Hippocampal Neuronal HT22 Cells Research Articles

Indirubin derivatives protect against endoplasmic reticulum stress-induced cytotoxicity and down-regulate CHOP levels in HT22 cells

Indirubin and its derivatives have been reported to exhibit anti-cancer and anti-inflammatory activities. Recently, some of its derived analogs have been shown to have neuroprotective potential. Endoplasmic reticulum (ER) stress has been demonstrated to contribute to the pathogenesis of various neurodegenerative diseases, whereas the effects of indirubin derivatives on ER stress-induced cell death have not been addressed. In the present study, a series of 44 derivatives of indirubin was prepared to search for a novel class of neuroprotective agents against ER stress-induced neuronal death. The MTT reduction assay indicated that tunicamycin (TM), an inducer of ER stress, significantly decreased the viability of hippocampal neuronal HT22 cells. Among the compounds tested, eight showed significant inhibitory activity against TM-induced cell death. Western blot analysis showed that application of these analogs to the cells simultaneously with TM reduced the TM-induced expression of CHOP, an established mediator of ER stress. Our results suggest that the preventive effect of these indirubin derivatives against ER stress-induced neuronal death may be due, at least in part, to attenuation of the CHOP-dependent signaling system.

Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke.

Reducing agent-free synthesis of curcumin-loaded albumin nanoparticles by self-assembly at room temperature

The purpose of this study was to prepare curcumin-loaded bovine serum albumin nanoparticles (CCM-BSA-NPs) by reducing agent-free self-assembly at room temperature. A 24 factorial design approach was used to investigate the CCM-BSA-NP preparation process at different pH values, temperatures, dithiothreitol amounts, and CCM/BSA mass ratios. Increasing the ionic strength enabled preparation of CCM-BSA-NPs at 25°C without reducing agent. CCM-BSA-NPs prepared under the optimized conditions at 25°C showed a particle size of 110±6nm, yield of 88.5%, and drug loading of 7.1%. The CCM-BSA-NPs showed strong antioxidant activity and neuroprotective effects in glutamate-induced mouse hippocampal neuronal HT22 cells. This study suggests that ionic strength can be a key parameter affecting the preparation of albumin-based NPs.

Structural characterization and biological properties of a new dinuclear oxidovanadium(IV) N-(phosphonomethyl)iminodiacetate complex with the 4-amino-2-methylquinolinium cation

The crystal and molecular structures of a new dinuclear oxidovanadium(IV) complex with the N-(phosphonomethyl)iminodiacetate (pmida) ligand and the 4-amino-2-methylquinolinium ([amqH]+) cation of the molecular formula [amqH]4[V2O2(pmida)2]6H2O have been determined. The phosphonate groups in the two pmida4− ligands form two bridges between adjacent V4+ atoms, resulting in the formation of a [V2O2(pmida)2]4− dimer with a V2O4P2 eight-membered ring. This is the first example of a quinoline derivative complex containing the [V2O2(pmida)2]4− ion. The susceptibility curve for the complex exhibits a maximum at approximately 10K, indicating the presence of antiferromagnetic interactions transmitted in the crystal lattice. Furthermore, the biological properties of the complex in the concentration range 1–100μM were investigated in relation to its cytoprotective activity against oxidative damage generated exogenously using hydrogen peroxide in the hippocampal neuronal HT22 cell line (MTT tests). The obtained results were subsequently referred to the [amqH][VO(nta)(H2O)]H2O analogue as well as trolox and ascorbic acid, used as known antioxidants. It has been established that the title compound effectively protects HT22 from oxidative damage and is a potentially good candidate for further evaluation of the mechanism of its action.

