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Background Benzo[a]pyrene (BaP) is neurotoxic and can cause neuronal damage by oxidative stress. Proanthocyanidin (PC) has antioxidant activity, and its mechanism may related to nuclear factor-erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway. Objective To explore potential protective effect of PC on hippocampal neuron injury induced by BaP oxidative stress. Methods Hippocampal neurons of neonatal SD rats delivered within 24 h were isolated and cultured, and cell activity was detected by cell counting kit-8 (CCK-8) method. According to the pre-experimental results, a control group and three BaP groups of 10, 20 and 40 µmol·L−1 were set up for Stage I experiment. The length of neurites and number of branches of hippocampal neurons in each group were observed by immunofluorescence method. Reactive oxygen species (ROS) fluorescence probe method was used to measure ROS levels in cells. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the mRNA and protein expression of Nrf2, Kelch-like epichlorohydrin associated protein-1 (Keap1), HO-1, B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in hippocampal neurons of each group, respectively. According to the results of Stage I experiment, three group were set up, including control group, BaP alone treatment group (BaP 20 µmol·L−1), and PC intervention group (BaP 20 µmol·L−1 + PC 2.5 µg·mL−1) for Stage II experiment, with the same protocol as Stage I. Results For Stage I experiment, compared with the control group, the 10, 20, and 40 µmol·L−1 BaP groups showed gradually shortened length of neurites [(177.60±3.49), (142.40±6.52), and (100.50±19.40) µm] (P<0.05) and decreased number of branches (8.00±1.00, 6.33±1.53, 4.33± 0.58) of hippocampal neurons (P<0.05); increased ROS production (2.38±0.33, 8.08±0.26, 9.86±0.19) (P<0.05); the qRT-PCR results showed that the mRNA expression levels of Nrf2 (0.35±0.03, 0.25±0.01, 0.13±0.03), Keap1 (0.70±0.01, 0.47±0.03, 0.15±0.02), HO-1 (0.77±0.02, 0.60±0.02, 0.32±0.01), and Bcl-2 (0.65±0.03, 0.47±0.02, 0.18±0.02) gradually decreased, and the mRNA expression level of Bax gradually increased (1.24±0.01, 2.25±0.15, 4.98±0.30) (P<0.05); the Western blotting results showed that the protein expression trends of Nrf2, Keap1, HO-1, Bcl-2, and Bax were consistent with the mRNA results. For Stage II experiment, compared with the BaP alone treatment group, the length of neurites in the PC intervention group became longer, (149.90±3.01) μm vs (202.00±4.45) μm (P<0.05), the number of branches increased, (4.67±0.58) vs (8.33±0.58) (P<0.05); the ROS production reduced, (10.81±0.63) vs (7.31±0.70) (P<0.05); the mRNA expression levels of Nrf2, Keap1, HO-1, and Bcl-2 increased (P<0.05), and the mRNA expression levels of Bax decreased (P<0.05); the Nrf2, Keap1, HO-1, and Bcl-2 protein expression levels increased (P<0.05), and Bax protein expression level decreased (P<0.05). Conclusion PC may exert neuroprotective effects by activating the Nrf2-HO-1 signaling pathway, inhibiting BaP-induced oxidative stress in neuronal cells, and reducing cytotoxicity.
