Apoptosis In Rat Hippocampal Neurons Research Articles

The miR-34b-5p-negative target Gnai2 aggravates fluorine combined with aluminum-induced apoptosis of rat offspring hippocampal neurons and NG108-15 cells.

It is known that fluorine and aluminum are commonly found in the environment and that long-term overexposure can adversely affect the organism's nervous system, damaging the structure and function of brain tissue. Our previous study showed that fluorine combined with aluminum (FA) could trigger apoptosis in vitro and cause spatial learning and memory impairment and differentially expressed miRNAs (including miR-34b-5p) in the hippocampi in vivo. However, the detailed mechanism is unclear. Learning memory damage is implicated in excessive hippocampal neuron apoptosis, and miR-34b-5p participates in regulating the hippocampal neuron apoptosis. Thus, in the current research, Sprague-Dawley (SD) rats were subjected to FA, and NG108-15 control cells and NG108-15 cells pretransfected with miR-34b-5p agomir or antagomir were exposed to FA. We found that FA triggered apoptosis of rat hippocampal neurons and NG108-15 cells, increased miR-34b-5p expression, and decreased Gnai2, PKA, ERK and CREB expression. Inhibition of miR-34b-5p alleviated FA-induced NG108-15 cell apoptosis and further increased Gnai2, PKA, ERK, and CREB expression, and vice versa. Furthermore, miR-34b-5p modulated the level of Gnai2 by directly targeting its 3'-untranslated region (UTR), as verified through the dual Luciferase reporter assay. These outcomes suggested that miR-34b-5p participated in FA-induced neuronal apoptosis by targeting Gnai2 negatively, thereby inhibiting the PKA/ERK/CREB signaling pathway.

Low expression of the intestinal metabolite butyric acid and the corresponding memory pattern regulate HDAC4 to promote apoptosis in rat hippocampal neurons

After intensive research on the gut-brain axis, intestinal dysbiosis is considered to be one of the important pathways of cognitive decline. Microbiota transplantation has long been thought to reverse the behavioral changes in the brain caused by colony dysregulation, but in our study, microbiota transplantation seemed to improve only behavioral brain function, and there was no reasonable explanation for the high level of hippocampal neuron apoptosis that remained. Butyric acid is one of the short-chain fatty acids of intestinal metabolites and is mainly used as an edible flavoring. It is commonly used in butter, cheese and fruit flavorings, and is a natural product of bacterial fermentation of dietary fiber and resistant starch in the colon, acting similarly to the small-molecule HDAC inhibitor TSA. The effect of butyric acid on HDAC levels in hippocampal neurons in the brain remains unclear. Therefore, this study used rats with low bacterial abundance, conditional knockout mice, microbiota transplantation, 16S rDNA amplicon sequencing, and behavioral assays to demonstrate the regulatory mechanism of short-chain fatty acids on the acetylation of hippocampal histones. The results showed that disturbance of short-chain fatty acid metabolism led to high HDAC4 expression in the hippocampus and regulated H4K8ac, H4K12ac, and H4K16ac to promote increased neuronal apoptosis. However, microbiota transplantation did not change the pattern of low butyric acid expression, resulting in maintained high HDAC4 expression in hippocampal neurons with continued neuronal apoptosis. Overall, our study shows that low levels of butyric acid in vivo can promote HDAC4 expression through the gut-brain axis pathway, leading to hippocampal neuronal apoptosis, and demonstrates that butyric acid has great potential value for neuroprotection in the brain. In this regard, we suggest that patients with chronic dysbiosis should pay attention to changes in the levels of SCFAs in their bodies, and if deficiencies occur, they should be promptly supplemented through diet and other means to avoid affecting brain health.

Effects of GABAB receptor positive allosteric modulator BHF177 and IRS-1 on apoptosis of hippocampal neurons in rats with refractory epilepsy via the PI3K/Akt pathway.

