Cell Line HT22 Research Articles

Agomelatine protects hippocampal neurons against doxorubicin induced toxicity

Doxorubicin (DOX) is a powerful chemotherapy drug used in numerous cancer treatments, known to causes neurotoxicity and impair cognitive function in cancer patients. The main mechanism thought to be responsible for the neuronal damage related to chemotherapy is oxidative stress. Agomelatine (AGM) has recently been identified as a neuroprotective agent against toxicity and the effect of AGM on DOX-induced neurotoxicity was investigated in this study. In order to evaluate the effect of AGM on oxidative stress and apoptosis, malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and Caspase-3 expression were analyzed in the HT-22 cell line. AGM considerably reduced oxidative damage due to DOX in vitro. MDA and Caspase-3 were considerably higher in the DOX group. These values were dramatically decreased by AGM. AGM greatly increased the CAT and SOD levels when compared to the DOX group. These results suggest that DOX therapy alters antioxidant status in hippocampus, a critical cognition-related region. So if we are aware of the functional effects of DOX, it is possible to avoid some of the disabling side effects of chemotherapy in cancer patients and may prevent these effects with antioxidant agents such as AGM

Effects of mitragynine on viability, proliferation, and migration of C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 immortalized mouse hippocampal neuron cell lines

Mitragynine (MG) is an indole alkaloid found in the extract of Mitragyna speciosa Korth native to Southeast Asia. Although MG is known for its pain-relieving and psychoactive effects, reports have suggested that it has therapeutic potential against neoplasms and psychiatric disorders. However, no evidence currently exists to support the effect of MG on brain tumors. This study aimed to investigate the antitumor effects of MG in C6 rat glioma and SH-SY5Y human neuroblastoma tumor cell lines compared with those in the non-tumor HT22 mouse hippocampal neuronal cell line. MTT assay for cell viability, clonogenic and wound healing assays for cell migration, Hoechst 33342/propidium iodide staining for nuclear morphology, and cell cycle distribution using flow cytometry were performed. MG at 125.47 μM (50 μg/ml) significantly reduced the viability of all cell lines, and the clonogenicity of C6 glioma cells began decreasing at 75.28 μM (30 μg/ml) of MG. Cell migration was inhibited in C6 and HT22 cells treated with 75.28 μM (30 μg/ml) of MG. Apoptotic nuclear condensation and fragmentation were observed in all cell lines treated with 125.47 μM (50 μg/ml) MG, whereas late-phase apoptotic cells were predominant in the group treated with 250.94 μM (100 μg/ml) of MG. The cell cycle assay results suggest that MG arrested the S phase in the C6 cell line and the G2/M phase in the HT22 cell lines. This study showed that MG induces cell death and cell cycle arrest, disrupting cell migration and reducing the clonogenicity of brain tumor cells.

Epigenome-wide DNA methylation analysis of myasthenia gravis.

Myasthenia gravis (MG) is a common neuromuscular junction (NMJ) disorder and autoimmune disease mediated by several antibodies. Several studies have shown that genetic factors play an important role in MG pathogenesis. To gain insight into the epigenetic factors affecting MG, we report here genome-scale DNA methylation profiles of MG. DNA was extracted from 8 MG patients and 4 healthy controls for genome-wide DNA methylation analysis using the Illumina HumanMethylation 850K BeadChip. Verification of pyrosequencing was conducted based on differential methylation positions (DMPs). Subsequently, C2C12 and HT22 cell lines (derived from mouse) were treated with demethylation drugs. Transcribed mRNA of the screened differential genes were detected using quantitative Real-time PCR. The control and MG group were compared, and two key probe positions were selected. The corresponding genes were CAMK1D and CREB5 (P<0.05). Similarly, the myasthenic crisis (MC) and non-MC group were compared and four key probe positions were selected. The corresponding genes were SAV1, STK3, YAP1 and WWTR1 (P<0.05). Subsequently, pyrosequencing was performed for verification, revealing that hypomethylation of CAMK1D was significantly different between the MG and control group (P<0.001). Moreover, transcription of CREB5, PKD, YAP1 and STK3 genes in the C2C12 cells was downregulated (P<0.05) after drug treatment, but only YAP1 mRNA was downregulated in HT22 cells (P<0.05). This is the first study to investigate genome-scale DNA methylation profiles of MG using 850K BeadChip. The identified molecular markers of methylation may aid in the prevention, diagnosis, treatment and prognosis of MG.

