Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations....Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.展开更多
Alzheimer’s disease is one of the most debilitating neurodegenerative diseases affecting the aging population. One of the neuropathological hallmarks of AD is an aggregation of the tau protein into neurofibrillary ta...Alzheimer’s disease is one of the most debilitating neurodegenerative diseases affecting the aging population. One of the neuropathological hallmarks of AD is an aggregation of the tau protein into neurofibrillary tangles(NFTs) in neurons and glial cells of the brain. Tau is a microtubuleassociated protein expressed mainly in neurons of the central nervous system[1].展开更多
BACKGROUND: Insulin receptor (IR) expression in the substantia nigra of patients with Parkinson disease (PD) is not only significantly lower than that in the substantia nigra of normal persons of the same age, but als...BACKGROUND: Insulin receptor (IR) expression in the substantia nigra of patients with Parkinson disease (PD) is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease.OBJECTIVE: To observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis of PC12.DESIGN: Controlled observation.SETTINGS: Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology, Huashan Hospital Affiliated to Fudan University.MATERIALS: PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Science. MPP+, MTT, HOECHST 33258 (Invitrogen Life Technologies), reverse transcription-polymerase chain reaction (RT-PCR) reagent (Takara Shuzo Co., Ltd.), flow cytometer (Bacton Dickionson, San Jose, CA), enzyme labelling instrument (Bio-Tek, Winooski, VT) and PCR circulation instrument (Takara Shuzo Co., Ltd) were used in this study.METHODS: This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. ① Cell culture and experimental grouping: PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP+ group and insulin group.② Detection of relative survival rate of cells: The relative survival rate of cells at different MPP+ final concentrations (0, 50, 100, 200, 300, 1 000 μmol/L) and at different culture time (0, 4, 8, 12, 18, 24 hours) in the 300 μmol/L MPP+ group and different concentrations of insulin (0, 15, 50, 100 nmol/L) in the insulin group was detected with MTT method according to the method from Hansen et al. ③ Observation of cell apoptosis: After stained by HOECHST 33258, the apoptotic cells were observed under the fluorescence miscroscope with the method from Chen et al. ④ Dection of apoptotic rate of cells: Apoptotic rate of cells was detected with flow cytometry according to the method from Zhang et al. ⑤ The expression of tyrosine hydroxylase (TH) mRNA in PC12 cells was detected with RT-PCR methods according to the modified method from Peng et al.MAIN OUTCOME MEASURES: Comparison of relative survival rate, apoptosis rate, the expression of IR mRNA and TH mRNA and cell apoptosis.RESULTS: ① After 12-hour incubation of 100, 200, 300 and 1 000 μmol/L MPP+, the relative survival rate of PC12 cells was (72.88±2.91)%, (60.64±0.81)%, (54.56±0.76)% and (16.89±2.83)%, respectively, which was significantly lower than that of blank control group (100%, P < 0.05); After 12, 18 and 24-hour incubation, the relative survival rate of PC12 cells was (54.56±0.76)%, (42.43±0.16)% and (23.56±0.17)% respectively, which was significantly lower than that of blank control group (100%, P < 0.05); When 15, 50 and 100 nmol/L insulin was pre-added to cells, the relative survival rate was (70.10±0.16)%, (78.01±2.43)% and (83.55±1.43)%, respectively, which was significantly higher than MPP+ alone [(54.56±0.76)%, P < 0.05]. ② Appototic bodies were rarely seen in the blank control group, but densely gathered in the MPP+ group and were significantly decreased in the insulin group. ③ Apoptosis rate of PC12 cells in the MPP+ group was significantly higher than that in the blank control group [(36.56±0.89)% vs. (2.34±0.23)%, P < 0.05], and that in the 15, 50, 100 nmol/L insulin group [(30.01±0.04)%, (24.23±0.37)%, (20.01±1.01)%, respectively] was significantly lower than that in MPP+ group (P < 0.05). ④ The TH mRNA expression in PC12 cells in MPP+ group was significantly lower than that in blank control group; The expression of TH mRNA in insulin group was gradually increased in an insulin dose-dependent manner. There were no significant changes in the expression of IR mRNA under different experimental conditions.CONCLUSION: Insulin can resist MPP+-induced apoptosis of PC12 cells, lessen the damage of PC12 cells, but does not change the gene expression of target cell insulin receptor.展开更多
BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE: To observe the effect of p...BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN: Randomized controlled study. SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University. MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate. The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items: At 1, 2 and 3 hours after reoxygenation, absorbance (A value) of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAIN OUTCOME MEASURES: Comparisons between quantity and activity of PC12 cells and mitochondrial membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS: ① Effect of phycocyanin on quantity and activity of PC12 cells: A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation (0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P < 0.05). A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture (P < 0.05). With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation (P < 0.05). ② Effect of phycocyanin on mitochondrial membrane potential of PC12 cells: Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation (1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P < 0.05); but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation (P < 0.05). With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation (P < 0.05). CONCLUSION: Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.展开更多
Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;ho...Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.展开更多
Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully...Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.展开更多
Summary:Dexmedetomidine(DEX),a potent and highly selective agonist for a2-adrenergic receptors(a2AR),exerts neuroprotective effects by reducing apoptosis through decreased neuronal Ca^2+influx.However,the exact action...Summary:Dexmedetomidine(DEX),a potent and highly selective agonist for a2-adrenergic receptors(a2AR),exerts neuroprotective effects by reducing apoptosis through decreased neuronal Ca^2+influx.However,the exact action mechanism of DEX and its effects on oxygen-glucose deprivation-reoxygenation(OGD/R)injury in vitro are unknown.We demonstrate that DEX pretreatment reduced OGD/R injury in PC12 cells,as evidenced by decreased oxidative stress,autophagy,and neuronal apoptosis.Specifically,DEX pretreatment decreased the expression levels of stromal interaction molecule 1(STIM1)and calcium release-activated calcium channel protein 1(Orail),and reduced the concentration of intracellular calcium pools.In addition,variations in cytosolic calcium concentration altered apoptosis rate of PC12 cells after exposure to hypoxic conditions,which were modulated through STIM 1/Orail signaling.Moreover,DEX pretreatment decreased the expression levels of Beclin-1 and microtubule-associated protein 1A/1B-light chain 3(LC3),hallmark markers of autophagy,and the formation of autophagosomes.In conclusion,these results suggested that DEX exerts neuroprotective effects against oxidative stress,autophagy,and neuronal apoptosis afier OGD/R injury via modulation of Caf-STIM1/Orai1 signaling.Our results offer insights into the molecular mechanisms of DEX in protecting against neuronal ischemia-reperfusion injury.展开更多
Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species...Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene is a novel compound that behaves as a free radical scavenger with excellent biology consistent. In the present study, we have synthesized and characterized a novel cystine C60 derivative for the first time, and investigated the effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by MTT, PI/Hoechst 33342 staining and flow cytometry analysis. These results suggested that cystine C60 derivative has the potential to prevent oxidative stress-induced cell death and has no evident toxicity.展开更多
As an aging-associated degenerative disease,Alzheimer’s disease is characterized by the deposition of amyloid beta(Aβ),oxidative stress,inflammation,dysfunction and loss of cholinergic neurons.Colla Corii Asini(CCA)...As an aging-associated degenerative disease,Alzheimer’s disease is characterized by the deposition of amyloid beta(Aβ),oxidative stress,inflammation,dysfunction and loss of cholinergic neurons.Colla Corii Asini(CCA)is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging.CCA might delay aging-induced degenerative changes in neurons.In the present study,we evaluated antioxidant activity,cytoprotective effects,and Aβremovability of enzyme-digested Colla Corii Asini(CCAD).Oxygen radical absorbance capacity(ORAC)activity assay showed that,as compared to gelatins from the skin of porcine,bovine and cold water fish,CCA exhibited the highest ORAC activity.The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage.Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008,2013,2017 and gelatin from cold water fish skin,CCA manufactured in 2008 presented the smoothest surface structure.We further tested the protective effects of CCAD(manufactured in 2008)and enzyme-digested gelatin from cold water fish skin(FGD)on hydrogen peroxide(H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells.Presto blue assay showed that both FGD and CCAD at 0.5 mg/m L increased cell viability in H2O2-treated neuronal-like PC12 cells.The protection of CCAD was significantly superior to that of FGD.Acetylcholinesterase(Ach E)assay showed that both FGD and CCAD inhibited Ach E activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1%and 74.5%of that in non-treated cells,respectively.The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine.Although CCAD inhibited Ach E activity in neuronal-like PC12 cells,CCAD prevented H2O2-induced abnormal deterioration of Ach E.ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ1–42-treated neuronal-like PC12 cells,suggesting that CCAD can enhance Aβclearance.Our results suggest that CCA might be useful for preventing and treating Alzheimer’s disease.展开更多
Alpinia oxyphylla,a traditional herb,is widely used for its neuroprotective,antioxidant and memory-improving effects.However,the neuroprotective mechanisms of action of its active ingredients are unclear.In this study...Alpinia oxyphylla,a traditional herb,is widely used for its neuroprotective,antioxidant and memory-improving effects.However,the neuroprotective mechanisms of action of its active ingredients are unclear.In this study,we investigated the neuroprotective effects of various organic extracts of Alpinia oxyphylla on PC12 cells exposed to hydrogen peroxide-induced oxidative injury in vitro.Alpinia oxyphylla was extracted three times with 95%ethanol(representing extracts 1–3).The third 95%ethanol extract was dried and resuspended in water,and then extracted successively with petroleum ether,ethyl acetate and n-butanol(representing extracts 4–6).The cell counting kit-8 assay and microscopy were used to evaluate cell viability and observe the morphology of PC12 cells.The protective effect of the three ethanol extracts(at tested concentrations of 50,100 and 200μg/mL)against cytotoxicity to PC12 cells increased in a concentration-dependent manner.The ethyl acetate,petroleum ether and n-butanol extracts(each tested at 100,150 and 200μg/mL)had neuroprotective effects as well.The optimum effective concentration ranged from 50–200μg/mL,and the protective effect of the ethyl acetate extract was comparatively robust.These results demonstrate that organic extracts of Alpinia oxyphylla protect PC12 cells against apoptosis induced by hydrogen peroxide.Our findings should help identify the bioactive neuroprotective components in Alpinia oxyphylla.展开更多
