal cell damage and cognitive impairment induced by SEV in neonatal rats by regulating NF-κB signaling pathway, alleviating inflammatory response, and inhibiting neuronal apoptosis.

Mechanism of Molecular Activity of Yolkin—a Polypeptide Complex Derived from Hen Egg Yolk—in PC12 Cells and Immortalized Hippocampal Precursor Cells H19-7

Food-derived bioactive peptides able to regulate neuronal function have been intensively searched and studied for their potential therapeutic application. Our previous study showed that a polypeptide complex yolkin, isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), improved memory and cognitive functions in rats. However, the mechanism activated by the yolkin is not explained. The goal of the present study was to examine what molecular mechanism regulating brain-derived neurotrophic factor (BDNF) expression is activated by the yolkin complex, using in vitro models of PC12 cell line and fetal rat hippocampal cell line H19-7. It was shown that yolkin increased the proliferative activity of rat hippocampal precursor cells H19-7 cells and upregulated the expression/production of BDNF in a cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB)-dependent manner. Additionally the upregulation of carboxypeptidase E/neurotrophic factor–α1 (CPE/(NF-α1) expression was shown. It was also determined that upregulation of CREB phosphorylation by yolkin is dependent on cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) signaling pathway activation. Moreover, the impact of yolkin on the level of intracellular Ca2+, nitric oxide, and activation of extracellular signal-regulated kinases 1/2 (ERK 1/2 kinase) was excluded. These results emphasize that yolkin can act comprehensively and in many directions and may participate in the regulation of neurons’ survival and activity. Therefore, it seems that the yolkin specimen can be used in the future as a safe, bioavailable, natural nutraceutical helping to improve the cognition of older people.

H2S Alleviates Propofol-Induced Impaired Learning and Memory by Promoting Nuclear Translocation of Nrf2 and Inhibiting Apoptosis and Pyroptosis in Hippocampal Neurons.

Repeated exposure to propofol can affect their learning and memory functions, but the mechanism remains unclear. The current study aimed to investigate the mechanism underlying the effect of hydrogen sulfide (H2S) on the alleviation of propofol-induced learning and memory impairment, mediated by promoting nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and inhibiting apoptosis and pyroptosis in hippocampal neurons. Rats used in this study were successively exposed to 200 mg/kg propofol for 8 consecutive weeks, followed by inhalation of 10, 40 or 80 ppm H2S. Subsequently, the effects of different concentrations of H2S on learning and memory were assessed using the water maze assay. Additionally, the effects of H2S on cell apoptosis and pyroptosis and nuclear translocation of Nrf2 in hippocampal neurons were also determined. Furthermore, NaHS (200 μmol/L) was used as an in vitro donor for H2S, and rescue experiments were carried out following Nrf2 knockdown in H19-7 cells. Moreover, Nrf2 function was inhibited following treatment with an intraperitoneal injection of ML385 (30 mg/kg) in the rats. The effects of H2S on reactive oxygen species (ROS) generation, cell apoptosis, and pyroptosis in propofol-treated and Nrf2-deficient H19-7 cells were also investigated. Exposure to propofol for 8 weeks affected the ability of the rats to find underwater platforms (p < 0.01). Further, the exposure induced cell apoptosis and NLR family pyrin domain containing 3 (NLRP3)-related pyroptosis (p < 0.01). Although inhalation of 10 ppm H2S did not attenuate the aforementioned effects (p > 0.05), exposure to 40 and 80 ppm H2S significantly alleviated propofol-induced injury in the hippocampal neurons (p < 0.01). However, the protective effect of 80 ppm H2S was more obvious as compared to that of the other two doses (p < 0.01). In addition, Nrf2 knockdown aggravated the propofol-induced cell pyroptosis and apoptosis as well as reversed the protective effect of H2S against these processes (p < 0.01). In vivo experiments in this study demonstrated that Nrf2 inhibition abrogated the protective effects of H2S inhalation against learning and memory impairment as well as propofol-induced cell apoptosis and pyroptosis in rats (p < 0.01). H2S could attenuate propofol-induced damage in hippocampal neurons by promoting the nuclear translocation of Nrf2 and inhibiting cell apoptosis and pyroptosis.