Neuroprotective carbazole alkaloids from Streptomyces sp. HS-NF-1322 by inhibition of ferroptosis

Three rare new carbazole alkaloids, antiostatin A7 (1), ( ± )-morindolestatin B (2) and morindolestatin C (3), were obtained from a fermentation broth of the soil-derived Streptomyces sp. HS-NF-1322 strain. Their structures were assigned by various spectroscopic techniques including HR-ESI-MS and 2D NMR. These new isolates, together with ( ± )-morindolestatin (4), were assayed for their neuroprotective effects on erastin-induced ferroptosis on the PC12 and HT-22 cell lines. All of these alkaloids significantly improved cell viability, and reduced ROS production of these neuronal cells. Notably, compound 1 dramatically protected PC12 cells from ferroptosis with a higher viability than the positive drug deferoxamine at 10 μM.

Paeoniae Radix Rubra extract attenuates cerebral ischemia injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway

Ethnopharmacological relevancePaeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. Aim of the studyThe present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. Materials and methodsThe neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. ResultsThe in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. ConclusionPRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

Taohong siwu decoction ameliorates cognitive dysfunction through SIRT6/ER stress pathway in Alzheimer's disease

Ethnopharmacological relevanceA growing number of people suffer from Alzheimer's disease (AD), but there is currently no effective treatment yet. Taohong Siwu Decoction (TSD) has been proved to take strong neuropharmacological activity on dementia, but the effect and mechanism of TSD against AD are still elusive. Aim of studyTo investigate whether TSD could be effective in ameliorating cognitive deficits through SIRT6/ER stress pathway. Materials and methodsHerein, the APP/PS1 mice, an AD model, and HT-22 cell lines were utilized. Different dosages of TSD (4.25, 8.50 and 17.00 g/kg/d) were administered to the mice for 10 weeks by gavage. Following the behavioral tests, oxidative stress levels were measured using malondialdehyde (MDA) and superoxide dismutase (SOD) kits. Nissl staining and Western blot analyses were used to detect the neuronal function. Then, immunofluorescence and Western blot analysis were applied to evaluate silent information regulator 6 (SIRT6) and ER Stress related protein levels in APP/PS1 mice and HT-22 cells. ResultsBehavioral tests revealed that APP/PS1 mice administered with TSD orally took more time in the target quadrant, crossed more times in the target quadrant, had a higher recognition coefficient, and spent more time in the central region. In addition, TSD could ameliorate oxidative stress and inhibit neuronal apoptosis in APP/PS1 mice. Furthermore, TSD could up-regulate the SIRT6 protein expression and inhibit ER sensing proteins expressions, such as p-PERK and ATF6, in APP/PS1 mice and Aβ1-42-treated HT22 cells. ConclusionAccording to the abovementioned findings, TSD could alleviate cognitive dysfunction in AD by modulating the SIRT6/ER stress pathway.

Sevoflurane induces neurotoxic effects on developing neurons through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway.

Children repeatedly exposed to anaesthesia have a high risk of cognitive impairment, but the mechanism of its regulation in this context is unknown. The objective of this study was to investigate the possible toxic mechanism of sevoflurane through the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway. The hippocampal neuronal HT22 cell line was used in this study. The intervention group was treated with the WNK1 inhibitor WNK-463, CaN inhibitor FK506 and Drp-1 inhibitor Mdivi-1 respectively in the medium for 30 min before sevoflurane anaesthesia. The sevofluane group and all intervention group treated with 4.1% sevoflurane for 6h. Compared with the control group, sevoflurane treatment decreased cell viability and increased cellular apoptosis. Our study found that WNK-463, FK506 and Mdivi-1 can all alleviate the sevoflurane-induced reduction in cell viability, decrease the cell apoptosis. In addition, WNK-463 pretreatment could inhibit the increase of WNK1 kinase and NKCC1 protein concentration caused by sevoflurane. Further, sevoflurane anaesthesia causes intracellular calcium overload, increases the expression of CaN and induces the dephosphorylation of Drp-1 protein at ser637, while CaN inhibitor FK506 pretreatment could reduce the dephosphorylation of Drp-1. Therefore, the WNK1/NKCC1/Ca2+ /Drp-1 signalling pathway plays an important role in sevoflurane-related neurotoxicity. Reducing intracellular calcium influx may be one of the important mechanism to ameliorate sevoflurane toxicity.

