Protective effect and mechanism of zonisamide on oxygen-glucose deprivation cell model of traumatic brain injury / 中华急诊医学杂志

Objective:To explore the protective effect of zonisamide (ZNS) on oxygen-glucose deprivation (OGD) cell model of traumatic brain injury (TBI), and its underlying mechanism.Methods:Human neuroblastoma cells (SH-SY5Y) were cultured in vitro and divided into the control group, OGD group, and drug administration group (OGD+ZNS group) according to the random number table method. The OGD method was used to establish a TBI cell model. After modeling, the cell activity, the release of lactate dehydrogenase (LDH), and β-galactosidase staining were detected to evaluate cell function and senescence. Additionally, mitochondrial morphology and potential membrane changes were observed using Mito Tracker Red and JC-1 mitochondrial membrane potential staining. ATP concentration was measured, and protein was extracted from SH-SY5Y cells and then subjected to Western blot analysis to detect endoplasmic reticulum stress-related markers, including glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), protein disulfide isomerase (PDI), and β-actin.Results:The OGD group had a significantly lower cell survival rate compared to the control group ( P<0.01), while the OGD+ZNS group had a significant higher cell survival rate than the OGD group ( P<0.01). The LDH release rate was significantly higher in the OGD group than in the control group ( P<0.01), while the OGD+ZNS group had a significant lower LDH release rate compared to the OGD group ( P<0.01). Moreover, the cell staining results indicated that compared to the control and OGD+ZNS groups, the cells in the OGD group exhibited significant damage and senescence with darker staining while the mitochondrial staining results demonstrated a significant reduction in mitochondrial linear junctions and decreased mitochondrial activity in the OGD group compared to the control and OGD+ZNS groups. Compared to the control and OGD+ZNS groups, the OGD group exhibited a significant reduction in mitochondrial staining red fluorescence, a significant increase in green fluorescence, and a significant decrease in mitochondrial membrane potential. The OGD group demonstrated a significant decrease in ATP concentration compared to the control group ( P<0.01), whereas the OGD+ZNS group exhibited a significant higher ATP concentration compared to the OGD group ( P<0.01). Western blot analysis revealed significant upregulation of GRP78, CHOP, and PDI in the OGD group compared to the control group (all P<0.05), while in the OGD+ZNS group, the expression levels of these proteins were significantly downregulated compared to the OGD group (all P<0.05). Conclusions:Zonisamide can protect OGD TBI cell model by preserving mitochondrial activity and inhibiting endoplasmic reticulum stress.

Mechanism of Dried Fruiting Bodies of Fomes officinalis Against Alzheimer's Disease： Based on Network Pharmacology and In Vitro Experimental Verification / 中国实验方剂学杂志

ObjectiveTo explore the possible mechanism of dried fruiting bodies of Fomes officinalis （FOA） against Alzheimer's disease （AD） based on network pharmacology and experimental verification. MethodThe effective components of FOA were retrieved from a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM） and previous reports. The targets of the components were searched from PharmMapper and TargetNet， and the targets related to AD from Gene Expression Omnibus （GEO）， DrugBank， among other databases. Thereby， the common targets of FOA and AD were obtained， and the protein-protein interaction （PPI） network and component-target network were established based on STRING and Cytoscape 3.7.1， followed by the topology analysis of the networks， and Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis of the common targets. The results were verified by the molecular docking and the in vitro cell experiment. ResultA total of 24 candidate components and 242 predicted targets of FOA， and 96 common targets of FOA and AD were screened out. The key components included ［2-（1-carboxyhexadecylamino）-2-aminosuccinic acid］， 3-keto-dehydrosulfurenic acid， and eburicoic acid， and the active targets were albumin （ALB）， acetylcholinesterase （AChE）， estrogen receptor 1 （ESR1）， cysteine aspartate-specific protease-3 （Caspase-3）， and beta-secretase1 （BACE1）. The common targets were involved in 392 GO terms， and the key terms were the β-amyloid metabolic process and cholinesterase activity. A total of 77 KEGG pathways were obtained， which mainly included estrogen signaling pathway， cholinergic synapse， and AD. The results of molecular docking showed that 7 components of FOA had high binding affinity to amyloid precursor protein （APP）， BACE1， AChE， and Caspase-3. The cell survival rate rose （P<0.01） and the mRNA and protein expression of APP， BACE1， AChE， and Caspase-3 reduced in FOA groups in a dose-dependent manner compared with those in the model group （P<0.05）. ConclusionThis study reveals for the first time that FOA has multi-component， multi-target， and multi-pathway characteristics in the treatment of AD， which serves as a reference for further explaining the mechanism of FOA against AD.