The present study was conducted to determine the effects of the γ-aminobutyric acid B (GABAB ) receptor positive allosteric modulator BHF177 on refractory epilepsy (RE). An RE rat model was initially established via treatment with lithium-pilocarpine. The RE rats were then treated with BHF177 or the GABAB receptor antagonist CGP46381, followed by recording of their seizure rate and assessment of their spatial learning in the Morris water maze test. Treatment of BHF177 reduced the seizure intensity, whereas this effect was revered upoj treatment with CGP46381. Immunohistochemistry revealed that BHF177 treatment diminished P-glycoprotein (P-gp) expression in the hippocampal tissues of RE rats. Next, we found that BHF177 activated GABAB receptor, resulting in upregulated expression of insulin receptor substrate 1 (IRS-1) and PI3K, as well as antiapoptotic factors (Bcl-2 and mTOR), along with suppression of the apoptosis factors Bax and cleaved caspase-3 in the hippocampal tissues. Further, activation of GABAB receptors by BHF177 alleviated the inflammatory response in hippocampal tissues of RE rats, as evidenced by reduced VCAM-1, ICAM-1, and tumor necrosis factor-α levels. Next, we treated primary cultured rat hippocampal neurons with BHF177 and the IRS-1 selective inhibitor NT157. BHF177 inhibited hippocampal apoptosis in rat hippocampal neurons by regulating the IRS-1/PI3K/Akt axis through crosstalk between GABAB and insulin-like growth factor-1 receptors. Collectively, our findings indicate that the BHF177 inhibited neuron apoptosis, thus protecting against RE through the IRS-1/PI3K/Akt axis, which may present a new therapeutic channel for RE.

Protective mechanism of FoxO1 against early brain injury after subarachnoid hemorrhage by regulating autophagy.

IntroductionEarly brain injury (EBI) plays a key role in the devastating outcomes after subarachnoid hemorrhage (SAH). Autophagy and apoptosis may share a common molecular inducer that regulates the process of cell death. FoxO1, as a key regulator of neuronal autophagy which is involved in apoptosis, has not been reported in SAH rats. This work was to investigate the protective and anti‐inflammatory effects of FoxO1 on EBI after SAH by regulating autophagy.MethodsIn this study, we constructed the SAH model. In experiment I, low dose (50 μl of 1 × 108 IU/ml) or high dose (50 μl of 1 × 1010 IU/ml) of FoxO1 gene overexpressed adenovirus vector was injected into the lateral ventricle of rats before SAH. In experiment II, we reported the effect of FoxO1 overexpress on nerve function recovery, oedema, BBB leakage, neuronal death in rats after SAH through autophagy regulation. Post‐SAH evaluation included neurological function score, brain water content, evans blue exosmosis, pathological changes, inflammatory response and apoptosis.ResultsThe experiment I showed that either low or high dose of ad‐FoxO1 could significantly improve nerve function, reduce cerebral water content and reduce blood‐brain barrier (BBB) destruction in rats, indicating that ad‐FoxO1 had a protective effect on brain injury in rats EBI after SAH. In addition, ad‐FoxO1 promoted autophagy in rat hippocampal tissue, as evidenced by accumulation of LC3II/I and Beclin‐1 and degradation of p62. Furthermore, ad‐FoxO1 inhibited the inflammatory response and apoptosis of rat hippocampal neurons after SAH. The experiment II showed that both ad‐FoxO1 and rapamycin attenuated the injury of nerve function in rats after SAH, and this synergistic effect further reduced cerebral edema and evansblue extravasation, decreased hippocampus neuronal cell apoptosis, and declined inflammatory response. However, this was contrary to the results of chloroquine. These findings suggested that FoxO1 regulated the neural function of EBI after SAH through the autophagy pathway.ConclusionsThe findings in this study was beneficial for identifying the novel therapeutic target for the treatment of SAH.

Oxytocin Attenuates Methamphetamine-Induced Apoptosis via Oxytocin Receptor in Rat Hippocampal Neurons.

Methamphetamine (METH) is a highly neurotoxic psychoactive substance that can directly damage the central nervous system through prolonged use. Oxytocin (OT) has attracted much attention because of its neuroprotective effect. The purpose of this study was to investigate whether OT is neuroprotective against METH-induced damage in rat hippocampal neurons. Our results revealed that pre-incubation with OT significantly prevented the damage of METH to hippocampal neurons, including the decrease of mitochondrial membrane potential and the increase of ROS (reactive oxygen species). OT pre-incubation attenuated the up-regulation of Cleaved-Caspase-3 expression and the down-regulation of Bcl-2/Bax expression induced by METH. Pre-incubation with OT prevented the decrease in oxytocin receptor density and P-CREB (phosphorylation of cAMP-response element binding) expression induced by METH in rat hippocampal neurons. Moreover, Pre-incubation of atosiban (ATO) significantly prevented these changes. In conclusion, our study proved that pre-administration of OT could significantly attenuate hippocampal neuron apoptosis induced by METH. Oxytocin receptor activation is involved in the preventive effect of OT on METH-induced apoptosis in rat hippocampal neurons.