LPS-Activated Microglial Cell-Derived Conditioned Medium Protects HT22 Neuronal Cells against Glutamate-Induced Ferroptosis.

Neuron-glia interactions are essential for the central nervous system's homeostasis. Microglial cells are one of the key support cells in the brain that respond to disruptions in such homeostasis. Although their participation in neuroinflammation is well known, studies investigating their role in ferroptosis, an iron-dependent form of nonapoptotic cell death, are lacking. To address this issue, we explored whether microglial (BV-2 cells) activation products can intensify, mitigate or block oxidative and/or ferroptotic damage in neuronal cells (HT22 cell line). Cultured BV-2 microglial cells were stimulated with 5-100 ng/mL lipopolysaccharide (LPS) for 24 h and, after confirmation of microglial activation, their culture medium (conditioned media; CM) was transferred to neuronal cells, which was subsequently (6 h later) exposed to glutamate or tert-butyl hydroperoxide (t-BuOOH). As a major finding, HT22 cells pretreated for 6 h with CM exhibited a significant ferroptosis-resistant phenotype characterized by decreased sensitivity to glutamate (15 mM)-induced cytotoxicity. However, no significant protective effects of LPS-activated microglial cell-derived CM were observed in t-BuOOH (30 µM)-challenged cells. In summary, activated microglia-derived molecules may protect neuronal cells against ferroptosis. The phenomenon observed in this work highlights the beneficial relationship between microglia and neurons, highlighting new possibilities for the control of ferroptosis.

Antioxidative polyphenols attenuate pyocyanin-induced ROS production in neuronal HT22 cell lines.

Pyocyanin, a secreted virulence factor, plays an essential role during Pseudomonas aeruginosa infection. Infection of the central nervous system by this bacterium results in high mortality, but the studies on its mechanism are still rather limited. In this study, we first evaluate the neuronal damage caused by pyocyanin exposure in neuronal HT22 cells. Pyocyanin leads to mitochondrial syndrome and antioxidant defense disruption, therefore increasing intercellular reactive oxygen species (ROS) production. Several typical superior antioxidant polyphenols effectively protect against pyocyanin-induced neuronal cell damage. These findings suggest the neuronal protective activity more or less relies on the structure, rather than the residues. Pre-incubation of catechin activates the essential pathway, indicating inverse correlation of ERK and AMPK phosphorylation participates in this process. These data outline a novel strategy to eliminate intracellular generated ROS. The investigated candidates could be potentially used as therapeutic agents against various ROS-related neurological diseases.

Neuroprotective and Anti-Neuroinflammatory Properties of Vignae Radiatae Semen in Neuronal HT22 and Microglial BV2 Cell Lines.

The important factors in the pathogenesis of neurodegenerative disorders include oxidative stress and neuron-glia system inflammation. Vignae Radiatae Semen (VRS) exhibits antihypertensive, anticancer, anti-melanogenesis, hepatoprotective, and immunomodulatory properties. However, the neuroprotective effects and anti-neuroinflammatory activities of VRS ethanol extract (VRSE) remained unknown. Thus, this study aimed to investigate the neuroprotective and anti-inflammatory activities of VRSE against hydrogen peroxide (H2O2)-induced neuronal cell death in mouse hippocampal HT22 cells and lipopolysaccharide (LPS)-stimulated BV2 microglial activation, respectively. This study revealed that VRSE pretreatment had significantly prevented H2O2-induced neuronal cell death and attenuated reactive oxygen species generations in HT22 cells. Additionally, VRSE attenuated the apoptosis protein expression while increasing the anti-apoptotic protein expression. Further, VRSE showed significant inhibitory effects on LPS-induced pro-inflammatory cytokines in BV2 microglia. Moreover, VRSE pretreatment significantly activated the tropomyosin-related kinase receptor B/cAMP response element-binding protein, brain-derived neurotrophic factor and nuclear factor erythroid 2-related factor 2, and heme oxygenase-1 signaling pathways in HT22 cells exposed to H2O2 and inhibited the activation of the mitogen-activated protein kinase and nuclear factor-κB mechanism in BV2 cells stimulated with LPS. Therefore, VRSE exerts therapeutic potential against neurodegenerative diseases related to oxidative stress and pathological inflammatory responses.