Neuroprotective effects of Chrysanthemum morifolium on cerebral ischemia- reperfusion injury contributes to the oxidative stress suppression and related Keap1/Nrf2 pathway

ABSTRACT Background Ischemic stroke, the cause of death and disability worldwide, is closely related to oxidative stress damage. Chrysanthemum has profound antiantioxidant activity. We aimed to verify whether Chrysanthemum morifolium extract (CME) influences brain injury in cerebral ischemia-reperfusion injury (CR/RI) model. Methods In vitro, rat hippocampal H19-7 neurons were pretreated with CME, CR/RI was simulated with oxygen glucose deprivation/reoxygenation (OGD/R). The cell viability, apoptosis, lactate dehydrogenase release, reactive oxygen species (ROS) generation, malonaldehyde (MDA) content and superoxide dismutase（SOD） activity were detected. In vivo, middle cerebral artery occlusion (MCAO) model rats were pre-administered with CME, and then behavioral test, triphenyltetrazolium chloride (TTC), hematoxylin-eosin staining (HE), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), ROS immunofluorescence, MDA and SOD activity were tested. Furthermore, Keap1/Nrf2 signaling of CME in CI/RI was investigated. Results In OGD/R induced in H19-7 cells, CME increased OGD/R-induced cell viability and reduced cell apoptosis, which was reversed by siNrf2 transfection . In MCAO rats, CME improved the neurological deficits and alleviated brain injury. However, co-treatment with MLK385 counteracted these neuroprotective effects of CME on MCAO rats. Conclusion CME could significantly reduce oxidative stress and nerve injury in vitro and in vivo models of CI/RI by regulating the Keap1/Nrf2 pathway.

Neuroprotective Effect of Stearidonic Acid on Amyloid β-Induced Neurotoxicity in Rat Hippocampal Cells.

Dietary intake of omega-3 fatty acids found in fish has been reported to reduce the risk of Alzheimer's Disease (AD). Stearidonic acid (SDA), a plant-based omega-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids. However, its role in neuronal degeneration is unknown. This study was designed to evaluate effects of SDA on Amyloid-β(A-β)-induced neurotoxicity in rat hippocampal cells. Results showed that SDA effectively converted to eicosapentaenoic acid (EPA) in hippocampal cells. Aβ-induced apoptosis in H19-7 cells was protected by SDA pretreatment as evidenced by its regulation on the expression of relevant pro- and anti-apoptotic genes, as well as the inhibition on caspase activation. SDA also protected H19-7 cells from Aβ-induced oxidative stress by regulating the expression of relevant pro- and anti-oxidative genes, as well as the improvement in activity of catalase. As for Aβ/LPS-induced neuronal inflammation, SDA pretreatment reduced the release of IL-1β and TNFα. Further, we found that the anti-Aβ effect of SDA involves its inhibition on the expression of amyloid precursor protein and the regulation on MAPK signaling. These results demonstrated that SDAs have neuroprotective effect in Aβ-induced H19-7 hippocampal cells. This beneficial effect of SDA was attributed to its antiapoptotic, antioxidant, and anti-inflammatory properties.

MiR-21 participates in the neuroprotection of diazoxide against hypoxic-ischemia encephalopathy by targeting PDCD4

ABSTRACT Background Hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of neonatal death and permanent neurological disability. Here, we designed to quest therapeutic effects of diazoxide (DZ) on HIE and its mechanism. Methods The cell model of HIE was established. CCK8 and flow cytometry were applied to test cell viability and apoptosis. RT-qPCR and western blotting was evaluated to the expression of miR-21, PDCD4, PI3K, and p-AKT/AKT. Commercial kits were employed to detect SOD, MDA, LDH. DCFH-DA was used to measure intracellular ROS. ELISA was performed to estimate IL-1β, IL-6 and TNF-α. Dual-luciferase reporter gene and RIP assay were applied to confirm the binding relationships between miR-21 and PDCD4. Results In H19-7 cells and PC12 cells stimulated by OGD, with low cell viability, high apoptosis, miR-21 high expression and PDCD4 low expression. However, the functions were all reversed by DZ administration. Furthermore, miR-21 inhibitor could abolish the beneficial effects of DZ on OGD-induced cells. Besides, miR-21 could interact with PDCD4. In addition, PDCD4 involved with the regulation of DZ to OGD-induced cells via PI3K/AKT pathway. Conclusion DZ enhanced miR-21 level and inhibited PDCD4 level via PI3K/AKT pathway to resisted HIE.