Dexmedetomidine reduces the apoptosis of rat hippocampal neurons via mediating ERK1/2 signal pathway by targeting miR-155

Rat hippocampal neurons were isolated and divided into Normal, oxygen glucose deprivation/reoxygenation (OGD/R), OGD/R + DEX, OGD/R + NC mimic, OGD/R + miR-155 mimic and OGD/R + DEX + miR-155 mimic groups. In OGD/R group, LDH, ROS and MDA levels and apoptosis rate was increased, with up-regulations of miR-155, Cyt c and Bax/Bcl-2 ratio, but decreases of SOD, GSH-Px and MMP levels, as well as down-regulations of p-ERK1/2/ERK1/2. As compared to the OGD/R group, parameters above in the OGD/R + DEX group were ameliorated evidently, while OGD/R + miR-155 mimic group manifested the opposite changes. Besides, miR-155 mimic could abolish the protective effect of DEX on the hippocampal neurons under OGD/R. DEX, via down-regulating the expression of miR-155, could activate the ERK1/2 pathway, thereby mitigating the apoptosis and oxidative stress injury and increasing the MMP, thereby protecting hippocampal cells from OGD/R injury.

Synergistic Effect and Mechanism of Apoptosis Induction by Morphine and the HIV-1gp120V3 Loop in Hippocampal Neurons.

HIV-associated neurocognitive disorders (HAND) are a collective name for neurological disorders associated with HIV-1 infection. The incidence and severity of HAND are increased by concomitant opioid use disorder, such as heroin and morphine abuse. Our previous study showed that the HIV-1 envelope protein gp120 and morphine synergistically induce apoptosis in rat hippocampal neurons. However, the underlying mechanism remains unclear. We hypothesized that morphine and gp120 activated the neuronal apoptosis signaling pathway via their typical membrane receptors. If they shared key signaling molecules, their induction of neuronal apoptosis could be inhibited by blocking these targets. We found that morphine and gp120V3 loop synergistically induced hippocampal neuron apoptosis, mediated by activating the extracellular signal-regulated kinase (ERK) pathway, increasing the intracellular Ca2 + concentration and expression of caspase-, and reducing the mitochondrial membrane potential. The ERK inhibitor PD98509 and the phosphatidylinositol 3-kinase activator IGF-1 blocked this effect. These results indicate that ERK plays a crucial role in the apoptosis of hippocampal neurons in HAND.

MiR-139-5p upregulation alleviated spontaneous recurrent epileptiform discharge-induced oxidative stress and apoptosis in rat hippocampal neurons via regulating the Notch pathway.

Epilepsy was characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. Previous studies suggested that microRNA (miR)-139-5p and the Notch pathway were implicated in epilepsy; however, their interaction remained vague. Rat primary hippocampal neurons were isolated and identified by immunofluorescence staining. The cells were then used for SREDs model construction and further subjected to flow cytometry for apoptosis detection. Contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), super oxidase dismutase (SOD) contents, and reactive oxygen species (ROS), and the level of mitochondrial membrane potential (MMP) were determined using commercial kits. Target gene and potential binding sites of miR-139-5p were predicted with TargetScan and confirmed by dual-luciferase reporter assay. Expressions of miR-139-5p, Notch pathway-related proteins and apoptosis-related proteins were measured by quantitative real-time polymerase chain reaction and western blot as needed. The results showed that the hippocampal neurons were microtubule-associated protein 2 (MAP2)-positive. miR-139-5p was downregulated in SREDs model cells. SREDs promoted apoptosis and increased the contents of LDH, MDA, and ROS and the level of MMP while reducing miR-139-5p expression and SOD content in cells, which was reversed by miR-139-5p overexpression. Notch-1 was recognized as the target gene of miR-139-5p, and its expression was negatively regulated by miR-139-5p. Besides, Notch-1 overexpression reversed the effects of miR-139-5p upregulation on the expressions of Notch pathway-related proteins and apoptosis-related proteins, cell apoptosis, oxidative stress and MMP in SREDs-treated cells. Our results indicated that miR-139-5p upregulation alleviated SREDs-induced oxidative stress and cell apoptosis via regulating the Notch pathway, which provides new insights into the role of miRNA in the occurrence and development of epilepsy.