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Background Benzo[a]pyrene (BaP) has neurotoxicity, which can induce the loss of hippocampal neurons in humans and animals and lead to spatial learning and memory dysfunction, but its mechanism is still unclear. Objective To observe the ferroptosis of mouse hippocampal neuron HT22 cells induced by 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), an active metabolite of BaP, and to explore its potential mechanism, so as to provide a basis for the study of BaP neurotoxicity mechanism. Method Mouse hippocampal neuron HT22 cells were selected and divided into four groups: solvent control group and low, medium, and high concentration BPDE exposure groups (0.25, 0.50, and 0.75 μmol·L−1). Cell survival was detected by CCK8 method. Cell morphology and ultrastructure were observed under light and electron microscopes. The levels of reactive oxygen species (ROS) and Fe2+ were detected by fluorescence probe method. Iron, 4-hydroxynonenoic acid (4-HNE), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-PX) levels were detected with commercial kits. The expression levels of acyl-CoA synthase long chain family member 4 (ACSL4), cyclooxygenase 2 (COX2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were detected by Western blotting. After interventions with ferroptosis inhibitors 20 μmol·L−1 deferoxamine (DFO) and 10 μmol·L−1 ethyl 3-amino-4-cyclohexylaminobenzoate (Fer-1), the cell survival rate of each BPDE exposure group and the changes of the ferroptosis characteristic indicators and protein expression levels were observed. Results With the increase of BPDE concentration, the survival rate of HT22 cells decreased gradually, and the survival rate of each BPDE group was significantly lower than that of the solvent control group (P<0.01). Under light microscope, the number of cells in the high concentration BPDE group was significantly reduced, and atrophic cells and reduced synapses were recorded. Under electron microscope, the HT22 cells in the high concentration BPDE group showed mitochondrial shrinkage, decreased crista, and increased mitochondrial membrane density. Compared with the solvent control group, the levels of intracellular lipid ROS, Fe2+, 4-HNE, and MDA significantly increased in the high concentration group (P<0.01), the GSH and GSH-PX levels were significantly decreased (P<0.01), the protein expression levels of ASCL4 and COX2 were significantly increased (P<0.01), and the protein expression levels of SCL7A11 and GPX4 were significantly decreased (P<0.01). The ferroptosis inhibitors DFO and Fer-1 significantly reversed the cell survival rate (P<0.01), the ferroptosis characteristic indicators (ROS, Fe2+, 4-HNE, MDA, GSH, and GSH-PX levels) (P<0.01), and the expression levels of ferroptosis-related proteins (ACSL4, COX2, SLC7A11, and GPX4) (P<0.01) in the high concentration BPDE group. Conclusion BPDE can induce ferroptosis in mouse hippocampal neuron HT22 cells, which may be related to the inhibition of SLC7A11/GSH/GPX4 axis and the induction of iron metabolism disorder.
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Objective:To observe the ferroptosis in hippocampal neurons of rats with multiple cerebral concussion (MCC) .Methods:Ninety clean grade male Wistar rats with a body mass of (250±10) g were randomly divided into control group (12 rats) and model group (78 rats) according to the random number table method. The rat of MCC model was prepared by hitting the frontotemporal lobe of rats with free fall method once a day for 3 consecutive days.Then the MCC model rats were randomly divided into 12 h, 24 h, 48 h, 72 h, 7 d and 14 d groups with 12 rats in each group. The balance beam experiment was used to detect the motor coordination function of the rats. The levels of interleukin-β(IL-1β), interleukin-6(IL-6), serum glutathione (GSH), neuron-specific enolase (NSE) in serum of rats were detected by ELISA.The content of iron ion in hippocampus was detected by colorimetry. The mRNA and protein levels of glutathione peroxidase 4(GPX4), ferritin heavy (FTH) and ferritin light(FTL) in the hippocampus were detected by RT-PCR and Western blot. Prussian blue staining was used to observe the iron deposition in brain tissue.The ultrastructural changes of hippocampal neurons and mitochondria were observed by transmission electron microscope. SPSS 24.0 statistical software was used for one-way ANOVA among groups, and LSD test was used for multiple pairwise comparison.Results:In the balance beam experiment, the passing time and motor coordination score of rats in each group were significantly different ( F=30.08, 60.34, both P<0.05). The passing time and motor coordination score of rats in the 48 h group ((87.00±4.74) s, (4.75±0.43)) were