Autophagy response in chronic neuronal oxidative stress

AbstractAge‐associated neurodegenerative diseases like Alzheimer's and Parkinson's disease (PD) but also glaucoma is generally accompanied by intracellular stress, malignant accumulations of dysfunctional proteins and a disturbance of the proteostasis. Over time adaptation and increased resilience are measures to make neurons more stable against age‐ and disease‐related challenges. The hippocampal cell line HT22 has been used to illustrate the adaptive ability in response to oxidative stress by enhancing autophagic activity via especially the process of BAG3‐mediated selective macroautophagy.We extended the resistance protocol and generated a novel HT22 cell line that is resistant to thapsigargin, depleting intracellular Ca2+ stores and a potent inducer of ER stressor.We observed a significantly increased LC3B‐II and p62 flux in thapsigargin‐resistant (TgR) compared with HT22‐WT | ells at both, basal and starved conditions. Intriguingly, when looking at the process of ER‐phagy manifold changes are occurring in TgR cells.We suggest that the observed changes in autophagy‐related process, including ER‐phagy, may promote resistance against age‐related Ca2+ disturbance and Ca2+ stress in neuronal cells. Taken together, neuronal cell lines selected for a particular resistance allow molecular insights into neuronal adaptability.

β‐Lactolin Improves Mitochondrial Dysfunction in Aβ‐treated Mouse Hippocampal Neuronal Cells and a Human iPSC‐Derived Neuronal Cell Model of Alzheimer’s Disease

AbstractBackgroundMitochondrial dysfunctions are a key hallmark of Alzheimer’s disease (AD). β‐Lactolin, a whey‐derived glycine–threonine–tryptophan–tyrosine tetrapeptide, has been previously reported to prevent AD‐like pathologies in an 5×FAD mouse model via regulation of microglial functions. However, the direct effect of β‐lactolin on neuronal cells and neuronal mitochondrial functions remains unknown. Here, we investigated the effects of β‐lactolin on mitochondrial dysfunctions in amyloid β (Aβ)‐treated mouse hippocampal neuronal HT22 cells and human induced‐pluripotent cell (hiPSC)‐derived AD model neurons.MethodMouse hippocampal neuronal HT22 cell line, and hiPSC‐derived AD model neuron (PSEN1 P117L mutation) and its wild‐type control were used in this study. HT22 cells were treated with β‐lactolin (1–100 nM) for 1 hr, and Aβ42 (1–10 μM) was added for the next 23 hrs. Oxygen consumption rate was measured using extracellular flux analyzer. Intracellular ATP level was determined by luciferase activity. Cells were stained with MitoTracker, JC‐1, or MitoSOX fluorescent dye, and then mitochondrial morphologies, membrane potential, and oxidative stress, respectively, were examined by high content image analysis. Cell viability was measured by MTT assay. Gene expression levels were evaluated by RT‐PCR. hiPSC‐derived wild‐type and AD neurons were cultured for 14 days under β‐lactolin treatment, and mitochondrial morphologies and membrane potentials were analyzed.ResultHT22 cells treated with both β‐lactolin and Aβ exhibited increased oxygen consumption rate and cellular ATP concentrations compared to cells treated with Aβ alone, suggesting that β‐lactolin improves mitochondrial respiration and energy production. In high content image analysis, β‐lactolin attenuated Aβ‐induced mitochondrial fragmentation, membrane potential decrease, and excess oxidative stress, eventually preventing neuronal cell death. Treatments with β‐lactolin increased gene expression of mitofusin‐2, which contribute to mitochondrial fusion events. Finally, β‐lactolin attenuated impairment in both mitochondrial morphologies and membrane potentials in hiPSC‐derived AD model neurons, suggesting β‐lactolin shows a suppressing effect on human AD pathologies.Conclusionβ‐lactolin improved AD‐related neuronal mitochondrial dysfunctions and suppressed neuronal cell death in both mouse and human AD model neuronal cells. The dual function of β‐lactolin on both neuron and microglia marks an advantage in AD prevention.

MiR-29c Inhibits TNF-α-Induced ROS Production and Apoptosis in Mouse Hippocampal HT22 Cell Line.