Antidepressant-Like Effects of Chaihu Shugan Powder () on Rats Exposed to Chronic Unpredictable Mild Stress through Inhibition of Endoplasmic Reticulum Stress-Induced Apoptosis.

To investigate the antidepressant-like effects of Chaihu Shugan Powder (CSP, ) and to explore its underlying mechanisms. Thirty-two Sprague-Dawley rats were randomly divided into control (CON), chronic unpredictable mild stress (CUMS), fluoxetine (FLU), and CSP groups, 8 rats in each group. All of the rats except for those in the control group were subjected to 3 consecutive weeks of CUMS to establish the depression model. The open field test (OFT), forced swimming test (FST), and sucrose preference test were used to assess the anti-anxiety and antidepressant effects of CSP. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling was used to determine the apoptosis rate in the hippocampal tissues. The mRNA and protein levels of glucose-regulated protein (GRP) 78, spliced X-box-binding protein (XBP)-1, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and c-Jun N-terminal kinase (JNK) in the hippocampus of rats were evaluated by real-time PCR and Western blot analysis, respectively. Administration of CSP alleviated anxiety and depression-like behavior in CUMS rats, as revealed by enhanced time and distance in the center of the OFT (P<0.05), an increased preference for sucrose, and longer swimming time and shorter immobility time during the FST (all P<0.05). In addition, CSP treatment significantly reduced the rate of apoptosis in rat hippocampal neurons (P<0.05). The mRNA and protein expression levels of GRP78, spliced XBP-1, and CHOP were down-regulated along with the expression of caspase-12 and cleaved caspase-12 proteins (all P<0.05), whereas total and phosphorylated JNK1 protein levels did not differ significantly between control and CSP-treated rats. CSP can improve depression-like behavior in rats exposed to CUMS, possibly by suppressing CHOP and caspase-12 mediated apoptosis in the rat hippocampus.

Theaflavins alleviate sevoflurane-induced neurocytotoxicity via Nrf2 signaling pathway

Aim: Sevoflurane could induce apoptosis of rat hippocampal neurons, while theaflavins (TFs) have antioxidant and anti-inflammatory properties. This study aims to explore whether TFs could alleviate sevoflurane-induced neuronal cell injury.Materials and methods: Cells were treated by concentration gradient of sevoflurane and TFs. Cell viability, level of reactive oxygen species (ROS) and apoptosis rate were determined by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Quantitative PCR (qPCR) and western blot were performed to determine mRNA and protein expressions.Results: TFs promoted viability of cells under the treatment of sevoflurane, while it suppressed apoptosis and down-regulated ROS level in a concentration-dependent manner. TFs could also down-regulate expression levels of caspase-3 and caspase-9 and cytosol and intranuclear nuclear factor E2-related factor 2 (Nrf2) in rat hippocampal nerve cells, while it up-regulated those of heme oxygenase 1 (HO-1), NADPH quinine oxidoreductase 1 (NQO1), glutamate cysteine ligase (GCL) and peroxiredoxin 1 (Prx1).Conclusions: Our study suggests that TFs exert protective effects on sevoflurane-induced neurocytotoxicity and therefore could be used as a potential drug for treatment of neuronal injury.