Protective Mechanism of Banxia Houputang on Lipopolysaccharide-induced Neuroinflammatory Injury / 中国实验方剂学杂志

ObjectiveTo study the inhibitory effect of Banxia Houputang （BHT） on lipopolysaccharide （LPS）-induced inflammation of microglia （BV2） cells and the neuroprotective effect on human neuroblastoma （SH-SY5Y） cells. MethodAfter the neuroinflammatory model was constructed by LPS inducing BV2 cells， model group （LPS 100 µg·L-1）， administration groups （LPS+1 g·L-1 BHT， LPS+2 g·L-1 BHT， LPS+5 g·L-1 BHT， LPS+10 g·L-1 BHT）， and blank group were given DEME medium at the same volume. In addition， neuronal apoptosis model was established by co-culture of LPS-induced BV2 cell inflammation medium and SH-SY5Y cells （LPS-DMEM） and was administrated according to the above grouping. Cell viability was detected by Cell Counting Kit-8 （CCK-8） assay. The content of nitric oxide （NO） and that of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were determined by Griess aasay and enzyme-linked immunosorbent assay （ELISA）， respectively. The mRNA levels of TNF-α， IL-1β， interleukin-4 （IL-4）， nitric oxide synthase （iNOS）， and interleukin-10 （IL-10） were measured by real-time polymerase chain reaction （Real-rime PCR）. Western blot was used to detect the expression levels of signal transducer and activator of transcription 3 （STAT3）， Janus kinase 2 （JAK2） and nuclear factor kappa-B （NF-κB p65）， protein kinase B （Akt）， inhibitor of nuclear factor κB α （IκBα）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2 associated X protein （Bax）. ResultCompared with blank group， LPS increased the NO release， levels of TNF-α， IL-1β， IL-6， and iNOS and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3， decreased the content of IL-4 and IL-10 in BV2 cells， and induced apoptosis of co-cultured SH-SY5Y cells （P<0.01）. Compared with model group， BHT reduced the content of NO， TNF-α， IL-1β， and iNOS （P<0.01） and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3 （P<0.01）， elevated the content of IL-4 and IL-10 （P<0.01）， and inhibited the apoptosis of SH-SY5Y cells induced by LPS-DMEM （P<0.01）. ConclusionThis experiment reveals that BHT inhibited LPS-induced inflammation in BV2 cells by regulating Akt/NF-κB/JAK2/STAT3 signaling pathway and showed neuroprotective effects on SH-SY5Y cells.

Inhibitory effect of ketogenic diet on neuroblastoma in BALB/c-nu mouse models / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of ketogenic diet (KD) on growth of neuroblastoma in mice.@*METHODS@#BALB/c-nu mouse models bearing neuroblastoma xenografts were established by subcutaneous injection of human neuroblastoma cell line (SH-SY5Y). When the tumor volume reached 250 mm3, the mice were randomized into SD group with standard diet and PBS treatment, KD group with ketogenic diet and PBS treatment, and CP+KD group with ketogenic diet and cyclophosphamide (60 mg·kg·day) treatment, =8. The tumor volume, body weight, blood glucose, ketone body (β-Hydroxybutyrate) levels, and hepatic steatosis in the mice were assessed. The expressions of caspase-3 and caspase-8 were detected by Western blotting, and Ki67 expresison was detected using immunohistochemistry (IHC). Transmission electron microscopy (TEM) was employed for the autophagosomes, and the autophagic protein Beclin1, LC3A/B and P62 were detected by IHC and Western blotting.@*RESULTS@#On day 28 post tumor cell injection, the mice in KD and CP+KD groups could prolong the overall survival rates than that in SD group ( < 0.001). On day 22 post the injection, the tumor volume in KD group was smaller than that in SD group ( < 0.05); on 16, 19, and 22 day post the injection, the tumor volume in CP+KD group was smaller than that in SD group ( < 0.01). The mice in SD group showed greater body weight on day 19 and higher blood glucose level on day 13 post the injection than those in the other two groups ( < 0.05). Blood ketone level and hepatic steatosis score were higher and glucose ketone index (GKI) was lower in KD and CP+KD groups than those in SD group (all < 0.05). The expressions of Ki67 and apoptotic proteins were detected in the tumor tissues of all groups. TEM revealed more autophagosomes in the tumor tissues of KD group than that of SD group. P62 expression was lowered ( < 0.01) and Beclin1 and LC3A/B expressions were up-regulated in the tumor tissues of KD group ( < 0.05), which is consisitent with IHC.@*CONCLUSIONS@#KD has a strong anti-tumor effect in the xenograft mouse model possibly by regulating cell autophagy.