Oleuropein isolated from Fraxinus rhynchophylla inhibits glutamate-induced neuronal cell death by attenuating mitochondrial dysfunction

Glutamate-induced neurotoxicity is related to excessive oxidative stress accumulation and results in the increase of neuronal cell death. In addition, glutamate has been reported to lead to neurodegenerative diseases, including Parkinson's and Alzheimer's diseases.It is well known that Fraxinus rhynchophylla contains a significant level of oleuropein (Ole), which exerts various pharmacological effects. However, the mechanism of neuroprotective effects of Ole is still poorly defined. In this study, we aimed to investigate whether Ole prevents glutamate-induced toxicity in HT-22 hippocampal neuronal cells. The exposure of the glutamate treatment caused neuronal cell death through an alteration of Bax/Bcl-2 expression and translocation of mitochondrial apoptosis-inducing factor (AIF) to the cytoplasm of HT-22 cells. In addition, glutamate induced an increase in dephosphorylation of dynamin-related protein 1 (Drp1), mitochondrial fragmentation, and mitochondrial dysfunction. The pretreatment of Ole decreased Bax expression, increased Bcl-2 expression, and inhibited the translocation of mitochondrial AIF to the cytoplasm. Furthermore, Ole amended a glutamate-induced mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria, regulating the phosphorylation of Drp1 at amino acid residue serine 637. In conclusion, our results show that Ole has a preventive effect against glutamate-induced toxicity in HT-22 hippocampal neuronal cells. Therefore, these data imply that Ole may be an efficient approach for the treatment of neurodegenerative diseases.

Structure, Physicochemical and Biological Properties of an Aqua (2,2′,2′′‐Nitrilotriacetato)‐oxidovanadium(IV) Salt with 4‐Methylpyridinium Cation

The crystal structure of a nitrilotriacetate (nta) oxidovanadium(IV) salt with 4‐methylpyridinium cation, [4‐Me(Py)H]+, of [4‐Me(Py)H][VO(nta)(H2O)] stoichiometry was determined. The complex comprises a discrete mononuclear [VO(nta)(H2O)]– coordination entity that can be rarely found among other known compounds containing nitrilotriacetate oxidovanadium(IV) moieties. The complex was characterized by spectroscopic (IR and EPR) methods, magnetic measurements, and thermogravimetry (TG‐FTIR). The stability of the title compound in aqueous solutions was investigated by using the potentiometric titration method. Furthermore, spectrophotometric (UV/Vis) studies have revealed that the compound is capable to scavenge the superoxide free radicals (O2•–) as well as stable organic radicals i.e. 2,2′‐azinobis(3‐ethylbenzothiazoline‐6 sulfonic acid) cation radical (ABTS+•) and 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH•). Finally, biological properties of the complex studied were investigated in relation to its cytoprotective activity against the oxidative damage generated exogenously by using hydrogen peroxide in the HT22 hippocampal neuronal cell line (the MTT assay). Additionally, the biological action of the compound towards two human osteosarcoma HOS and MG‐63 cell lines (the MTT and BrdU tests) as well as the untransformed human osteoblast hFOB 1.19 cell line was tested.

Cleistocalyx nervosum var. paniala berry fruit protects neurotoxicity against endoplasmic reticulum stress-induced apoptosis

Oxidative and endoplasmic reticulum (ER) stresses cause neuronal damage leading to neurodegenerative disorders. Cleistocalyx nervosum var. paniala (CNP) berry fruit has been shown to possess powerful antioxidant properties. Here, we investigated the neuroprotective effect of CNP extract against glutamate-mediated oxidative/ER stress-induced cell death in mouse hippocampal neuronal HT22 cells. CNP extract was clarified for its radical scavenging activities, total phenolic and anthocyanin contents. The key anthocyanin cyanidin-3-glucoside was used as a marker to standardize the extract used in the study. We found that pretreated cells with CNP extract (0.05–1 μg/ml) prevented neuronal cell death in response to 5 mM glutamate evaluated by cell viability MTT, LDH and apoptosis/necrosis Annexin V/propidium iodide co-staining assays. For mechanistic approach, glutamate-induced cell death through reactive oxygen species (ROS)-mediated ER stress pathways, indicating the increase of ROS and ER stress signature molecules including calpain, caspases-12 and C/EBP homologous proteins (CHOP). CNP extract inhibited ROS production. Moreover, the extract also suppressed the specific-ER stress apoptotic proteins level in glutamate-induced cells by upregulating the gene expression of cellular antioxidant enzymes (SODs, CAT, GPx and GSTs). Taken together, our results provide information about and the molecular mechanism of CNP extract as a promising neuroprotectant and antioxidant.