Sevoflurane Post-treatment Mitigates Oxygen-glucose Deprivationinduced Pyroptosis of Hippocampal Neurons by Regulating the Mafb/DUSP14 Axis.

Ischemic brain injury often results in irreversible pyroptosis of neurons. Sevoflurane (Sevo) post-treatment exerts an alleviative role in neuroinflammation. This work evaluated the mechanism of Sevo post-treatment in oxygen-glucose deprivation (OGD)-induced pyroptosis of rat hippocampal neurons. Rat hippocampal neuron cell line H19-7 cells were treated with OGD, followed by posttreatment of 2% Sevo. The expression patterns of Mafb ZIP Transcription Factor B (Mafb) and dual- specificity phosphatase 14 (DUSP14) were determined via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods. H19-7 cell viability and the release of lactate dehydrogenase (LDH) were examined via the cell counting kit-8 and LDH assay kits. Levels of pyroptosis-related proteins and cytokines NOD-like receptor family, pyrin domain containing 3 (NLRP3), N-term cleaved Gasdermin-D (GSDMD-N), cleaved-caspase-1, interleukin (IL)-1β, and IL-18 were also examined. The binding relation between Mafb and the DUSP14 promoter was detected. Besides, the roles of Mafb/DUSP14 in OGD-induced pyroptosis of rat hippocampal neurons were investigated through functional rescue experiments. Mafb and DUSP14 expression levels were decreased in OGD-induced hippocampal neurons. Sevo post-treatment up-regulated Mafb and DUSP14, facilitated H19-7 cell viability, inhibited LDH release, and reduced levels of NLRP3, GSDMD-N, cleaved-caspase-1, IL-1β, and IL-18. Mafb increased DUSP14 expression via binding to the DUSP14 promoter. Repressing Mafb or DUSP14 exacerbated pyroptosis of hippocampal neurons. Sevo post-treatment increased Mafb and DUSP14 expressions, which repressed OGDinduced pyroptosis of hippocampal neurons.

Immunoregulatory and neuroprotective activity of ovocystatin

IntroductionOvocystatin has beneficial properties for cognitive function in young rats and might prevents aging-related cognitive impairment in older animals, as well as reduces memory decline in APP/PS1 mice model.ObjectivesOur study aimed at assessing the impact of ovocystatin on microglia activation and neurogenesis.MethodsImmunoactivation: Mouse wild type microglia were stimulated with ovocystatin at dose of 100 micrograms/ml. The effect of ovocystatin on nitric oxide production and interleukin 1 beta secretion were determined. Neurogenesis: Primary rat hippocampal neurons of H19-7 cell line was used. The impact of ovocystatin on proliferation, nitric oxide production, and expression of markers of neurogenesis: microtubule-associated protein 2 (MAP2, isoforms A/B and C/D) and Synapsin 1, were determined.ResultsIt was shown that ovocystatin does not stimulate microglial cells to produce inflammatory mediators. Whereas, no toxic effect of ovocystatin (1-100 ug/ml) on H19-7 cells viability, and dose-dependent down-regulation of proliferation were demonstrated. It was also shown that in primary hippocampal neurons of H19-7 cells incubated with ovocystatin (100 micrograms/ml), the expression level of MAP2 C/D (75kDa) - characteristic form of immature neurons is unchanged. However, the increased expression of MAP2 A/B protein (280 kDa) – characteristic for mature neurons was observed after 6 and 24h incubation with ovocystatin. Relatively to MAP2 A/B, increased expression of synapsin 1 was observed.ConclusionsThe ovocystatin might be a potential activator of molecular mechanisms in primary hippocampal neurons, participating in regulation of neurogenesis. Nevertheless, further studies are needed.DisclosureNo significant relationships.

Early Developmental PMCA2b Expression Protects From Ketamine-Induced Apoptosis and GABA Impairments in Differentiating Hippocampal Progenitor Cells.