Esomeprazole and pantoprazole enhance the antiproliferative effects of cisplatin on the human neuroblastoma SH-SY5Y cell line

Background: Proton pump inhibitors (PPIs) largely used a drug to treat gastroesophageal disease such as gastric ulcers. Moreover, in recent years, several studies suggest that PPIs have an important anti-cancer effect in monotherapy and or combination with chemotherapy. The aim of this study was to investigate whether esomeprazole and pantoprazole exhibit anti-cancer effect alone or could enhance chemosensitivity on the human neuroblastoma cell line SH-SY5Y to cisplatin.Methods: The human neuroblastoma SH-SY5Y cells were cultured and treated with different concentrations of esomeprazole, pantoprazole, and cisplatin alone. Also, these cells exposed to cisplatin+ esomeprazole and cisplatin + pantoprazole combinations, respectively and incubated 24 h. The antiproliferative activities of the (PPIs) alone or in a combination of cisplatin was evaluated using the XTT colorimetric assay.Results: According to experimental data, neither PPIs showed no cytotoxicity on the human neuroblastoma cell line SH-SY5Y at all concentrations. However, when combined with cisplatin separately, they were found to have significant antiproliferative effects on the human neuroblastoma SH-SY5Y cell lines when compared to cell lines treated with cisplatin alone (p<0.05).Conclusions: Taken together, the inhibition of V-ATPase via esomeprazole and pantoprazole might enhance the chemosensitivity of cisplatin on the human neuroblastoma cell line SH-SY5Y. However, further studies are needed to be able to utilize PPIs in human neuroblastoma cells.

Protective Effect of Syringaresinol on Excitatory Damage Induced by Sodium Glutamate in SH-SY5Y Cells / 中国实验方剂学杂志

Objective: To establish a model for the injury of human neuroblastoma cell (SH-SY5Y) induced by sodium glutamate, and to observe the protective effect of syringaresinol on cell damage from Viscum liquidambaricolum hayataon, and to explore its mechanism. Method: Construction of SH-SY5Y cell injury model using sodium glutamate.The experiment was divided into normal cell group, injury model group (sodium glutamate 50 mmol·L-1, sodium glutamate 50 mmol·L-1 + DMSO),syringaresinol experimental group (6.25, 12.5, 25 μmol·L-1), by cell counting, cell morphology observation, Annexin V-FITC/PI apoptosis detection, ROS reactive oxygen species detection, mitochondrial membrane potential, and Western blot, evaluation of syringaresinol on glutamate-induced neuronal excitability injury neuroprotective activity. Result: Compared with normal group, the cell survival rate of the model group was significantly decreased (PPPPP-1) showed a concentration-dependent increase in cells. Survival rate (PPPPPConclusion: Syringaresinol has significant protective activity against excitatory damage induced by sodium glutamate in SH-SY5Y neurons, the mechanism may be through anti-oxidative stress, repairing mitochondrial function and DNA damage to significantly reduce sodium glutamate-induced neuronal apoptosis.

Expression of p75 neurotrophin receptor in human neuroblastoma cells under condition of oxygen-glucose deprivation and its significance / 第二军医大学学报

Objective: To explore the expression and effect of p75 neurotrophin receptor (p75NTR) in human neuroblastoma cell line (SH-SY5Y cells) under the condition of oxygen-glucose deprivation (OGD). Methods: The OGD model of SH-SY5Y cells was established by glucose-free and serum-free culturing using tri-gas incubator, and then was assigned to 3 groups, including serum-free regular culturing group (control group), OGD group and OGD + LM11A-31 (a competitive blocker of p75NTR) group. After 12 h of culturing, the cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release activity was determined by LDH activity assay kit, cell apoptosis proportion was detected by flow cytometry, and p75NTR protein expression was detected by Western blotting. Results: The OGD model of SH-SY5Y cells was successfully established. Twelve hours after cell culture, the cell viability of the control, OGD and OGD + LM11A-31 groups was (94.80 ± 4.06)%, (50.34 ± 5.55)% and (64.68 ± 4.59)%, the LDH release activities were (46.93 ± 5.49) U/L, (353.09 ± 30.67) U/L and (282.20 ± 25.60) U/L, the proportions of apoptosis cells were (1.82 ± 0.45)%, (14.98 ± 2.59)% and (7.36 ± 1.98)%, and the relative expression levels of p75NTR were 0.06 ± 0.01, 0.41 ± 0.02 and 0.19 ± 0.03, respectively, and the differences were all significant (F=67.94, 142.10, 36.28 and 221.20, all P<0.05). Post hoc analysis showed that the cell viability of the OGD group was significantly lower than that of the control group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD group were significantly higher (Bonferroni test, all P' < 0.05). After treatment with LM11A-31, the cell viability of the OGD + LM11A-31 group was significantly higher than that of the OGD group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD LM11A-31 group were significantly lower (Bonferroni test, all P' < 0.05). Conclusion: The expression of p75NTR is increased in human neuroblastoma cell line SH-SY5Y cells under OGD condition, which may promotes neuronal injury and apoptosis.