significantly higher than those in the control group ((35.13±6.99) s, (0.75±0.23)) (both P<0.05). There was significant difference in the total iron ion content, Fe 2+ content and Fe 3+ content in hippocampus of rats in each group ( F=25.20, 94.42, 40.25, all P<0.05), and the content of Fe 2+ in hippocampus of 48 h group was significantly higher than that of the control group ((10.17±0.05) ng/μL, (8.65±0.01) ng/μL)( P<0.05). In the results of RT-PCR and Western blot, the mRNA and protein levels of GPX4, FTH and FTL in hippocampus of each group were significantly different ( F=37.94, 82.09, 49.01, 71.63, 28.94, 15.78, all P<0.05). The mRNA level and protein level of GPX4 ((1.09±0.01), (0.23±0.01) )and FTL ((1.60±0.03), (0.64±0.02)) in 24 h group were significantly higher than those of the control group (GPX4: (1.00±0.02), (0.17±0.01)), FTL: ((1.00±0.04), (0.32±0.01))(all P<0.05). The mRNA level and protein level of FTH ((0.24±0.03), (0.07±0.01)) in 24 h group were significantly lower than those of the control group((1.00±0.01), (0.67±0.03))(both P<0.05). The results of electron microscope showed that the hippocampal neuronal cells of the model rats were reduced, the nucleolus was broken and the nuclear membrane was shrunk in varying degrees, the mitochondria were swollen and deformed and there were vacuoles, and the cristae in the mitochondria decreased or disappeared. Conclusion:The levels of inflammatory reaction and oxidative stress in the multiple cerebral concussion model rats increase, and the hippocampal neurons show the characteristics of ferroptosis, especially at 24 h and 48 h.
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To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.
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Animals , Humans , Rats , Catalytic Domain , Down-Regulation , HEK293 Cells , Mitochondria , Protein Phosphatase 2 , tau ProteinsABSTRACT
Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-β oligomers (AβOs) and the intracellular accumulation of neurofibrillary tangles formed by hyperphosphorylated tau protein. In this paper, an in vitro pathological model of AD based on neuronal network chip and its real-time dynamic analysis were presented. The hippocampal neuronal network was cultured on the microelectrode array (MEA) chip and induced by AβOs as an AD model to simultaneously record two firing patterns from the interneurons and pyramidal neurons. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. This biosensor enabled the detection of the AβOs toxicity responses, and the identification of connectivity and interactions between neuronal networks, which can be a novel technique in the research of AD pathological model .
Subject(s)
Humans , Alzheimer Disease , Amyloid beta-Peptides , Neurofibrillary Tangles , tau ProteinsABSTRACT
Objective:To observe the effect of isopimpinellin on primary hippocampal neuron cells γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and receptor genes expressions, in order to explore its hypnotic mechanism. Method:The primary hippocampal neurons of neonatal Sprague-Dawley rats were cultured in vitro. And subsequent experiments were conducted in the optimal state of cell growth, and the purity was identified by immunohistochemistry of neuron-specific enolase. Hippocampal neurons were randomly divided into five groups, namely blank control group, diazepam group (25 mg·L-1), and low-dose (5 mg·L-1), moderate-dose (10 mg·L-1) and high-dose (20 mg·L-1) isopimpinellin groups. Early apoptosis of hippocampus neuron cells were detected using flow cytometry technique after 24 h administration, and the changes in the levels of GABA and 5-HT were detected using enzyme-linked immunosorbent. The changes in mRNA expressions of receptor genes relating to gamma-aminobutyric acid type A receptor(GABAA) genes GABRA1,GABRA5,GABBR1, gamma-aminobutyric acid type B receptor genes (GABAB) GABRB2, 5-hydroxytryptamine 1A receptor (5-HT1A)5-HT1A(A),5-HT1A(B),5-HT1A(C) were detected by real-time quantitative PCR(Real-time PCR). Result:On the 7th day, the hippocampal neurons grew in a good condition, and the purity was above 90%. Apoptosis rates of hippocampal neurons in the low-dose and moderate-dose groups were significantly lower than that in the blank control group (PP1,GABRA5,5-HT1A(A),5-HT1A(C) in the moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PP1,5-HT1A(B) in the low-dose, moderate-dose and high-dose isopimpinellin groups were significantly higher than those in the blank control group (PPConclusion:The hypnotic mechanism of isopimpinellin may be related to the inhibition of hippocampal neuron apoptosis, the increase of the content of inhibitory neurotransmitter GABA, and the up-regulation of GABA and 5-HT-related receptor genes.