Selenium suppresses glutamate-induced cell death and prevents mitochondrial morphological dynamic alterations in hippocampal HT22 neuronal cells

BackgroundPrevious studies have indicated that selenium supplementation may be beneficial in neuroprotection against glutamate-induced cell damage, in which mitochondrial dysfunction is considered a major pathogenic feature. However, the exact mechanisms by which selenium protects against glutamate-provoked mitochondrial perturbation remain ambiguous. In this study glutamate exposed murine hippocampal neuronal HT22 cell was used as a model to investigate the underlying mechanisms of selenium-dependent protection against mitochondria damage.ResultsWe find that glutamate-induced cytotoxicity was associated with enhancement of superoxide production, activation of caspase-9 and -3, increases of mitochondrial fission marker and mitochondrial morphological changes. Selenium significantly resolved the glutamate-induced mitochondria structural damage, alleviated oxidative stress, decreased Apaf-1, caspases-9 and -3 contents, and altered the autophagy process as observed by a decline in the ratio of the autophagy markers LC3-I and LC3-II.ConclusionThese findings suggest that the protection of selenium against glutamate stimulated cell damage of HT22 cells is associated with amelioration of mitochondrial dynamic imbalance.

Ginsenoside Rd promotes non-amyloidogenic pathway of amyloid precursor protein processing by regulating phosphorylation of estrogen receptor alpha

AimsPrevious study demonstrated that Ginsenoside Rd. (GS-Rd) could improve cognitive and memory function in animal model of Alzheimer's disease. This study was aimed to investigate whether GS-Rd could improve non-amyloidogenic pathway by activating estrogen receptor (ER). Main methods10mg/kg GS-Rd in ovariectomy (OVX)+GS-Rd group and equivalent volume of saline in sham operated group and OVX group were administrated intraperitoneally for two months, respectively. The Morris Water Maze was used to examine cognitive function of rats, with sAPPα and Aβ levels in the hippocampi measured. The culture medium of HT22 hippocampal neuronal cells were incubated with GS-Rd, ER antagonist ICI182.780, MAPK inhibitor PD98059, or PI3Kinhibitor LY294002, respectively. sAPPα levels was measured, and expression of α-secretase, sAPPα, β-secretase, Aβ, phosphorylation form of AKT (p-AKT), total AKT, p-ERK, total ERK, p-ERα, total ERα, p-ERβ and total ERβ were examined by Western blot to explore the estrogenic-like activity of GS-Rd. Key findingsGS-Rd attenuate cognitive and memory impairment, increased levels of sAPPα and reduced extracellular Aβ of OVX rats. In HT22, GS-Rd could upregulate sAPPα level, which can be inhibited by inhibitor of MAPK and PI3K pathway. In addition, inhibitor of estrogen receptor prevented GS-Rd triggered release of sAPPα and activation of MAPK and PI3K pathways. GS-Rd could increase expression of α-secretase and sAPPα, while decrease expression of β-secretase and Aβ. Besides, GS-Rd promoted phosphorylation of estrogen receptor alpha at Ser118 residue. SignificanceOur findings show that GS-Rd enhances learning and memory function of OVX rats by activating estrogen-like activity.

Mimetics of brain-derived neurotrophic factor loops 1 and 4 are active in a model of ischemic stroke in rats.