Regulation of p38 mitogen-activated protein kinase pathway by West Nile virus in human neuroblastoma cells / 第二军医大学学报

Objective: To explore the regulation of p38 mitogen-activated protein kinase (MAPK) pathway by West Nile virus (WNV) in human neuroblastoma SH-SY5Y cells and the contributions of p38 MAPK to WNV replication as well as stress and inflammatory response related molecule expression. Methods: Total and phosphorylated p38 MAPK levels were analyzed in SH-SY5Y cells incubated with WNV for short (5, 15, 30 and 60 min) and long (12, 24, 48 and 60 h) durations by Western blotting. Dynamic changes of CCAAT/enhancer-binding protein homologous protein (CHOP), interleukin 6 (IL-6), activating transcription factor 6α (ATF6α) and interferon-stimulated gene 15 (ISG15) mRNA expression in WNV infected cells were detected by qRT-PCR. In response to WNV infection, WNV RNA level and CHOP, IL-6, ATF6α and ISG15 mRNA levels were assessed in SH-SY5Y cells transfected with p38 MAPK siRNA. Results: Incubation with WNV for short durations enhanced p38 MAPK phosphorylation compared to the untreated control. The p38 MAPK signaling pathway was activated at 12 h and 24 h in WNV-infected SH-SY5Y cells, but down-regulated at 48 h and 60 h. WNV infection led to increased mRNA expression of CHOP, IL-6 and ISG15 and reduced ATF6α mRNA. In comparison with control siRNA transfection, the levels of WNV RNA (P<0.05) and ATF6α mRNA (P<0.01) were increased and CHOP mRNA level was decreased (P<0.05) in WNV-infected SH-SY5Y cells with the p38 MAPK siRNA transfection. Conclusion: The p38 MAPK pathway is activated during early stage of WNV infection and such activation may negatively regulate WNV replication. WNV-induced stress response molecules CHOP and ATF6α and proinflammatory cytokine IL-6 production by SH-SY5Y cells are coupled with the regulation of p38 MAPK pathway.

Effect of silencing Fisl gene on METH-induced proliferative capacity, mitochondrial membrane potential and ultrastructure of SH-SY5Y cells cultured in vitro / 中国药理学通报

Aim To evaluate the role of fisson I (Fisl) in methamphetamine (METH)-induced injur)' of human neuroblastoma (SH-SY5Y) cells cultured in vitro. Methods SH-SY5Y cells cultured in vitro were divided into different groups by the group design method∗. unsilent groups, silent negative groups and silent groups. Different concentrations of METH induced SH-SY5 Y cells in each group for 24 hours. The expression level of Fisl was detected by Western blot. The effect of METH on the proliferative capacity of SH-SY5Y cells was analyzed by CCK-8 cytotoxicity proliferation assay. The MMP level of METH on SH-SY5Y cells was detected by mitochondrial membrane potential detection kit (JC-1). The effect of METH on the mitochondrial ultrastructure of SH-SY5Y cells was observed by transmission electron microscopy. Results In unsilent group, silent negative group and silent group, the expression level of Fisl increased (P < 0. 05) and the proliferative capacity decreased (P < 0. 05) , and the MMP levels decreased (P <0. 05) with the increase of the concentration of SH-SY5Y cells induced by METH. Compared with the same concentration in unsi-lent group and silent negative group, in silent group, the expression level of Fisl in SH-SY5Y cells de-creased (P < 0. 05) , the proliferative capacity increased (P<0. 05) , and the MMP level increased (P < 0. 05). Compared with control group, 2. 0 mmol • L"1 METH induced unsilent groups, silent negative groups and silent groups, and transmission electron microscopy showed the increase in the mitochondrial small globular structure (P < 0. 01). Conclusion Fisl may play a key role in METH-induced injury of SH-SY5Y cells cultured in vitro.