Effects of ELK-1/JNK/c-Fos on apoptosis of rat hippocampal neurons cultured in vitro with Zuogui Jiangtang Jieyu Formula in simulated diabetes mellitus complicated with depression

To study the effects of Zuogui Jiangtang Jieyu Formula (ZGJTJYF, the Chinese Medicine) on hippocampal neuron apoptosis in diabetes mellitus complicated with depression (DD). The primary cultured hippocampal neurons were treated with high glucose (150 mmol/L) and corticosterone (200 micromol/L) to establish the cell model of DD in vitro. The cultured hippocampal neurons were randomly divided into five groups: blank serum group, normal group, Zuogui Jiangtang Jieyu recipe drug-containing serum group, positive drug (metformin + fluoxetine) drug-containing serum group and model group (three compound holes in each group). The model group and the normal group were given the same amount of culture medium, and the other groups were given the corresponding serum with 10% volume fraction for 18 hours. Hoechst staining, high content cell imaging and RT-PCR were used to detect the apoptosis of hippocampal neurons and the expressions of apoptosis-related ETS-like 1 transcription factor(ELK-1), C-Jun N-terminal kinase(JNK) and c-Fos proteins and genes. Compared with the blank group, the apoptotic number of hippocampal neurons in the model group was increased significantly, and the expression levels of ELK-1, JNK and c-Fos were increased significantly (P＜0.05). Compared with the model group, the local bright spots of hippocampal neurons in the Zuogui Jiangtang Jieyu recipe-containing serum group and the positive drug-containing serum group were decreased significantly, and the number of apoptotic cells was decreased significantly. The expressions of JNK, c-fos protein and mRNA were down-regulated significantly (P＜ 0.05), and the neural network and dendritic junction were improved significantly. Zuo Gui Jiang Tang Jie Yu Formula can reverse the expressions of ELK-1, JNK and c-Fos signals in hippocampal neurons under DD environment and play an anti-apoptotic effect.

Influence of mTOR signaling pathway on ketamine-induced injuries in the hippocampal neurons of rats

ABSTRACTObjective: To explore the influences of mammalian target of rapamycin (mTOR) signaling pathway on ketamine-induced apoptosis, oxidative stress and Ca2+ concentration in the hippocampal neurons of rats.Methods: The primary hippocampal neurons isolated from fetal Sprague Dawley rats were treated with ketamine (0, 50, 100 and 500 μM) for 4 days to observe its effect on mTOR signaling pathway and apoptosis of rat hippocampal neurons. Then, the hippocampal neurons were divided into C (Control), R (Rapamycin, an inhibitor of mTOR signaling pathway), K (Ketamine) and R + K (Rapamycin + Ketamine) groups to detect the apoptosis, reactive oxygen species (ROS) production, and Ca2+ concentration via the terminal transferase uridyl nick end labelling (TUNEL) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) method and Fluo-3 acetoxymethyl ester (Fluo-3AM) staining, respectively. The expressions of mTOR signaling pathway and apoptosis-related proteins in hippocampal neurons were examined by qRT-PCR and Western blot.Results: Ketamine could dose-dependently promote the apoptosis of rat hippocampal neurons with upregulation of p-mTOR and its downstream regulators (p-4E-BP-1 and p-p70S6K). However, ketamine-induced apoptosis in hippocampal neurons was reversed significantly by the administration of rapamycin, as evident by the decrease in expressions of pro-apoptotic proteins (Bax and cleaved Caspase-3) and the increase in anti-apoptotic protein (Bcl-2). Meanwhile, the ROS generation and Ca2+ concentration was inhibited accompanied with reduced malonildialdehyde levels but elevated superoxide and glutathione peroxidase activities.Conclusion: Inhibition of mTOR signaling pathway protected rat hippocampal neurons from ketamine-induced injuries via reducing apoptosis, oxidative stress, as well as Ca2+ concentration.Abbreviations: mTOR: mammalian target of rapamycin; SD: Sprague-Dawley; SPF: Specific-pathogen free; ROS: reactive oxygen species; TUNEL: terminal transferase uridyl nick end labelling; DCFH-DA: Dichloro-dihydro-fluorescein diacetate; Fluo-3A: Fluo-3 acetoxymethyl ester; NMDAR: non-competitive N-methyl-D-aspartame glutamate receptor; 4E-BP1: 4E binding protein 1; p70S6K: p70 S6 Kinase; PCR: Polymerase chain reaction; MDA: malonildialdehyde; GSH-PX: glutathione peroxidase; ANOVA: One-way Analysis of Variance.

P38mitogen-activated protein kinase gene silencing rescues rat hippocampal neurons from ketamine-induced apoptosis: An invitro study.