BackgroundTwo dimeric dipeptides, bis-(N-monosuccinyl-l-seryl-l-lysine)hexamethylenediamide (GSB-106) and bis-(N-monosuccinyl-l-methionyl-l-serine) heptamethylenediamide (GSB-214), were designed based on the brain-derived neurotrophic factor (BDNF) loop 4 and loop 1 β-turn sequences, respectively. Earlier, both of these dipeptides were shown to exhibit neuroprotective activity in vitro (10−5–10−8 M). The present study aimed to investigate the mechanisms of action of these peptides and their neuroprotective activity in an experimental stroke model.MethodsWe used western blot and HT-22 hippocampal neuronal cell line to investigate whether these peptides induced phosphorylation of the TrkB receptor and the AKT and ERK kinases. Rat middle cerebral artery occlusion (MCAO) was used as a stroke model. GSB-106 and GSB-214 were administered intraperitoneally (0.1 mg (1.3×10−7 mol)/kg) 4 hours after MCAO and daily for 7 days. The cerebral infarct volumes were measured with 2,3,5-triphenyltetrazolium chloride staining 21 days after MCAO.ResultsBoth compounds were shown to elevate the TrkB phosphorylation level while having different post-receptor signaling patterns. GSB-106 activated the PI3K/AKT and MAPK/ERK pathways simultaneously, whereas GSB-214 activated the PI3K/AKT only. In experimental stroke, the reduction of cerebral infarct volume by GSB-106 (∼66%) was significantly greater than that of GSB-214 (∼28% reduction), which could be explained by the fundamental role of the MAPK/ERK pathway in neurogenesis and neuroplasticity. Notably, between these two dipeptides, only GSB-106 exhibited antidepressant activity, as was found previously.ConclusionThe results provided support for the beneficial pharmacological properties of BDNF loop 4 mimetic GSB-106, thereby suggesting a potential role for this dipeptide as a therapeutic agent.

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Synthesis and evaluation of multifunctional ferulic and caffeic acid dimers for Alzheimer’s disease

In this study, a series of novel ferulic and caffeic acid dimers was designed and synthesised, and their multifunctional properties against Alzheimer’s disease (AD) were evaluated. Results showed that our multifunctional strategy was great supported by enhancing the inhibition of Aβ1–42 self-induced aggregation. Moreover, 7b also had potent protective effects against glutamate-induced cell death without significant cell toxicity in mouse hippocampal neuronal HT22 cells and 10c effectively scavenged diphenylpicrylhydrazyl free radicals. Collectively, these data strongly encourage further optimisation of 7b as a new hit to develop multifunctional agents for the treatment of AD.

Iron overload-induced calcium signals modulate mitochondrial fragmentation in HT-22 hippocampal neuron cells.

Iron is necessary for neuronal functions; however, excessive iron accumulation caused by impairment of iron balance could damage neurons. Neuronal iron accumulation has been observed in several neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Nevertheless, the precise mechanisms underlying iron toxicity in neuron cells are not fully understood. In this study, we investigated the mechanism underlying iron overload-induced mitochondrial fragmentation in HT-22 hippocampal neuron cells that were incubated with ferric ammonium citrate (FAC). Mitochondrial fragmentation via dephosphorylation of Drp1 (Ser637) and increased apoptotic neuronal death were observed in FAC-stimulated HT-22 cells. Furthermore, the levels of intracellular calcium (Ca(2+)) were increased by iron overload. Notably, chelation of intracellular Ca(2+) rescued mitochondrial fragmentation and neuronal cell death. In addition, iron overload activated calcineurin through the Ca(2+)/calmodulin and Ca(2+)/calpain pathways. Pretreatment with the calmodulin inhibitor W13 and the calpain inhibitor ALLN attenuated iron overload-induced mitochondrial fragmentation and neuronal cell death. Therefore, these findings suggest that Ca(2+)-mediated calcineurin signals are a key player in iron-induced neurotoxicity by regulating mitochondrial dynamics. We believe that our results may contribute to the development of novel therapies for iron toxicity related neurodegenerative disorders.

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI’s isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity.

Tat-NOL3 protects against hippocampal neuronal cell death induced by oxidative stress through the regulation of apoptotic pathways.

Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide(H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein(Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species(ROS), DNA fragmentation and reduced mitochondrial membrane potential(ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3and-8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury.