The effects of resveratrol on mitochondrial biogenesis dysfunction induced by fluoride in human neuroblastoma SH-SY5Y cells / 中华劳动卫生职业病杂志

Objective@#To explore the role of mitochondrial biogenesis and the neuroprotective mechanism of resveratrol in fluoride neurotoxicity.@*Methods@#SH-SY5Y cells in exponential phase were treated with different concentrations (20, 40, 60 mg/L) of sodium fluoride (NaF) for 24 h. Co-treatment with 60 mg/L NaF, 20 μmol/L resveratrol (RSV) was administrated in the resveratrol intervene trial. Western blotting was used to determine the expression levels of mitochondrial biogenesis key regulating factor of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) , nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in SH-SY5Y cells. The mRNA levels of PGC-1α, NRF1 and TFAM were determined by Quantitative Real-time PCR in SH-SY5Y cells, as well as the relative mitochondrial DNA (mtDNA) contents and mRNA expression of mitochondrial respiratory chain complexes subunit CO1 and ATP8. Flow cytometry was used to determine mitochondrial membrane potential in SH-SY5Y cells.@*Results@#Both the protein and mRNA levels of PGC-1α, NRF1 and TFAM were decresed after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05) . The relative mtDNA contents and mRNA expression of complexes subunit CO1 and ATP8 were also significantly decreased compared with control (P<0.05) . Mitochondrial membrane potential were also significantly decreased after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05) . Compared with 60 mg/L NaF group, the protein and mRNA levels of PGC-1α, NRF1 and TFAM in 20 μmol/L RSV+60 mg/L NaF group were significantly increased (P<0.05) . The relative mtDNA contents, mitochondrial membrane potential and mRNA levels of complexes subunit CO1 and ATP8 in 20 μmol/L RSV+60 mg/L NaF group were also significantly higher than that in 60 mg/L NaF group (P<0.05) .@*Conclusion@#Resveratrol may alleviate the fluoride-induced mitochondrial biogenesis dysfunction in SH-SY5Y cells.

The effects of co-culture of L-iminoethyl ornithine and excessive fluorine on the expression of nitric oxide/endothelial nitric oxide synthase in human neuroblastoma SH-SYSY cells / 中华地方病学杂志

Objective To investigate the effects of endothelial nitric oxide synthase (eNOS) specific inhibitor L-iminoethyl ornithine hydrochloride (L-NIO) for all the fluorine in vitro cultivation of SH-SY5Y cells apoptosis,eNOS mRNA and protein expression,and effect of nitric oxide (NO) content and nitric oxide synthase (NOS) activity change.Methods The SH-SY5Y cells cultured in vitro were divided into control group,low fluoride group,high fluoride group,L-NIO group,low fluoride with L-NIO group,high fluoride with L-NIO group,n =3.The control group added equal volume culture liquid with the experimental group,the concentration of sodium fluoride (NaF) in the low fluoride group and the high fluoride group were 0.2 and 2.0 mmol/L,respectively.The L-NIO group added 3 μmol/L L-NIO,the low fluoride with L-NIO group and the high fluoride with L-NIO group were added to 0.2 mmol/L NaF and 3 μmol/L L-NIO,2.0 mmol/L NaF and 3 μmol/L L-NIO,respectively.The incubation time was 48 h.The expression level of eNOS protein in cells was detected by Western blotting.The expression level of eNOS mRNA in cells was detected by Real-time fluorescence quantitative PCR method.The apoptosis of cells was detected by flow cytometry,NO content and NOS activity in cell culture liquid were detected by nitrate reductase and colorimetric assay.Results Compared with the control group (1.000 ± 0.026),the expression of eNOS protein in the low and high fluoride groups (1.108 ± 0.071,1.349 ± 0.057) increased (P ＜ 0.05),and the L-NIO group (0.755 ± 0.148) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group (0.802 ± 0.115) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (0.988 ± 0.135) decreased (P ＜ 0.05).Compared with the control group (1.000 ±0.018),the expression of eNOS mRNA in the low and high fluoride groups (1.809 ± 0.099,2.416 ± 0.295) increased (P ＜ 0.05),the L-NIO group (0.609-± 0.077) decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated(P ＜ 0.05),the low fluoride with L-NIO group (1.040 ± 0.034) decreased (P ＜ 0.05);compared with the high fluoride group,high fluoride with L-NIO group (1.233 ± 0.152) decreased (P ＜ 0.05).Compared with the control group [(1.66 ± 0.07)％],the cell apoptosis rate in the low and high fluoride groups [(8.81 ± 0.27)％,(17.60 ± 0.20)％] increased,L-NIO group [(1.03 ± 0.04)％] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group increased and the low fluoride with L-NIO group [(6.03 ± 0.10)％] decreased (P ＜ 0.05);compared with the high fluoride group,the high fluoride with L-NIO [(12.12 ± 0.08)％] decreased (P ＜ 0.05).Compared with the control group [(2.773 ± 0.145)μmol/L],the content of NO in the cell culture medium in the low and high fluoride groups [(8.251 ± 1.047),(14.287 ± 1.062) μmol/L] increased (P＜ 0.05),and the L-NIO group [(1.648 ± 0.155) μmol/L] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜ 0.05),the low fluoride with L-NIO group [(4.622 ± 0.252) μmol/L] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(7.899 ± 0.385) μmol/L] decreased (P ＜ 0.05).Compared with the control group [(0.507 ± 0.041) U/ml],the activity of NOS in the cell culture medium in the low and high fluoride groups [(0.772 ± 0.032),(2.258 ± 0.062) U/ml] increased (P ＜ 0.05),and the L-NIO group [(0.346 ±0.015) U/ml] decreased (P ＜ 0.05);compared with the low fluoride group,the high fluoride group was elevated (P ＜0.05),the low fluoride with L-NIO group [(0.637 ± 0.026) U/ml] decreased (P ＜ 0.05);compared with the the high fluoride group,high fluoride with L-NIO group [(1.161 ± 0.071) U/ml] decreased (P ＜ 0.05).Conclusions Excessive fluoride can lead to overexpression of eNOS protein and mRNA in SH-SY5Y cells,increase of apoptosis rate,increase the content of NO in cell culture and enhance the activity of NOS.After co-culture of L-NIO and fluorine,it can antagonize the damage of fluorine to SH-SY5Y cells and play a certain neuroprotective effect.