Ketamine (KTM) is an anesthetic drug with several advantages, including the elevation of cardiac output and blood pressure. However, KTM may also induce the apoptosis of hippocampal neurons. Notably, p38 mitogen-activated protein kinase (p38MAPK) has previously been studied for its role in neuronal injury. Therefore, the present study evaluated the effect of lentivirus-mediated p38MAPK gene silencing on KTM-induced apoptosis of rat hippocampal neurons. Hippocampal neurons were extracted from neonatal Sprague-Dawley rats, and then treated with KTM, p38MAPK-short hairpin RNA or SB203580 (an inhibitor of p38MAPK). Next, the expression levels of p38MAPK and apoptosis-associated genes, including caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), were detected. In addition, cell viability and apoptosis were determined using an MTT assay and flow cytometry, respectively. Finally, telomerase activity of hippocampal neurons was detected by ELISA. The results revealed that silencing of p38MAPK in KTM-treated cells decreased the expression levels of p38MAPK, caspase-3 and Bax, and the extent of p38MAPK phosphorylation, while it increased the expression of Bcl-2. Furthermore, silencing p38MAPK promoted cell viability, cell cycle progression and the telomerase activity of hippocampal neurons, and inhibited the apoptosis of hippocampal neurons. Taken together, the results suggested an inhibitory role of lentivirus-mediated p38MAPK gene silencing on KTM-induced apoptosis of rat hippocampal neurons. Thus, p38MAPK gene silencing may serve as a potential target for preventing the KTM-induced apoptosis of hippocampal neurons.

Effect of cyclooxygenase‑2 inhibition on the development of post‑traumatic stress disorder in rats.

Post-traumatic stress disorder (PTSD) is characterized by re-experiencing of a traumatic event, avoidance of trauma-associated stimulation, general changes in mood and cognition, and hyper arousal symptoms. Cyclooxygenase is involved in the production of prostaglandins and thromboxanes, and its inducible form cyclooxygenase-2(COX-2), an important mediator of cell injury in inflammation, is primarily expressed in leukocytes and brain cells. The present study investigated the expression of COX-2 in the hippocampi of rats with PTSD and evaluated the effect of COX-2 inhibition on PTSD. Adult male Wistar rats were randomly divided into three groups: Control (n=20), PTSD (n=20) and intervention group (PTSD+COX-2 inhibitor treatment, n=20). The expression of COX-2 was detected by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blotting. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining was used to observe the apoptosis of rat hippocampal neurons. Tumor necrosis factor α (TNF-α), interleukin (IL)-6 and prostaglandin E2 (PGE2) levels were analyzed by ELISA. Nitric oxide (NO) was detected using the Griess test. The behavioral and cognitive function of rats in the PTSD group was significantly decreased compared with the control group, while the behavioral and cognitive function of rats in the intervention group were improved. The COX-2 mRNA and protein expression levels in hippocampi of rats in the PTSD group were higher than in the control and intervention group. The apoptosis of hippocampus in rats with PTSD was significantly increased compared with the control group and following treatment with COX-2 inhibitor, apoptosis was decreased. In addition, compared with the control group and intervention group, the levels of TNF-α, IL-6, PGE2 and NO in hippocampi of rats were increased in the PTSD group. The present study indicated that COX-2 may be involved in the pathogenesis of PTSD, and inhibition of its expression serves a neuroprotective role in hippocampi of PTSD rats.

Enantioselective apoptosis and oxidative damage induced by individual isomers of profenofos in primary hippocampal neurons

ABSTRACTThe purpose of this study was to investigate the apoptosis-related cytotoxic effects and molecular mechanisms of individual isomers of profenofos (PFF) on primary hippocampal neurons at 1.0 to 20 mg L−1. The cell viability and lactate dehydrogenase (LDH) efflux indicated that (−)­PFF exposure was associated with more toxic effects than (+)­PFF above the concentration of 5 mg L−1 (P < 0.5). Flow cytometric results showed that the percentages of apoptotic cells incubated with 20 mg L−1 (−)­PFF, (+)­PFF and rac-PFF for 24 h reached 23.4%, 9.2% and 14.2% (P < 0.01), respectively. Hippocampal neurons incubated with (−)­PFF, (+)­PFF and rac-PFF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) and a dose-dependent inhibition of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, implying that the defense system of the tests induces oxidative damage. A statistically significant difference was observed between the two enantiomers at 5 mg L−1 and above. Moreover, the results showed that (−)­PFF exposure caused a significant loss in mitochondrial transmembrane potential (MMP), an upregulation of Ca2+ and Bax protein expression, a downregulation of Bcl-2 protein expression, and the activation of caspase-3 and caspase-9 in a dose-dependent manner; (+)­PFF and rac-PFF exhibited these effects to a lesser degree. All results suggest that PFF induced apoptosis in rat hippocampal neurons via the mitochondria-mediated pathway, and oxidative stress is one of the factors of PFF-induced apoptosis. In addition, (−)­PFF appears to play an important role in oxidative stress and apoptosis, indicating that enantioselectivity should be considered when assessing ecotoxicological effects and health risks of chiral pesticides.