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.

Serotonin Type 3 Receptor Is Potentially Involved in Cellular Stress Induced by Hydrogen Peroxide.

Depression is a disease with several molecular mechanisms involved, such as problems in the serotonergic pathway. This disease is very complex and prevalent, and thus important to deeply study and aim to overcome high rates of relapse and therapeutic failure. In this study, two cellular lines were used (HT-22 and SH-SY5Y cells) to gain insight about the role of the serotonin type 3 (5-HT3) receptor in cellular stress induced by hydrogen peroxide and/or corticosterone. In research, these compounds are known to mimic the high levels of oxidative stress and dysfunction of the hypothalamus–hypophysis–adrenal axis by the action of glucocorticoids, usually present in depressed individuals. The receptor 5-HT3 is also known to be involved in depression, previously demonstrated in studies that highlight the role of these receptors as promising targets for antidepressant therapy. Indeed, the drugs used in this work (mirtazapine, scopolamine, and lamotrigine) interact with this serotonergic receptor. Thus, by using cell morphology, cell viability (neutral red and MTT), and HPLC assays, this work aimed to understand the role of these drugs in the stress induced by H2O2/corticosterone to HT-22 and SH-SY5Y cell lines. We concluded that the antagonism of the 5-HT3 receptor by these drugs may be important in the attenuation of H2O2-induced oxidative stress to the cells, but not in the corticosterone-induced stress.

Microglial exosomal miR-466i-5p induces brain injury via promoting hippocampal neuron apoptosis in heatstroke.

BackgroundBrain injury is the main cause of poor prognosis in heatstroke (HS) patients due to heat-stress-induced neuronal apoptosis. However, as a new cross-talk way among cells, whether microglial exosomal-microRNAs (miRNAs) are involved in HS-induced neuron apoptosis has not been elucidated.MethodsWe established a heatstroke mouse model and a heat-stressed neuronal cellular model on HT22 cell line. Then, we detected neuron apoptosis by histopathology and flow cytometry. The microglial exosomes are isolated by standard differential ultracentrifugation and characterized. Recipient neurons are treated with the control and HS exosomes, whereas in vivo, the exosomes were injected into the mice tail vein. The internalization of HS microglial exosomes by neurons was tracked. Apoptosis of HT22 was evaluated by flow cytometry and Western blot in vitro, TUNEL assay, and immunohistochemistry in vivo. We screened miR-466i-5p as the mostly upregulated microRNAs in HS exosomes by high-throughput sequencing and further conducted gene ontology (GO) pathway analysis. The effect and mechanism of HS exosomal miR-466i-5p on the induction of neuron apoptosis are demonstrated by nasal delivery of miR-466i-5p antagomir in vivo and transfecting miR-466i-5p mimics to HT22 in vitro.ResultsHS induced an increase in neurons apoptosis. Microglial exosomes are identified and taken up by neurons, which induced HT22 apoptosis in vivo and vitro. HS significantly changed the miRNA profiles of microglial exosomes based on high-throughput sequencing. We selected miR-466i-5p as a target, and upregulated miR-466i-5p induced neurons apoptosis in vivo and vitro experiments. The effects are exerted by targeting Bcl-2, activating caspase-3 to induce neurons apoptosis.ConclusionsWe demonstrate the effect of microglial exosomal miR-466i-5p on neurons apoptosis and reveal potentially Bcl-2/caspase-3 pathway in heatstroke.

Transcriptome analysis in an AEG‑1‑deficient neuronal HT22 cell line.