PMCA2 is not expressed until the late embryonic state when the control of subtle Ca2+ fluxes becomes important for neuronal specialization. During this period, immature neurons are especially vulnerable to degenerative insults induced by the N-methyl-D-aspartate (NMDA) receptor blocker, ketamine. As H19-7 hippocampal progenitor cells isolated from E17 do not express the PMCA2 isoform, they constitute a valuable model for studying its role in neuronal development. In this study, we demonstrated that heterologous expression of PMCA2b enhanced the differentiation of H19-7 cells and protected from ketamine-induced death. PMCA2b did not affect resting [Ca2+]c in the presence or absence of ketamine and had no effect on the rate of Ca2+ clearance following membrane depolarization in the presence of the drug. The upregulation of endogenous PMCA1 demonstrated in response to PMCA2b expression as well as ketamine-induced PMCA4 depletion were indifferent to the rate of Ca2+ clearance in the presence of ketamine. Yet, co-expression of PMCA4b and PMCA2b was able to partially restore Ca2+ extrusion diminished by ketamine. The profiling of NMDA receptor expression showed upregulation of the NMDAR1 subunit in PMCA2b-expressing cells and increased co-immunoprecipitation of both proteins following ketamine treatment. Further microarray screening demonstrated a significant influence of PMCA2b on GABA signaling in differentiating progenitor cells, manifested by the unique regulation of several genes key to the GABAergic transmission. The overall activity of glutamate decarboxylase remained unchanged, but Ca2+-induced GABA release was inhibited in the presence of ketamine. Interestingly, PMCA2b expression was able to reverse this effect. The mechanism of GABA secretion normalization in the presence of ketamine may involve PMCA2b-mediated inhibition of GABA transaminase, thus shifting GABA utilization from energetic purposes to neurosecretion. In this study, we show for the first time that developmentally controlled PMCA expression may dictate the pattern of differentiation of hippocampal progenitor cells. Moreover, the appearance of PMCA2 early in development has long-standing consequences for GABA metabolism with yet an unpredictable influence on GABAergic neurotransmission during later stages of brain maturation. In contrast, the presence of PMCA2b seems to be protective for differentiating progenitor cells from ketamine-induced apoptotic death.

MiR-212-3p attenuates neuroinflammation of rats with Alzheimer's disease via regulating the SP1/BACE1/NLRP3/Caspase-1 signaling pathway.

Alzheimer’s disease (AD) ranks as the leading cause of dementia. MicroRNA (miR)-212-3p has been identified to exert neuroprotective effects on brain disorders. The current study analyzed the protective role of miR-212-3p in AD rats through regulating the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)/Caspase-1 signaling pathway. The AD rat model was established through injection of amyloid-β 1-42 (Aβ1-42), followed by the Morris water maze test. The morphology and functions of neurons were observed. Furthermore, miR-212-3p, NLRP3, cleaved Caspase-1, gasdermin D N-terminus, interleukin (IL)-1β, and IL-18 expressions were measured. H19-7 cells were treated with Aβ1-42 to establish the AD cell model, followed by an assessment of cell viability and pyroptosis. Downstream targets of miR-212-3p and specificity protein 1 (SP1), as well as beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), were predicted by databases and testified using dual-luciferase and chromatin immunoprecipitation assays. miR-212-3p was weakly expressed in AD rats. miR-212-3p overexpression was linked to improved learning and memory capacities of AD rats and reduced neuronal pyroptosis linked to neuroinflammation attenuation. In vitro, miR-212-3p improved viability and suppressed pyroptosis of neurons through inhibiting NLRP3/Caspase-1. Overall, miR-212-3p inhibited SP1 expression to block BACE1-induced activation of NLRP3/Caspase-1, thereby attenuating neuroinflammation of AD rats.