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Objective: To investigate the neuroprotective effect of modified Yuejuwan and Ganmai Dazao Tang (YJGZ) on glutamate-induced cell injury of mouse hippocampal neuron cell line (HT22). Method: The HT22 cell was cultured aseptically, and the cell injury model was established with high concentration of glutamate acid, YJGZ aqueous extract and drug-containing serum were prepared. Then experiment was divided into normal group, model group, YJGZ drug-containing serum groups (1%, 5%, 10%), YJGZ aqueous extract group (166 mg·L-1), and nimodipine group (100 μmol·L-1). Methye thiazolye telrazlium (MTT) was used to detect the cell survival rate of each group, the level of lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the protein expression of hippocampal N-methyl-D-aspartate receptor subunit 2B protein (NR2B), cyclic adenosine response element binding protein (CREB), phosphorylated cyclic adenosine response element binding protein (p-CREB), extracellular regulated protein kinase (ERK),and phosphorylated extracellular regulated protein kinase (p-ERK). Result: As compared with normal group, the cell survival rate was significantly decreased in model group (PPPPPPPPPPConclusion: YJGZ aqueous extract has a significant protective effect on glutamate-induced HT22 cell injury.
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Objective: To preliminary study on the feasibility of constructing three-dimensional (3D) hippocampal neural network in vitro by using microfluidic technology. Methods: A network patterned microfluidic chip was designed and fabricated by standard wet etching process. The primary hippocampal neurons of neonatal Sprague Dawley rats were isolated and cultured, and then inoculated on microfluidic chip for culture. Immunofluorescence staining was used to observe the growth of hippocampal neurons at 3, 5, and 7 days of culture and electrophysiological detection of hippocampal neuron network at 7 days of culture. Results: The results showed that the number of hippocampal neurons increased gradually with the prolongation of culture time, and the neurite of neurons increased accordingly, and distributed uniformly and regularly in microfluidic chip channels, suggesting that the 3D hippocampal neuron network was successfully constructed in vitro. Single and multi-channel spontaneous firing signals of hippocampal neuronal networks could be detected at 7 days of culture, suggesting that neuronal networks had preliminary biological functions. Conclusion: Patterned microfluidic chips can make hippocampal neurons grow along limited paths and form 3D neuron networks with corresponding biological functions such as signal transduction, which lays a foundation for further exploring the function of neuron networks in vitro.
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Objective: To investigate the protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation (ZGF) on hippocampal neuron of neurovascular unit (NVU) induced by autophagy in diabetes mellitus with depression (DD). Methods Hippocampal neurons, astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was employed to identify primitive cells. The DD model of NVU was established by applying intervention of high glucose (150 mmol/L) and cortisone (200 μmol/L) after co-culture system was built in vitro. The cultured cells were randomly divided into normal group, blank serum group, model group, and medicated serum of ZGF group. Intracellular calcium levels (Ca2+) on hippocampal neuron of NVU was detected by Furo-3/AM staining. The expression of autophagy marker Beclin-1, LC3-II, and GluR2/Ca2+/mTOR signaling pathway key proteins was detected by high content analysis. The apoptosis of hippocampal neurons was observed by Tunel staining. Results:Compared with the normal group, Ca2+ levels was remarkably increased, expression of autophagy marker Beclin-1 and LC3-II was dramatically up-regulated, proteins expression of GluR2 and mTOR was obviously down-regulated while AMPK up-regulated, and the apoptosis in hippocampal neuron of NVU was significantly increased in model and blank serum group. Compared with the model group, intracellular calcium levels was obviously decreased, expression of autophagy marker Beclin-1 and LC3-II was down-regulated, GluR2/Ca2+/mTOR signaling pathway proteins expression of GluR2, mTOR was up-regulated while AMPK down-regulated, furthermore the apoptosis of hippocampal neurons was significantly decreased in ZGF group. Conclusion: ZGF has significant protective effects on hippocampal neuron of NVU induced by autophagy in DD, and its mechanism is related to the GluR2/Ca2+/mTOR signaling pathway and apoptosis.