The role of neuroglobin in oxygen-glucose deprivation and reoxygenation-induced mitochondrial ;depolarization and reactive oxygen species production in SH-SY5Y cells / 中华内科杂志

Objective To investigate the role of neuroglobin ( NGB) in oxygen-glucose deprivation and reoxygenation ( OGD/R ) induced mitochondrial depolarization and reactive oxygen species ( ROS ) production in a human neuroblastoma cell line (SH-SY5Y).Methods SH-SY5Y cells were transfected with lentivirus to establish a stable cell line of NGB knockdown ( KD).After treated with OGD/R, cells were collected at different time points to analyze NGB mRNA and protein levels.Furthermore, cells were stained with JC-1 and DCFH-DA to evaluate mitochondrial depolarization and ROS production by inverted fluorescence microscope.Also, to determine the neurotoxicity , we measured the lactate dehydrogenase ( LDH) level in the cell culture medium.Results After the treatment of OGD/R, the NGB mRNA and protein started to elevate and peak at 4 h and 8 h (2.04 ±0.35 fold,1.69 ±0.18 fold).Compared with the vector group , NGB KD group had much more mitochondrial depolarization [ JC-1 red/green ( 1.10 ±0.10 ) vs (1.46 ±0.11),P<0.05] and ROS production [DCFH-DA fluorescence (36.30 ±5.32) vs (16.26 ± 2.97),P<0.05].Furthermore, NGB KD groups had a higher level of LDH release [(63.42 ±6.14)%vs (49.65 ±5.09 )%, P <0.05 ].Conclusions NGB plays an important role in the homeostasis of mitochondria.Knockdown of NGB results in increased mitochondrial depolarization , ROS production and neurotoxicity under hypoxia circumstances.

The expression and correlation between neural nicotinic acetylcholine receptor subunit α3 and mitogen-activated protein kinase cell signaling transduction pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride / 中华地方病学杂志

Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.

Protective effect of the traditional Chinese medicine prescription, Jiu Nao Yi Zhi water extract, on human neuroblastoma SH-SY5Y cell line / 中国比较医学杂志

Objective To observe the protective effect of the traditional Chinese medicine prescription, Jiu Nao Yi Zhi water extract, on human neuroblastoma SH-SY5Y cell line, its effect on expression of insulin signal transduction pathway, and to explore the related mechanisms.Methods SH-SY5Y cells cultured in vitro, were divided into control group, Jiu Nao Yi Zhi No.1 prescription group and No.3 prescription group.The doses were 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 1 mg/mL.The thiazolyl blue ( MTT) metabolic rate of each group was determined.The dose of 0.125 mg/mL was chosen for cell immunofluorescence analysis, and to observe the expression of insulin receptor substrates-1 ( IRS-1 ) , cAMP response element binding protein ( CREB ) , and the factors of insulin signal transduction pathway.Results Compared with the control group, MTT metabolic rates of the Jiu Nao Yi Zhi groups were significantly increased (P<0.05), and the cell morphology was much better in those groups, cell body more plump, well-adherent and neurite extensions were observed.The expressions of IRS-1 and CREB were higher than that in the control group.Conclusions The traditional Chinese medicine prescription Jiu Nao Yi Zhi water extract can protect neurons by promoting nerve cell growth, and improving the expression of IRS-1 and CREB, the factors of insulin signal transduction pathway.