Astrocyte elevated gene-1 (AEG-1) is a key regulatory factor of progression in multiple types of tumor and neurodegenerative disease development. AEG-1 is associated with glutamate excitotoxicity due to its reported function of repressing excitatory amino acid transporter 2 expression in astrocytes. Although the function of AEG-1 has been demonstrated in neurological disorders, such as Alzheimer's disease and amyotrophic lateral sclerosis, the underlying mechanism of neuronal AEG-1 function remains unclear. The aim of the present study was to clarify the function and related mechanism of AEG-1 in neurons. A stable AEG-1-deficient HT22 neuronal cell line was constructed using CRISPR/Cas9 gene-editing technology. Reverse transcription-quantitative PCR and western blotting were carried out to analyze the knockdown efficiency of AEG-1-deficient HT22 cell line. RNA Sanger sequencing analysis was performed in AEG-1-deficient HT22 cells and wild-type HT22 cells without knockout (KO). Results from RNA sequencing revealed that AEG-1 modulated neuronal morphology and development by regulating the expression of numerous genes, such as ubiquitin C, C-X-C motif chemokine ligand 1, MMP9, Notch1, neuropilin 1 and ephrin type-A receptor 4. In addition, AEG-1 deficiency impacted several signaling pathways by mediating cell survival differentiation, apoptosis, and migration; this included the TNF-α pathway, the NF-κB pathway, the MAPK signaling pathway, the Notch signaling pathway and Axon guidance. Downregulation in cellular ion homeostasis, including ion channel function and neurotransmitter release, were observed after knocking out AEG-1 expression. Collectively, the present study provides insights into AEG-1-dependent gene regulation and signaling pathway transduction in neurons. The results of the present study may be applied for improving the understanding of AEG-1-associated central nervous system diseases.

Effects of 5-Aza on neurogenesis contribute to learning and memory in the mouse hippocampus

Background5-Aza-2′-deoxycytidine (5-Aza-CdR) is a demethylating agent that has various biological effects related to DNA methylation. DNA methylation plays important roles in learning and memory. We have reported that 5-Aza-CdR improved the performance of mice in the water maze and step-down tests. Some behaviours have been well recognized to be mediated by neurogenesis in the hippocampus. The Notch signalling pathway plays a key role in adult hippocampal neurogenesis. In this study, we examined whether 5-Aza-CdR (DNA methyltransferase inhibitor) affects neurogenesis and Notch1 expression. MethodsThe learning and memory behaviour of mice was evaluated by a conditioned avoidance learning 24 h after 5-Aza-CdR treatment. The mRNA and protein expression levels of Notch1 and HES1 were measured by real-time PCR and Western blotting. The 5-bromo-2′-deoxyuridine (BrdU)-positive cells and the expression of Notch1 in the hippocampal DG were observed through laser confocal microscopy. To further clarify whether 5-Aza-CdR affects behaviour through neurogenesis, the expression level of Notch1, cell viability and cell cycle were analysed using the HT22 cell line. ResultsThe behaviour in conditioned avoidance learning was improved, while neurogenesis and the Notch1 pathway were increased in the hippocampus of mice that were injected with 5-Aza-CdR. In vitro experiments showed that 5-Aza-CdR increased the expression of the Notch1 pathway and upregulated S-phase in the cell cycle and cell viability. ConclusionsOur results suggest that the effect of 5-Aza-CdR on behaviour may be related to an increase in neurogenesis with upregulation of the Notch1 pathway in the hippocampus.

Up-and-coming anti-epileptic effect of aloesone in Aloe vera: Evidenced by integrating network pharmacological analysis, in vitro, and in vivo models.

Background: Aloe vera is a medically valuable plant with anti-epileptic activity; however, its mechanism of action remains unknown. In this study, network pharmacological, in vitro, and in vivo experiments were carried out to explore the potential anti-epileptic components and targets of Aloe vera. Methods: The main active components of Aloe vera were identified by searching the Traditional Chinese Medicine System Pharmacology database. Targets of Aloe vera were predicted using SwissTargetPrediction, whereas information about the epilepsy disease targets was obtained from Gene Cards. The protein–protein interaction network and core targets were screened according to the topological structure and CytoNCA plugin. The glutamate-induced HT22 cell line and pentylenetetrazol-induced seizure rats were used to confirm the effect of aloesone by detecting reactive oxygen species (ROS) and apoptosis, and predicting the targets. Results: A total of 14 core active components were selected based on the screening criteria of oral bioavailability ≥30% and drug-likeness ≥ 0.10. Four compounds, namely linoleic acid, aloesone, isoeleutherol glucosiden qt, and anthranol, demonstrated the potential ability of crossing the blood-brain barrier. A total of 153 targets associated with epilepsy were predicted for the four compounds. Moreover, after network analysis with CytoNCA, 10 targets, namely, MAPK1, SRC, MARK3, EGFR, ESR1, PTGS2, PTPN11, JAK2, PPKCA, and FYN, were selected as the core genes, and SRC, which has been predicted to be the target of aloesone and anthranol, exhibited the highest subgraph centrality value. In vitro experiments confirmed that aloesone treatment significantly inhibited the glutamate-induced neuronal injury by reducing the intracellular ROS content and the early phase of apoptosis. Additionally, treatment with 50 mg/kg aloesone resulted in anti-seizure effects by reducing the seizure score and prolonging the latent period in acute and chronic rats. Furthermore, aloesone treatment increased the phosphorylation of c-SRC at Y418 and reduced the phosphorylation at Y529, simultaneously activating c-SRC. Conclusion: Integrating network pharmacology with in vitro and in vivo experiments demonstrated that aloesone, which inhibited seizure by activating c-SRC, is a potential anti-seizure compound present in Aloe vera.