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Objective: To evaluate the role of dexmedetomidine in protective effects of propofol on newborn rats hippocampal synapses and the influence of the Bcl-2 expression. Methods: Neurons were separated and purificated and identificated by microtubule associated protein-2 (MAP-2) antibody. Rats were divided into normal control group, propofol group(add 12 tng/L propofol), and propofol + dexmedetomidine group (add 12 mg/L propofol and 0.25 mg/L dexmedetomidine). The expression of Bcl-2 test by Real-time PCR and Western blotting; cell synapse was detected by transmission electron microscope. Results: Rat hippocampal neurons were separated successfuly. Hippocampal nerve cells in control group were given priority to with multiple processes of pyramidal cell, the cell body was triangular or elliptic, large cell body bright and pervious to light quality, had a halo around, abundant cytoplasm, nuclear. Compared with the control group, in propofol group the Bcl-2 expression decreased obviously. Compared with the propofol group, the expression of Bcl-2 increased obviously (P<0.05). Conclusion: Dexmedetomidine can influence the expression of Bcl-2 so as to play a protective role of hippocampal neurons.
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Objective To observe the effects of Zuogui Jiangtang Jieyu Prescription (ZGJTJYP) on pathological morphology of hippocampal neurons in rats of diabetes mellitus with depression (DD); To discuss its protective mechanism of action. Methods DD rat model was established by tail vein injection of STZ combined with chronic stress. Totally seventy-two SPF rats were randomly divided into normal group, model group, positive medicine group, ZGJTJYP low-, medium-, and high-dose groups, with 12 cases in each group, and were given corresponding medicine. After the last administration, blood glucose and behavioral changes were measured in each group. HE staining, Nissl staining, and Golgi staining were used to observe the changes of pathological morphology in rat hippocampus. The expression of Caspase-3 in hippocampus was detected by immunohistochemistry. Results Compared with the normal group, blood glucose level was significantly increased in the model group; the number of neurons in the hippocampus decreased in the model group; the apoptosis increased in the model group; the neuronal soma showed deformation and reduced dendritic spines and other pathological damages in the model group. After intervention of ZGJTJYP, hyperglycemia and depression-like status of the model rats were relieved; the number of hippocampal neurons increased significantly; neuronal morphology basically returned to normal. The expression of Caspase-3 in ZGJTJYP high-and medium-dose groups was significantly lower than that in model group. Conclusion ZGJTJYP can significantly improved blood glucose and learning and memory abilities in DD rats, which may be related to slowing hippocampal injury and apoptosis, and restoring neuronal quantity and morphology.
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Objective To investigate the potential mechanism of POD in the elder rat model in different depths of anesthesia. Method 120 elderly rats were randomly divided into the A1,A2,A3,B1,B2 and B3 groups. The incidence of POD in the elderly rats was assessed in different depths of anesthesia ,and then the death of neurons,and the expressions of Bid,Bim,Puma and Caspase?3 were detected by Annexin/PI ,real?time PCR and Western blot assay ,respectively. Results Compared with the A1 and A3 group ,the incidence of POD in the elderly rats was decreased and the death of neurons ,and the expressions of Bid ,Bim ,Puma and Caspase?3 were decreased in Group A2(P<0.05). Compared with the preoperative condition,the incidence of POD in the elder?ly rats was increased and the death of neurons,and the expressions of Bid,Bim,Puma and Caspase?3 were in?creased in all groups (P < 0.05). Additionally,similar results were found in the group of inhalational anesthesia. Conclusion The depth of BIS 60 ~ 75/BIS 30 ~ 45 in the elderly rats lead to the increase in the incidence of POD,and that might result from the apoptosis of neurons and the increases of Bid,Bim,Puma and Caspase?3.