Study on the Effect of Astragalus Polysaccharide on the Proliferation of Human Neuroblastoma SH-SY5Y Cells / 中国药房

OBJECTIVE:To investigate the inhibitory effect of astragalus polysaccharide(APS)on the proliferation of human neuroblastoma SH-SY5Y cells. METHODS:After the cells were cultured with 0(blank control),25,50 and 100 mg/ml APS for 6,12 and 24 h,MTT method was used to determine cell viability and calculate inhibition rate. Following cell cultured with 0 (blank control),25,50 and 100 mg/ml APS for 24 h,Hoechst 33258 fluorescent staining was performed,and then cell nucleus morphology was observed under the fluorescence microscope;flow cytometer was used to detect the distribution of cell cycles and apoptosis;western blot was employed to determine the expression of extracellular regulated protein kinases (ERK) 1/2 protein in cells. Enzyme linked immunosorbent assay (ELISA) was conducted to determine the contents of interleukin 2 (IL-2),IL-6 and IL-12 in the cells. RESULTS:Compared to the blank control,those cultured with 100 mg/ml APS for 6 h,50 and 100 mg/ml APS for 12 h and 25,50 and 100 mg/ml APS for 24 h demonstrated higher inhibition rate. After the cells were cultured with 50 and 100 mg/ml APS for 24 h,those in G0/G1 phase increased and those in G2/M and S phases decreased,and the contents of IL-2 and IL-6 increased. After cells were cultured with 25,50 and 100 mg/ml APS for 24 h,the apoptosis rate was higher,densely hyperchromat-ic fragments in cell nuclei and apoptotic bodies appeared,the phosphorylation level of ERK1/2 protein in the cells was lower,and the content of IL-12 was higher. There was statistically significance (P<0.01 or P<0.05). CONCLUSIONS:APS can inhibit the proliferation of SH-SY5Y cells by arresting cell cycle and inducing cell apoptosis through a mechanism which may be correlated to the decrease in the phosphorylation of ERK1/2 and increase in cytokine.

Influence of different concentrations of dbcAMP in differentiation potentiality of SH-SY5Y cells to GABAergic-like cells / 吉林大学学报(医学版)

Objective To establish a nerve stem cell model possessing the potentiality of differentiating intoγ-aminobutyric acid neuron-like cells(GABAergic-like cells),and provide eligible investigative vector for study on GABAergic neurons degenerative diseases. Methods Dibutyryl cyclic adenosine monophosphate (dbcAMP)was applied to induce human neuroblastoma SH-SY5Y cells,and the SH-SY5Y cells were divided into control group (0 mmol·L-1 dbcAMP),0.3 mmol·L-1 dbcAMP group,0.6 mmol·L-1 dbcAMP group,1.0 mmol·L-1 dbcAMP group and 2.0 mmol·L-1 dbcAMP group;the morphological changes of SH-SY5Y cells were observed.The Image-Pro Plus 5.0 software was used to measure the length of neurites of the neuron-like cells, and the percentage of the SH-SY5Y cells with neurites longer than 30 μm was calculated. The immunofluorescence cytochemistry technique was utilized to test GAD65 positive cells,and the positive rate of immune response was calculated.Results The results of light microscope observation showed that the cells in control group were polygonal,circular or shuttle type with smooth membrane and clear boundaries. With the increasing of the concentrations of dbcAMP and the prolongation of time,the morphology of SH-SY5Y cells’bodies became smaller with longer processes in dbcAMP groups.The cells interwined each other and showed mature neuron phenotype in 1.0 mmol·L-1 dbcAMP group.Compared with control group(31.4%±4.2%),the percentages of the cell with neurites longer than 30 μm in 0.3, 0.6, 1.0, and 2.0 dbcAMP groups (40.1%± 5.7%, 47.5%± 6.2%, 73.1%±3.2%,and 74.3%± 6.1%)72 h after induction were significantly increased(P<0.05 or P<0.01). Compared with control group (10.2%± 2.1%), the GAD65 positive expression rates in 0.3, 0.6, 1.0,and 2.0 mmol·L-1,dbcAMP groups(22.1%±2.4%,46.9%±3.2%,70.7%±3.4%,and 72.3%±3.7%)72 h after induction were significantly increased(P<0.05 or P<0.01).Conclusion The SH-SY5Y cells have the potentiality of differentiating into GABAergic-like cells, and 1.0 mmol · L-1 is the optimal concentration of dbcAMP.

A2B Adenosine Receptors in SH-SY5Y Cells-Functional Approaches.