Integrating network pharmacology and in vitro model to investigate hippocampal neurotoxicity induced by atrazine

Atrazine (ATR), a commonly applied herbicide in agriculture, has been found to cause hippocampal injury in rodents. However, the underlying toxicological targets and mechanisms are unclear. In this study, network pharmacology analysis and in vitro model were integrated to investigate the effect and mechanism of ATR-induced hippocampal neurotoxicity. In total, 71 targets of hippocampal neurotoxicity induced by ATR were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment (KEGG) enrichment analysis suggested that these targets were related to multiple GO terms and signaling pathways. To further investigate the underlying mechanisms, the top 10 hub targets were screened and included tumor protein p53 (Tp53), caspase 3 (Casp3), prostaglandin-endoperoxide synthase 2 (Ptgs2), cAMP-responsive element-binding protein 1 (Creb1), estrogen receptor 1 (Esr1), Jun proto-oncogene (Jun), brain-derived neurotrophic factor (Bdnf), catalase (Cat), sirtuin 1 (Sirt1) and Fos proto-oncogene (Fos). Moreover, the cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay showed that ATR had time and dose-dependent cytotoxicity on H19-7 cells. TUNEL staining revealed that ATR increased the apoptotic ratio. In addition, Real-time quantitative polymerase chain reaction (RT-qPCR) results indicated that the mRNA expression levels of all hub targets showed significant changes, except Esr1 and Jun. Our study demonstrated that ATR mainly acted on multiple targets and signaling pathways to exert its hippocampal neurotoxicity. These results provided initial evidence for the further exploration of the toxicological mechanism of ATR.

Astrocyte-derived exosomes carry microRNA-17-5p to protect neonatal rats from hypoxic-ischemic brain damage via inhibiting BNIP-2 expression

Exosomes play critical roles in neurogenesis. This study aims to explore the mechanism of astrocyte-derived exosomes in neonatal rats with hypoxic-ischemic brain damage (HIBD). Astrocytes were collected and astrocyte-derived exosomes were isolated and identified. Neonatal rats were pre-treated with exosomes and then subjected to HIBD induction. Then the neurobehaviors, neuronal apoptosis, inflammation and oxidative stress in rat brain were measured. Differentially expressed microRNAs (miRNAs) in rat brain before and after HI procedure were analyzed. H19-7 cells were subjected to oxygen and glucose deprivation (OGD) for in vitro studies. Target relation between miR-17-5p and BNIP2 was identified. Gain- and loss-of functions of miR-17-5p and BNIP2 were conducted to identify their roles in viability, apoptosis, oxidative stress and inflammation of OGD-treated cells. Collectively, astrocyte-derived exosomes improved neurobehaviors, and reduced cerebral infarction, neuronal apoptosis, oxidative and inflammation in vivo and in vitro. The exosomes carried miR-17-5p bound to BNIP2 and negatively regulated BNIP2 expression in OGD-treated cells. Over-expression of miR-17-5p increased viability, and decreased OGD-induced apoptosis, oxidative stress and inflammation of H19-7 cells, which were reversed by over-expression of BNIP2. Taken together, the study suggested that astrocyte-derived exosomes could carry miR-17-5p to protect neonatal rats from HIBD via inhibiting BNIP-2 expression.

Bcl-2 Overexpression Induces Neurite Outgrowth via the Bmp4/Tbx3/NeuroD1 Cascade in H19-7 Cells.

Bcl-2 is overexpressed in the nervous system during neural development and plays an important role in modulating cell survival. In addition to its anti-apoptotic function, it has been suggested previously that Bcl-2 might act as a mediator of neuronal differentiation. However, the mechanism by which Bcl-2 might influence neurogenesis is not sufficiently understood. In this study, we aimed to determine the non-apoptotic functions of Bcl-2 during neuronal differentiation. First, we used microarrays to analyze the whole-genome expression patterns of rat neural stem cells overexpressing Bcl-2 and found that Bcl-2 overexpression induced the expression of various neurogenic genes. Moreover, Bcl-2 overexpression increased the neurite length as well as expression of Bmp4, Tbx3, and proneural basic helix-loop-helix genes, such as NeuroD1, NeuroD2, and Mash1, in H19-7 rat hippocampal precursor cells. To determine the hierarchy of these molecules, we selectively depleted Bmp4, Tbx3, and NeuroD1 in Bcl-2-overexpressing cells. Bmp4 depletion suppressed the upregulation of Tbx3 and NeuroD1 as well as neurite outgrowth, which was induced by Bcl-2 overexpression. Although Tbx3 knockdown repressed Bcl-2-mediated neurite elaboration and downregulated NeuroD1 expression, it did not affect Bcl-2-induced Bmp4 expression. While the depletion of NeuroD1 had no effect on the expression of Bcl-2, Bmp4, or Tbx3, Bcl-2-mediated neurite outgrowth was suppressed. Taken together, these results demonstrate that Bcl-2 regulates neurite outgrowth through the Bmp4/Tbx3/NeuroD1 cascade in H19-7 cells, indicating that Bcl-2 may have a direct role in neuronal development in addition to its well-known anti-apoptotic function in response to environmental insults.