Folate deprivation modulates the expression of autophagy- and circadian-related genes in HT-22 hippocampal neuron cells through GR-mediated pathway

Folic acid (FA) is an extremely important nutrient for brain formation and development. FA deficiency is highly linked to brain degeneration and age-related diseases, which are also associated with autophagic activities and circadian rhythm in hippocampal neurons. However, little is known how autophagy- and circadian-related genes in hippocampal neurons are regulated under FA deficiency. Here, hippocampal neuroncells (HT-22) were employed to determine the effect of FA deprivation (FD) on the expression of relevant genes and to reveal the potential role of glucocorticoid receptor (GR). FD increased autophagic activities in HT-22 cells, associated with significantly (P<0.05) enhanced GR activation indicated by higher ratio of GR phosphorylation. Out of 17 autophagy-related genes determined, 8 was significantly (P<0.05) up-regulated in FD group, which includes ATG2b, ATG3, ATG4c, ATG5, ATG10, ATG12, ATG13 and ATG14. Meanwhile, 4 out of 7 circadian-related genes detected, Clock, Cry1, Cry2 and Per2, were significantly (P<0.05) up-regulated. The protein content of autophagy markers, LC3A and LC3B, was also increased significantly (P<0.05). ChIP assay showed that FD promoted (P<0.05) GR binding to the promoter sequence of ATG3 and Per2. Moreover, MeDIP analysis demonstrated significant (P<0.05) hypomethylation in the promoter sequence of ATG12, ATG13 and Per2 genes. Together, we speculate that FD increases the transcription of autophagy- and circadian-related genes through, at least partly, GR-mediated pathway. Our results provide a basis for future investigations into the intracellular regulatory network in response to folate deficiency.

Neuroprotective effects of pterostilbene against oxidative stress injury: Involvement of nuclear factor erythroid 2-related factor 2 pathway

Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) regulates multiple anti-oxidative enzymes and has neuroprotective effects. Pterostilbene (PTE) is a natural anti-oxidant found in blueberries. Its non-metabolized form exhibits high distribution in the brain after dietary administration. In this study, we aimed to explore the potential of PTE in protecting murine hippocampal neuronal HT22 cells against glutamate-induced oxidative stress injury and possible underlying mechanisms. PTE was nontoxic and induced the nuclear translocation of Nrf2 when HT22 cell cultures were incubated with different concentrations of PTE. Further, PTE displayed a dose-dependent neuroprotective effect, as indicated by increased cell viability and a reduction in lactate dehydrogenase (LDH) release after glutamate treatment. Nrf2 siRNA treatment inhibited PTE-induced neuroprotective effects. Moreover, the levels of nuclear Nrf2 and downstream heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) were elevated after PTE treatment. The PTE-induced elevation of nuclear Nrf2, as well as the increases in HO-1 and NQO1 levels, was abolished by Nrf2 siRNA. PTE treatment reduced the production of reactive oxygen species (ROS) and significantly enhanced the activities of the cellular anti-oxidants glutathione (GSH) and superoxide dismutase (SOD), indicating an attenuation of glutamate-induced oxidative stress. These changes in ROS and GSH and SOD activity were reversed by Nrf2 siRNA. Our results indicate that PTE treatment attenuates glutamate-induced oxidative stress injury in neuronal cells via the Nrf2 signaling pathway.

Influence of Primary Ligands (ODA, TDA) on Physicochemical and Biological Properties of Oxidovanadium (IV) Complexes with Bipy and Phen as Auxiliary Ligands.

The influence of the oxydiacetate (ODA) and thiodiacetate (TDA) ligands on the physicochemical and biological properties of the oxidovanadium(IV) ternary complexes of the VO(L)(B) type, where L denotes ODA or TDA and B denotes 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen), has been investigated. The stability of the complexes in aqueous solutions, assessed based on the potentiometric titration method, increases in the following direction: VO(TDA)(bipy)<VO(ODA)(bipy)<VO(TDA)(phen)<VO(ODA)(phen). Furthermore, the influence of the TDA and ODA ligands on the antioxidant activity of the oxidovanadium(IV) complexes toward superoxide free radical (O2•-), 2,2'-azinobis(3-ethylbenzothiazoline-6 sulfonic acid) cation radical (ABTS+•) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) has been examined and discussed. The reactivity of the complexes toward O2•- increases in the following direction: VO(TDA)(phen)<VO(TDA)(bipy)≈VO(ODA)(bipy)<VO(ODA)(phen). The antioxidant activity against ABTS+• and DPPH• free radicals is higher for phen complexes, whereas the thiodiacetate complexes are more reactive than are the corresponding oxydiacetate ones. Finally, herein, the cytoprotective activity of the complexes against the oxidative damage generated exogenously by hydrogen peroxide in the hippocampal neuronal HT22 cell line (the MTT and LDH tests) is reported. In a low concentration (1μM), the cytoprotective action of thiodiacetate complexes is much higher than that of the corresponding oxydiacetate complexes. However, in the higher concentration range (10 and 100μM), the antioxidant activity of the complexes is overcompensated by their cytotoxic effect.