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Objective: To investigate the effects of Paeoniae Alba Radix extracts (PAREs) on 5-HT3R mediated ion channels in primarily cultured hippocampal neurons of rats with depression. Methods: An animal model of depression was successfully developed and evaluated in rats. PARE was used for drug intervention. Serum in each group was collected, inactivated and then added into the primary hippocampal neurons for 24 h. The protein expression levels of 5-HT3AR and 5-HT3BR in the neurons of each group were examined by Western blotting (WB). The 5-HT3R channel current was recorded by a whole-cell patch clamp. Results: Compared with normal rats, the rats with depression had significantly reduction in total distance of the open-field test (OFT) and sucrose preference ratio (P < 0.01). The hippocampal neurons treated with serum of depressive rats had significantly increased protein expression of 5-HT3AR and 5-HT3BR (P < 0.05) and current density value (P < 0.05) compared to those treated with normal rat serum. Compared with the depressive rats, the rats treated with PARE and fluoxetine had significantly increased OFT (P < 0.05) and sucrose preference ratio (P < 0.01). The primary hippocampal neurons cultured with serum from PARE and fluoxetine-treated rats had significantly reduction in protein expression of 5-HT3AR and 5-HT3BR (P < 0.05, 0.01, 0.001) and current density value (P < 0.01). Conclusion: PARE can reduce the 5-HT3R ion channel current density in the rats with depression. This may be its central mechanism in treating depression.
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Objective To explore the impacts and potential mechanisms of MitoQ on isoflurane-induced injury of primary cultured hippocampal neurons in newborn rats.Methods Fifteen healthy SPF Sprague-Dawley rats of both sex were randomly divided into three groups (n =5 each)u-sing a random number table:control group (group C),multiple exposures to isoflurane anesthesia group (group I)and multiple exposures to isoflurane anesthesia+MitoQ group (group IM).On post-natal days 7,14 and 21,1.5% isoflurane was inhaled for 2 h in group I.MitoQ was intraperitoneally administered in a volume of 0.4 ml/kg before isoflurane anesthesia in group IM,while a mixture of oxygen and air was inhaled instead of isoflurane in group C.HE staining was carried out on postnatal day 28 to observe the morphological changes in hippocampal CA1 region of rat neural cell structures. Hippocampal neuron cells were dissected from clean Sprague-Dawley rats born in 24 h.After primary culture for seven days,MTT assay and TUNEL assay was respectively performed to measure the cell viability and apoptosis of hippocampal neurons.The malondialdehyde (MDA)content and superoxide dismutase (SOD)activity were detected respectively using the thiobarbituric method and xanthinoxi-dase method.Mitochondrial membrane potential (MMP)was measured by rhodamine 123 staining, intracellular levels of reactive oxygen species (ROS)were tested by DCFH-DA staining.Western blot was used to analyze the protein levels of Bax,Bcl-2 and caspase-3.Results Compared with group C, group I decreased the number of neural cells and the cell survival rate;the apoptotic rate was signifi-cantly increased;MDA contents and ROS production were significantly increased;SOD activity and MMP level were significantly decreased;the expression of Bax and caspase-3 were significantly in-creased,while the expression of Bcl-2 was significantly decreased (P < 0.05 ).Compared with the group I,the damaged neural cells were decreased,the cell survival rate was significantly increased, the apoptotic rate was significantly decreased in group IM;MDA contents and ROS production were significantly decreased;SOD activity and MMP level were significantly increased;the expression of Bax and caspase-3 were significantly decreased,while the expression of Bcl-2 was significantly in-creased (P < 0.05 ).Conclusion Antioxidant MitoQ attenuates isoflurane-induced neuron damage, which may be associated with the inhibition on oxidative stress and mitochondrial dysfunction.