LONG NONCODING RNA UPREGULATES ADAPTER SHCA PROTEIN EXPRESSION TO PROMOTE COGNITIVE IMPAIRMENT AFTER CARDIAC ARREST AND RESUSCITATION.

Aim: More patients are resuscitated from cardiac arrest and cardiopulmonary resuscitation (CA/CPR) due to advances in medical care. However, the burden now lies with post-cardiac arrest cognitive impairment in CA/CPR survivors. Based on our previous study, we aimed to further confirm the correlation between the long noncoding RNA-promoting ShcA (lncRNA-PS)/Src homology and collagen A (ShcA) axis and CA/CPR-induced cognitive impairment in molecular, cellular, and tissue levels. Methods and Results: The in vivo experiments were based on a mouse model of CA/CPR, while oxygen-glucose deprivation and reoxygenation was used as a cell model in vitro. Conditional ShcA suppression in neurons of the hippocampal CA1 region was achieved by cyclization recombinase of bacteriophage P1 recognizing DNA fragment locus of x-over P1 site (Cre/LoxP recombination system). Genetic manipulation of HT22 was achieved by lentivirus targeting lncRNA-PS and ShcA. Neurological function score was remarkably decreased, and cognitive function was affected after restoration of spontaneous circulation. LncRNA-PS and ShcA overexpression after CA/CPR, mainly happened in neurons of hippocampal CA1 region, was observed by in situ hybridization and immunofluorescence. Neuronal ShcA knockdown in hippocampal CA1 region before CA/CPR attenuated cognitive impairment after CA/CPR. ShcA deficiency protected HT22 cell line against oxygen-glucose deprivation and reoxygenation by inhibiting inflammation and apoptosis. In vitro upregulation of lncRNA-PS elevated ShcA expression, which was reversed by knockdown of ShcA. Conclusions: This study revealed that lncRNA-PS/ShcA axis is critically involved in the pathogenesis of cognitive impairment after CA/CPR. By inhibiting ShcA expression in neurons of the hippocampal CA1 region could improve the survival outcomes in mice after CA/CPR.

Rhoifolin attenuates damage to hippocampal neuronal culture model of acquired epilepsy in vitro by regulating NF-κB/iNOS/COX-2 axis

To assess the effect of Rhoifolin (ROF [apigenin 7-O-enneohesperidoside]) on the damage to hippocampal neu-ronal culture model of acquired epilepsy (AE) and investigate its possible mechanisms. A hippocampal neuronal culture model of AE was established through incubating HT-22 cells with MgCl2 free medium. 3-(4,5-Dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the effect of ROF on cell viability and apoptosis exposed to epilepsy. The oxidative stress and secretion of inflammatory cytokines were measured by reverse transcription-quantitative polymerase chain reaction and enzyme-linked-immunosorbent serologic assay, respectively. Immunoblot assays were performed to determine the protein expression levels of nuclear factor kappa B/nitric oxide synthases/cyclooxygenase-2 (NF-κB/iNOS/COX-2) axis. ROF increases viability and reduces apoptosis of AE medium-treated HT-22 cell line. ROF relieves oxidative stress in AE medium-treated HT-22 cell line. ROF decreases the levels of pro-inflammatory cytokines in AE medium-treated HT-22 cell line. The functional effects of ROF on AE medium-treated HT-22 cell line is through inhibiting NF-κB/iNOS/COX-2 axis. ROF increased viability, decreased apoptosis, suppressed oxidative stress, and reduced pro-inflammatory cytokine levels in an epilepsy model in vitro by inhibiting NF-κB/iNOS/COX-2 axis. ROF might serve as a potential drug for epilepsy treatment.