Prolonged Effects of Silver Nanoparticles on p53/p21 Pathway-Mediated Proliferation, DNA Damage Response, and Methylation Parameters in HT22 Hippocampal Neuronal Cells

It is widely accepted that silver nanoparticles (AgNPs) are toxic to biological systems. However, little is known about their actions at molecular level and the cytophysiological effects after AgNP removal. As nanoparticles are suggested a promising tool to transport drugs to the brain for use in neurological conditions, we used HT22 mouse hippocampal neuronal cells as a model to study AgNP-mediated effects after their removal from the cell culture medium. We selected a relatively low concentration of AgNPs, 5 μg/ml, treated the cells for 48 h, and evaluated AgNP-induced cytophysiological effects after 96 h of AgNP removal. AgNP removal did not result in cytotoxicity. In contrast, AgNPs modulated HT22 cell cycle and proliferation and induced oxidative stress and 53BP1 recruitment, which were accompanied by elevated levels of p53 and p21. AgNP-associated diminution in lamin B1 pools did not significantly affect the structure of the nucleus. No disruption in F-actin dynamics was observed upon AgNP treatment. Moreover, we showed for the first time that AgNPs stimulated changes in DNA methylation: the augmentation in 5-methylcytosine (5-mC) and DNMT1, DNMT2, DNMT3a, and DNMT3b levels were observed. The upregulation of DNMT2 may be a part of cellular stress response to AgNP treatment. Taken together, AgNP removal resulted in p53/p21-mediated inhibition of cell proliferation, oxidant-based DNA damage response, and changes in DNA methylation patterns, which suggests that more attention should be paid to the possible outcomes in individuals exposed to nano-sized biomaterials.

Sildenafil Ameliorates Advanced Glycation End Products-Induced Mitochondrial Dysfunction in HT-22 Hippocampal Neuronal Cells.

ObjectiveAccumulation of advanced glycation end-products (AGE) and mitochondrial glycation is importantly implicated in the pathological changes of the brain associated with diabetic complications, Alzheimer disease, and aging. The present study was undertaken to determine whether sildenafil, a type 5 phosphodiesterase type (PDE-5) inhibitor, has beneficial effect on neuronal cells challenged with AGE-induced oxidative stress to preserve their mitochondrial functional integrity.MethodsHT-22 hippocampal neuronal cells were exposed to AGE and changes in the mitochondrial functional parameters were determined. Pretreatment of cells with sildenafil effectively ameliorated these AGE-induced deterioration of mitochondrial functional integrity.ResultsAGE-treated cells lost their mitochondrial functional integrity which was estimated by their MTT reduction ability and intracellular ATP concentration. These cells exhibited stimulated generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential, induction of mitochondrial permeability transition, and release of the cytochrome C, activation of the caspase-3 accompanied by apoptosis. Western blot analyses and qRT-PCR demonstrated that sildenafil increased the expression level of the heme oxygenase-1 (HO-1). CoPP and bilirubin, an inducer of HO-1 and a metabolic product of HO-1, respectively, provided a similar protective effects. On the contrary, the HO-1 inhibitor ZnPP IX blocked the effect of sildenafil. Transfection with HO-1 siRNA significantly reduced the protective effect of sildenafil on the loss of MTT reduction ability and MPT induction in AGE-treated cells.ConclusionTaken together, our results suggested that sildenafil provides beneficial effect to protect the HT-22 hippocampal neuronal cells against AGE-induced deterioration of mitochondrial integrity, and upregulation of HO-1 is involved in the underlying mechanism.