The diversity in intracellular signalling downstream of adenosine receptors is dependent on the receptor subtype activated by adenosine. A1 and A2A are considered high affinity receptors while A2B and A3 are considered as low affinity receptors. Despite the apparent widespread distribution of A2B in every cell of every species, the receptor number and low affinity for adenosine and its analogue, makes it the least wellcharacterised adenosine receptor. A2B adenosine receptors are classically coupled to Gs protein and elevation of cAMP levels in target cells, although, A2B have also been known to couple to Gq and not just Gs proteins, and also to couple to Mitogen activated protein kinases (MAPK). In the present study, changes in intracellular calcium concentration ([Ca2+]i) and MAPK phosphorylation following subtype-specific and non-specific agonists and antagonists stimulation were investigated in human neuroblastoma SH-SY5Y cell line. In conclusion, our results indicate that SH-SY5Y cells express A2B adenosine receptors that are coupled to stimulation of Extracellular regulated kinase (ERK1/2) mitogen activated protein (MAP) kinases.

Neuroprotective Effect of Convolvulus pluricaulis Methanol Extract on Hydrogen Peroxide Induced Oxidative Stress in Human IMR32 Neuroblastoma Cell Line.

Aims: The present study aimed to evaluate and ascertain the protective role of methanolic/ethanolic/water extracts of Convolvulus pluricaulis against H2O2 induced cytotoxicity in IMR32 Neuroblastoma cell line as model system and identify the factor responsible for the protective effect. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar & Department of Biotechnology, DAV College, Amritsar, PuCPab, between August 2010 and March 2012. Methodology: Firstly, cytotoxic dose of H2O2 and non-toxic dose of methanolic, ethanolic and water extracts of C. pluricaulis (CP-MEx, CP-EEx and CP-WEx respectively) was determined by MTT assay. Protective effect of CP-MEx, CP-EEx and CP-WEx was determined using quercetin as a positive control. The expression of IMR32 cytoskeletal marker, Neurofilament (NF-200) and stress markers, Heat shock protein (HSP70) and (glucose regulated protein 75, Grp75) Mortalin studied by immunofluorescence and RTPCR results. The level of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, direct scavenger of free radicals, Glutathione and lipid peroxidation were analysed by their standard procedures. Results: The results showed that quercetin, CP-MEx, CP-EEx and CP-WEx displayed cytoprotective activity in IMR32 cells. Out of tested extracts CP-MEx significantly decreased hydrogen peroxide-induced cell death. Significant decrease in NF-200, HSP70 and Mortalin expression was observed in CP-MEx+H2O2 treated cultures as compared to H2O2 treated. Catalase, superoxide dismutase, glutathione peroxidase, Glutathione levels significantly increased in Quercetin and CP-MEx treated cultures. Lipid peroxidation was significantly decreased in both Quercetin and CP-MEx treated cultures. Conclusions: The present work establishes the protective effect of CP-MEx on IMR 32 Human Neuroblastoma cell line which is as much as by quercetin. The cytoprotective effect of CP-MEx was due to induction of antioxidant machinery of the cell hence holds therapeutic value in the treatment and/or prevention of neurodegenerative disorders of oxidative stress.

Protective Effect of Coriolus versicolor Cultivated in Citrus Extract Against Nitric Oxide-Induced Apoptosis in Human Neuroblastoma SK-N-MC Cells

Nitric oxide (NO) is a reactive free radical and a messenger molecule in many physiological functions. However, excessive NO is believed to be a mediator of neurotoxicity. The medicinal plant Coriolus versicolor is known to possess anti-tumor and immune-potentiating activities. In this study, we investigated whether Coriolus versicolor possesses a protective effect against NO donor sodium nitroprusside (SNP)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. We utilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and caspase-3 enzyme activity assay in SK-N-MC cells. MTT assay showed that SNP treatment significantly reduces the viability of cells, and the viabilities of cells pre-treated with the aqueous extract of Coriolus versicolor cultivated in citrus extract (CVEcitrus) was increased. However, aqueous extract of Coriolus versicolor cultivated in synthetic medium (CVEsynthetic) showed no protective effect and aqueous citrus extract (CE) had a little protective effect. The cell treated with SNP exhibited several apoptotic features, while those pre-treated for 1 h with CVEcitrus prior to SNP expose showed reduced apoptotic features. The cells pre-treated for 1 h with CVEcitrus prior to SNP expose inhibited p53 and Bax expressions and caspase-3 enzyme activity up-regulated by SNP. We showed that CVEcitrus exerts a protective effect against SNP-induced apoptosis in SK-N-MC cells. Our study suggests that CVEcitrus has therapeutic value in the treatment of a variety of NO-induced brain diseases.

Biological Analysis of HSV-1 Immediate-early Proteins ICPO, ICP22,and ICP27 in Neuro-blastoma Cells / 中国病毒学·英文版

The three immediate-early proteins of HSV-1, ICPO, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.