Programming for increased expression of hippocampal GAD67 mediated the hypersensitivity of the hypothalamic\u2013pituitary\u2013adrenal axis in male offspring rats with prenatal ethanol exposure

An imbalance of excitatory and inhibitory signals in the brain has been proposed to be one of the main pathological features of various diseases related to hypothalamic–pituitary–adrenal axis (HPAA) dysfunction. Excessive glutamate release induces neuronal excitotoxicity, while glutamic acid decarboxylase (GAD) 67 promotes the transformation of excessive glutamate to γ-aminobutyric acid (GABA). Our previous studies demonstrated that prenatal ethanol exposure (PEE) causes foetal over-exposure to maternal corticosterone and hypersensitivity of the HPAA after birth, but its intrauterine programming mechanism is unknown. In this study, PEE was shown to lead to an enhanced potential excitatory ability of the hypothalamus and hypersensitivity of the HPAA, as well as mild abnormal hippocampal morphology, demethylation of the -1019 to -691-bp region in the hippocampal GAD67 promoter and upregulation of GAD67 expression accompanied by a reduction in glutamatergic neurons and increase in GABAergic neurons in PEE male offspring. Similar changes were also found in PEE male foetal rats. Furthermore, corticosterone increased the expression of the glucocorticoid receptor (GR) and GAD67 in foetal hippocampal H19-7 cells in a concentration-dependent manner, accompanied by demethylation of the GAD67 promoter, a decrease in glutamatergic neurons and increase in GABAergic neurons. The GR inhibitor, mifepristone, reversed the effects of corticosterone on H19-7 cells. These results suggested that PEE-induced excessive corticosterone can lead to upregulation of GAD67 through epigenetic modification mediated by the GR in the male foetal hippocampus, thereby weakening the negative regulation of the HPAA by the hippocampus and increasing the potential excitatory ability of the hypothalamus. These changes persisted until after birth, resulting in hypersensitivity of the HPAA. However, gender differences were observed in the hippocampal development, morphology and GAD67 expression associated with PEE. Programming for the increased expression of hippocampal GAD67 is a potential mechanism responsible for the hypersensitivity of the HPAA in PEE male rats.

Telmisartan Activates PPAR\u03b4 to Improve Symptoms of Unpredictable Chronic Mild Stress-Induced Depression in Mice

Major depression is a common mental disorder that has been established to be associated with a decrease in serotonin and/or serotonin transporters in the brain. Peroxisome proliferator-activated receptor δ (PPARδ) has been introduced as a potential target for depression treatment. Telmisartan was recently shown to activate PPARδ expression; therefore, the effectiveness of telmisartan in treating depression was investigated. In unpredictable chronic mild stress (UCMS) model, treatment with telmisartan for five weeks notably decrease in the time spent in the central and the reduced frequency of grooming and rearing in open filed test (OFT) and the decreased sucrose consumption in sucrose preference test (SPT) compared with the paradigms. Telmisartan also reversed the decrease in PPARδ and 5-HTT levels in the hippocampus of depression-like mice. Administration of PPARδ antagonist GSK0660 and direct infusion of sh-PPARδ into the brain blocked the effects of telmisartan on the improvement of depression-like behavior in these mice. Moreover, telmisartan enhanced the expression of PPARδ and 5HTT in H19-7 cells. In conclusion, the obtained results suggest that telmisartan improves symptoms of stress-induced depression in animals under chronic stress through activation of PPARδ. Therefore, telmisartan may be developed as a potential anti-depressant in the future.