Aging and amyloid \u03b2 oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons

BackgroundToll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.MethodsCa2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.ResultsLPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.ConclusionsAging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.

Protective Effects of Cerebrospinal Fluid With The Intake of Xixin Decoction on Hippocampal Neuron Apoptosis Induced by Aβ1-42

Objective: To study the protective effects of cerebrospinal fluid with the intake of Xixin Decoction (CSF) on As1-42-induced apoptosis in rat hippocampal neurons. Methods: Hippocampal neurons were extracted from Wistar newborn rats and cultured in vitro. Cultured neurons were divided into the following groups: the normal control group, the As model group, the low concentration treatment group, the medium concentration treatment group, and the high concentration treatment group. We analyzed cell viability using an MTT assay. Cell apoptosis was observed using transmission electron microscopy, and the rate of cell apoptosis was measured with flow cytometry using Annexin V/PI double staining kit. The gene expression and protein expression levels of Caspase-9, Bcl-2, and Bax in cells were measured using Real-time PCR, Western blot, and immunofluorescence techniques. Results: cerebrospinal fluid with the intake of Xixin Decoction was found to improve the survival rate of cells, improve cell morphology during apoptosis, and reduce the number of apoptotic cells. Compared with the control group, the expression levels of Caspse-9 and Bax were increased in the model group, while the Bcl-2 expression level was decreased. In addition, the expression levels of Caspase-9 and Bax were found to be reduced in each treatment group, compared with the model group. It was also quite evident that Bcl-2 expression levels were increased (P<0.05 or P<0.01). Conclusion: cerebrospinal fluid with the intake of Xixin Decoction exhibits a significant protective effect on cells against As1-42-induced apoptosis. Importantly, we demonstrate a dose-effect relationship, with the effects of cerebrospinal fluid with the intake of Xixin Decoction at a density of 20% showing the most significant results.

Expression changes of indoleamine 2, 3-dioxy-genase in the hippocampus of rat with post-traumatic stress disorder

Objective To observe the expression of indoleamine 2, 3-dioxy-genase(IDO) in hippocampus of rats with posttraumatic stress disorder (PTSD) and the protective effect of IDO inhibitor on neurons, and to explore the role of IDO in the pathogenesis of PTSD. Methods Adult male Wistar rats were randomly divided into the normal control group, PTSD model group and IDO inhibitor treatment group. The expression of IDO was detected by immunohistochemistry, RT-PCR and Western-blot. The apoptosis of rat hippocampal neurons was assayed by Tunel staining. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by ELISA. Moreover behavioral evaluation was performed, including central residence time, percentage of open arm residence time and stage latency. Results Comparing with the control group, PTSD rats showed decreased central residence time ((22.65±1.54)s), decreased percentage of open arm residence time((10.55± 1.96)%), prolonged stage latency ((56.38±4.21) s) (P <0.05), increased TNF-α ((8.58±0.6) pg/ml), IL-6 ((15.72±1.42)pg/ml) and IDO mRNA (0.8278±0.0796), increased IDO protein (1.2329±0.1148) expression and apoptosis rate((81.47±6.86)%) in hippocampus (P <0.05)(P <0.05). However, rats treated with IDO inhibitor showed increased central residence time((30.78±3.20)s), increased percentage of open arm residence time((10.55±1.96)%), shortened stage latency ((56.38 ±4.21)s), meanwhile reduced expression of TNF-α((3.69±0.41)pg/ml), IL-6((7.45±0.58)pg/ml), IDO mRNA(0.2236 ±0.0387) and IDO protein(0.4235±0.0411) was detected in hippocampus(P <0.05). Apoptosis rate ((42.54± 3.98)%) was also decreased in hippocampus(P <0.05). Conclusion The content of TNF-α, IL-6 and IDO are increased significantly in the hippocampus of PTSD rats. IDO may participate in the pathogenesis of PTSD, and the IDO inhibitor may play a neuroprotective role in hippocampus of PTSD. Key words: Post-traumatic stress disorder; Indoleamine 2, 3-dioxy-genase; Tumor necrosis factor-α; Interleukin-6

Autophagy activation prevents sevoflurane-induced neurotoxicity in H4 human neuroglioma cells.