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Objective Obstructive sleep apnea hypopnea syndrome (OSAHS) often causes damage to multiple systems, especially to the central nervous system, inducing cognitive dysfunction.This study aims to explore the possible correlation of the expressions of serum hypoxia-inducible factor-1α (HIF-1α) and brain-derived neurotrophic factor (BDNF) with cognitive impairment in rats under different hypoxia conditions.Methods Twenty-four Wistar rats were equally randomized into a normal control, a chronic intermittent hypoxia (CIH), and a chronic continuous hypoxia (CCH) group.The rats of the CIH group were placed in a hypoxia chamber filled with N2 and air, the oxygen concentration switched from (7±0.5)% to 21%, 1.5 minutes for each state and 4 minutes for each cycle, while those of the CCH group were placed in another hypoxia chamber with the oxygen concentration of (7±0.5)%, 8 hours a day and all for 30 days.Then we recorded the body weight of the rats, detected the expressions of serum HIF-1α and BDNF by ELISA, and observed the changes of behavior by Morris water maze test and those of the hippocampal morphological structure by HE staining.Results At 30 days after modeling, the body weight of the rats was significantly decreased in the CIH and CSH groups as compared with the normal control ([195.75±6.497] and [180.88±12.017] vs [218.63±15.287] g, P<0.05).Positioning navigation showed that the escape latency was significantly longer in the hypoxia models than in the controls (P<0.05), even longer in the CIH than in the CCH group (P<0.05).Spatial exploration test manifested a lower frequency of crossing the platform in the CIH and CCH groups than in the control ([2.63±1.45] and [3.22±1.30] vs [4.97±0.47] times, P<0.05).The expression levels of serum HIF-1α and BDNF were significantly higher in the CIH ([36.14±9.34] and [1625.34±332.44] pg/mL) and CCH ([27.27±6.88] and [1204.07±363.81] pg/mL) than in the normal control group ([14.11±4.06] and [1036.40±124.48] pg/mL) (P<0.05), even higher in the CIH than in the CCH group (P<0.05).HE staining exhibited scattered and disorderly arrangement of hippocampal neurons in the model rats, with unclear nuclear membrane, pyknosis of the nuclei, darkly stained cytoplasm, and some damaged cells.More obvious absence and vacuolization of some cells were observed in the rats of the CIH group.Conclusion Chronic hypoxia inhibits the growth and development of rats and induces cognitive dysfunction.High-level HIF-1α in chronic intermittent hypoxia indicates hypoxia-stress of the body, while compensatory increase of serum BDNF may be involved in neuronal cell damage regulation.
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Objective To study the effect and mechanism of endoplasmic reticulum stress on hippocampal neuron apoptosis and cognitive impairment in type 1 diabetic rats. Methods Ten rats were randomly selected as normal control group (Con group) from thirty Wistar rats, and the remaining twenty rats were intraperitoneally injected with streptozotocin to prepared for type 1 diabetic rat model. The successful model rats were randomly divided into diabetic group (DM group) and diabetic +Salubrinal group (DM+Sal group). Rats of DM+Sal group were injected by lateral ventricle with Salubrinal ( 75μmol/L, 1μL). Rats of Con group and DM group were given the equal volume of normal saline. The rat cognitive function was assayed by Morris water maze test. The hippocampal neuron apoptosis was detected by TUNEL and flow cytometry methods. The protein levels of endoplasmic reticulum stress markers, GRP78, CHOP and Caspase12, and the mRNA levels of GRP78 and CHOP, were examined by Western blot assay and Real-time PCR, respectively. Results Compared with the Con group, the cognitive function was significantly decreased in DM group, and the apoptotic rate of hippocampal neurons was increased. The protein levels of endoplasmic reticulum stress markers GRP78, CHOP and Caspase12 were significantly increased (P<0.05), and the mRNA levels of GRP78 and CHOP were also higher in DM group (P<0.05). After the injection with Salubrinal, the cognitive function was improved and hippocampal neuron apoptotic rate was decreased in DM+Sal group compared with those of DM group. Moreover, the protein levels of GRP78, CHOP and Caspase12 were significantly decreased (P<0.05) and the mRNA levels of GRP78 and CHOP were also lowered in DM+Sal group (P<0.05). Conclusion The cognitive impairment and the higher apoptotic rate appear in the diabetic rats, and endoplasmic reticulum stress may play an important role in it.
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Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention.