Signals mediating Klotho-induced neuroprotection in hippocampal neuronal cells

The erythropoietin (Epo) receptor (EpoR) is expressed in the brain and was shown to have neuroprotective effects against brain damage in animal models. A recent study indicated that EpoR and its activity are the downstream effectors of Klotho for cytoprotection in the kidney. Thus, we propose that Klotho can stimulate the expression of EpoR in neuronal cells to enhance Epo-mediated protection. H19-7 hippocampal neuronal cells were treated with recombinant Klotho. In H19-7 cells, Klotho increased the expression of both the EpoR protein and mRNA. Klotho also enhanced the transcription activity of the EpoR promoter in H19-7 cells. Moreover, Klotho augmented the Epo-triggered phosphorylation of Jak2 and Stat5 and protected H19-7 cells from hydrogen peroxide cytotoxicity. The silencing of EpoR abolished the protective effect of Klotho against peroxide-induced cytotoxicity. Finally, the silencing of GATA1 diminished the Klotho-induced increase in EpoR protein and mRNA expression as well as its promoter activity. In conclusion, Klotho increased EpoR expression in neuronal cells through GATA1, thereby enabling EpoR to function as a cytoprotective protein against oxidative injury.

Resveratrol protects β amyloid-induced oxidative damage and memory associated proteins in H19-7 hippocampal neuronal cells.

Resveratrol (trans-3, 5, 4'-trihydroxystilbene) is a polyphenolic phytoalexin known to exhibit antioxidant and neuroprotective effects in several experimental models. Amyloid β peptide (Aβ), a core component of extracellular senile plaques accumulates in the brains of patients with Alzheimer's disease and is related to the development of cognitive impairment and neuronal loss. The present study evaluates the neuroprotective action of resveratrol on Aβ-induced oxidative stress and memory loss. Cultured rat hippocampal H19-7 neuronal cell line was pretreated with 75 μM of resveratrol for 2 hrs followed by 25 μM of Aβ (1-40) for 24 hrs. H19-7 cells treated with Aβ exhibited increased lipid peroxide levels. Enzymatic antioxidants including superoxide dismutase, catalase, glutathione reductase, and non-enzymatic antioxidants such as tocopherol, ascorbic acid and glutathione were decreased in the Aβ treated group when compared to the control group. Aβ treatment also increased the expression of total tau as well as phosphorylated forms of tau (CP13, S202/205; PHF1, S396/404) and decreased the expression of insulin degrading enzyme (IDE), phosphoglycogen synthase kinase 3β involved in Aβ degradation and tau hyper phosphorylation. Expression of PSD-95 and Arc proteins, essential for synaptic maturity and plasticity, was decreased by Aβ treatment. Resveratrol treatment attenuated the accumulation of lipid peroxide levels, up-regulated the antioxidant activities and improved the expression of memory-associated proteins in Aβ treated H19-7 cells. These findings highlight the neuroprotective effect of resveratrol in preventing Aβ-induced oxidative damage and memory loss in vitro.

Neuroprotective effects of resveratrol against β‐amyloid induced oxidative damage and memory loss in rat hippocampal (H19‐7) cells (647.44)

Resveratrol is a polyphenolic phytoalexin known to exhibit neuroprotective effects in several experimental models. Accumulation of amyloid beta in the Alzheimer disease patient brain seems to be the cause of cognitive impairment and neuronal loss. The present study evaluates the neuroprotective action of resveratrol on β‐amyloid induced oxidative stress and memory loss in cultured rat hippocampal H19‐7 neuronal cell line pretreated with 75 μM of resveratrol for 2 hrs followed by 25 μM of Aβ (1‐40) for 24 hrs. The lipid peroxidase levels were significantly increased in amyloid β treated H19‐7 cells. The enzymic antioxidants like superoxide dismutase, catalase, glutathione reductase, and non‐enzymic antioxidants like tocopherol, ascorbic acid and glutathione were decreased. amyloid β treatment also increased tau phosphorylation and decreased expressions of certain proteins like Insulin Degrading Enzyme (IDE), glycogen synthase kinase 3beta and p62, which are involved in Amyloid beta degradation and PSD‐95, Arc proteins essential for synaptic maturity and plasticity. Resveratrol treatment attenuated the lipid peroxide levels, up‐regulated the antioxidant activities and increased the expression of proteins involved in neuroprotection in H19‐7 cells. These findings suggest the neuroprotective effect of resveratrol in improving the β‐ amyloid induced oxidative damage and memory loss in vitro.