The inhaled anesthetic sevoflurane may induce cognitive impairment in both animals and humans. Previous study has shown that sevoflurane triggers ER stress and may lead to apoptosis in rat hippocampal neurons. In this study, we examined whether sevoflurane caused autophagy and its contributions to sevoflurane induced neuronal cell injury. H4 human neuroglioma cells were exposed to 4.1% sevoflurane for 6 h. Cell viability and apoptosis ratio were assessed using a CCK8 kit and flow cytometry, respectively. Autophagosomes in the cells were detected using GFP-LC3 plasmid transfection or transmission electronic microscopy. The expression of LC3B, p62/SQSTM, C/EBP homologous protein (CHOP) and glucose-related protein 78 (GRP78) was assessed with Western blotting. Sevoflurane treatment induced apoptosis and markedly increased the LC3-II level and GFP-LC3 puncta number, decreased p62 expression in H4 cells. Activation of autophagy by rapamycin (1 μmol/L) significantly reduced sevoflurane-induced apoptosis and increased cell viability, whereas inhibition of autophagy with 3-MA (5 mmol/L) caused the opposite effects. Furthermore, sevoflurane treatment markedly increased the expression of CHOP and GRP78, two hallmark proteins of ER stress. Inhibition of ER stress by 4-phenylbutyrate (500 μmol/L) abrogated sevoflurane-induced autophagy and apoptosis, and improved the viability. Moreover, sevoflurane-stimulated expression of CHOP and GRP78 was inhibited by rapamycin, but further enhanced by 3-MA. Sevoflurane treatment induces ER stress and activates autophagy, which antagonizes sevoflurane-induced apoptosis in H4 human neuroglioma cells. The results suggest that autophagy may be a potential therapeutic target in preventing sevoflurane-induced neurotoxicity.

Puerarin reduces apoptosis in rat hippocampal neurons cultured in high glucose medium by modulating the p38 mitogen activated protein kinase and c-Jun N-terminal kinase signaling pathways

ObjectiveTo investigate the neuroprotective effect of puerarin on rat hippocampal neurons cultured in high glucose medium, and to examine the role of the p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways in this effect. MethodsPrimary cultures of hippocampal neurons were prepared from newborn Sprague Dawley rats. Neuron-specific enolase immunocytochemistry was used to identify neurons. The neurons were cultured with normal medium (control group) or with high-glucose medium (high-glucose group), and puerarin (puerarin group), a p38 MAPK inhibitor (SB239063; p38 MAPK inhibitor group) or a JNK inhibitor (SP600125; JNK inhibitor group) were added. After 72 h of treatment, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was performed to detect apoptosis, and western blotting was used to assess protein levels of p-p38, p38, p-JNK and JNK. ResultsIn the high-glucose group, the neuronal apoptosis rate and the p-p38/p38 and p-JNK/JNK ratios were higher than in the control group. The p38 MAPK and JNK inhibitors prevented this increase in the apoptosis rate. The apoptosis rates in the puerarin group, the p38 MAPK inhibitor group and the JNK inhibitor group were significantly decreased compared with the high-glucose group. Moreover, protein levels of p-p38 and p-JNK were significantly reduced, and the p-p38/p38 and p-JNK/JNK ratios were decreased in the puerarin group compared with the high-glucose group. In addition, compared with the high-glucose group, p-p38 levels and the p-p38/p38 ratio were reduced in the p38 MAPK inhibitor group, and p-JNK levels and the p-JNK/JNK ratio were decreased in the JNK inhibitor group. ConclusionPuerarin attenuates neuronal apoptosis induced by high glucose by reducing the phosphorylation of p38 and JNK.