Subject(s)
Animals , Mice , Cell Line , Cytoplasmic Dyneins , Genetics , Metabolism , Hippocampus , Metabolism , Pathology , MAP Kinase Kinase Kinases , Genetics , Metabolism , MAP Kinase Signaling System , Mice, Knockout , Neurodegenerative Diseases , Genetics , Metabolism , Pathology , Proto-Oncogene Proteins B-raf , Genetics , Metabolism , ras Proteins , Genetics , MetabolismABSTRACT
Objective To investigate the effect of different concentrations of dexmedetomidine on the apoptosis of hippocampal neurons in neonatal rat induced by propofol in vitro. Methods Hippocampal neurons of primary cultured neonatal SD rat were divided randomly into three groups. Group C (control group)was normal cultured without any treatment for 12 h; group P (Propofol group)was incubated with 12 μg/mL propofol for 12 h and group DP (Dexmedetomidine + propofol group)was incubated with 0.002 5 ~ 25 μg/mL dexmedetomidine for 30 min, and then further incubated with 12 μg/mL propofol for 12 h. Results Compared with that of group C, the apoptosis rate of hippocampal neurons increased in group P and DP (P < 0.05 or P < 0.01); Compared with that of group P, the apoptosis rate of neurons decreased with the increase of dexmedetomidine concentration in group DP (P<0.05 or P<0.01). The result of transmission electron microscope indicated that compared with group C , group P showed obvious neuronal damage; the nerve cells damage alleviated in group DP, which were negatively associated with the concentration. Conclusions With the concentration ranging from 0.002 5 to 25 μg/mL, dexmedetomidine set pre-incubation and breeding can reduce apoptosis of hippocampus neuron of neonatal rats induced by propofol and the effect is concentration dependent.
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Objective To explore the role of tetramethylpyrazine (TMP) in protection of homocysteine (HCY)-induced apoptosis of hippocampal neurons in mice. Methods One hundred healthy adult Kunming mice of clean grade were randomly divided into 5 groups, 20 for each group. HCY was injected into lateral ventricle using stereotaxic technique. FJB staining was used to detect neural apoptosis and the Morris water maze test was applied to assess memory and cognitive abilities. Results FJB staining showed that apoptosis of hippocampal neurons in the mice treated with TMP at different doses decreased significantly as compared with that in HCY group, especially at a dose of 20 g/L. Conclusions TMP can alleviate HCY-induced damage of the hippocampal neurons in mice and improve memory and cognitive functions, which suggests TMP has a potential clinical value in prevention and treatment of Alzheimer′s disease.
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ABSTRACT:Objective To study the BDNF/TrkB signaling pathway in the epilepsy model of hippocampal neurons and the regulatory effect of on it.Methods The primary hippocampal neurons cultured in vitro for 7 days were randomly divided into seven groups:control group,epilepsy group,control+BDNF group,epilepsy+BDNF group,control + miR-185 ? group,epilepsy + miR-185 ? group,and epilepsy + miR-185 ? + BDNF group.We constructed miR-185 ? lentivirus vector and observed the changes of BDNF/TrkB pathway expression after transfaction of miR-185 ? by immunohistochemistry,patch clamp technique and Western blot technique.Results Compared with the control+BDNF group,the phosphorylated TrkB (pTrkB)/TrkB value was significantly lower in epilepsy+BDNF group (P < 0.05 )and control group (P < 0.001 ).Compared with the epilepsy group,the phosphorylated TrkB (pTrkB)/TrkB value was significantly higher in epilepsy+BDNF group (P <0.05).Compared with the epilepsy+miR-185 ? +BDNF group,the phosphorylated TrkB (pTrkB)/TrkB value was significantly lower in epilepsy + BDNF group and epilepsy + miR-185 ? group (P < 0.001 ).BDNF could promote the signaling conduction and miR-185 ? could remove the inhibition of BDNF/TrkB signaling.Conclusion BDNF can activate the BDNF/TrkB signaling pathway and transfection with miR-185 ? can relieve the inhibition of BDNF/TrkB signaling pathway of epileptic state by up-regulating the expression of TrkB.