BACKGROUND:Previous studies have shown that rifampicin exhibits neuroprotective effects,but the precise mechanisms remain unclear.Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical sc...BACKGROUND:Previous studies have shown that rifampicin exhibits neuroprotective effects,but the precise mechanisms remain unclear.Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger.OBJECTIVE:To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN,TIME AND SETTING:A repeated measure,cell-based study was performed at the Department of Neurology,Second Affiliated Hospital,Sun Yat-sen University,China between December 2007 and November 2008.MATERIALS:PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School,Sun Yat-sen University,China.Rotenone and rifampicin were purchased from Sigma,USA.METHODS:PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco's modified Eagle's medium/Nutrient Mix F12(DMEM/F12) supplemented with 10% fetal bovine serum.The cells were assigned to six groups according to various treatment conditions:control,cultured with normal media;rifampicin group,treated with 300 μmol/L rotenone for 26 hours;rotenone group,treated with 2.5 μmol/L rotenone for 24 hours;rifampicin pretreatment groups,pretreated with 100,200,and 300 μmol/L rifampicin for 2 hours,respectively,followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES:Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry,respectively,using rhodamine123 staining.Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2',7'-dichlorofluorescin-diacetate staining,and intracellular reduced glutathione was measured with a microplate reader.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry.RESULTS:Increased apoptosis in rotenone-induced,differentiated,PC12 cells was accompanied by the loss of mitochondrial transmembrane potential,the formation of reactive oxygen species,and reduced glutathione depletion(P<0.01).Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin(P<0.05 or P<0.01).CONCLUSION:Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.展开更多
Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was co...Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.展开更多
Objective:To explore the possible neuroprotective activities of Humulus japonicus extract against Parkinson's disease(PD)in a cellular model.Methods:PD was modeled in PC12 cells using 6-hydroxydopamine(6-OHDA).The...Objective:To explore the possible neuroprotective activities of Humulus japonicus extract against Parkinson's disease(PD)in a cellular model.Methods:PD was modeled in PC12 cells using 6-hydroxydopamine(6-OHDA).The cell activity,intracellular levels of reactive oxygen species(ROS),anti-oxidative and anti-apoptotic effects,and other related indicators and related signaling pathways were evaluated to elucidate the neuroprotective effects of Humulus japonicus extract.Results:Humulus japonicus extract exhibited anti-oxidative and anti-apoptotic effects in 6-OHDA-stimulated PC12 cells.It also reduced oxidative stress-induced ROS accumulation;upregulated antioxidant enzymes,such as glutathione,catalase,heme oxidase-1,and 8-oxguanine glycosylase 1;promoted cell survival by decreasing BAX and increasing Bcl-2 and sirtuin 1 expression via the MAPK and/or Nrf2 signaling pathways.Conclusions:Humulus japonicus extract has antioxidative and anti-apoptotic effects and could be developed as a promising candidate for preventing and treating oxidative stress-related neurodegenerative diseases.展开更多
In our previous study, defatted walnut meal hydrolysate(DWMH) could attenuate D-galactose-induced acute memory deficits in vivo, and six potent active peptides including WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG ...In our previous study, defatted walnut meal hydrolysate(DWMH) could attenuate D-galactose-induced acute memory deficits in vivo, and six potent active peptides including WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG and ADIYTEEAGR were identified. The aim of this study was to investigate the possible mechanism underlying their neuroprotective effects on glutamate-induced apoptosis in PC12 cells and their digestive stability. Results showed that all these peptides could attenuate the reduction of cell viability caused by glutamate in PC12 cells, especially WSREEQEREE and ADIYTEEAGR. The addition of Arg residue in WSREEQEREE and ADIYTEEAGR might be the potential reason for their stronger protective effects. Additionally, these two peptides possibly protected PC12 cells against glutamate-induced apoptosis via activating intracellular antioxidant defence(superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px)) through Kelch-like ECH-associated protein 1(Keap1) inhibition, inhibiting ROS production, Ca;influx and mitochondrial membrane potential(MMP) collapse as well as regulating the expression of apoptosis-related proteins(Bax and Bcl-2). This might be due to the presence of Trp, Tyr and Arg in these two peptides. However, encapsulation of WSREEQEREE and ADIYTEEAGR should be considered based on their digestive sensibility during in vitro gastrointestinal digestion.展开更多
Aluminum has been associated with neurodegenerative diseases.ALA(α-linolenic acid),an essential dietary component for human health,possesses prominent biological activities.Herein,we aim to explore the neuroprotectiv...Aluminum has been associated with neurodegenerative diseases.ALA(α-linolenic acid),an essential dietary component for human health,possesses prominent biological activities.Herein,we aim to explore the neuroprotective effects of ALA on aluminum toxicity and reveal the underlying mechanism.Results show that aluminum chloride(denoted as Al)enabled cell viability decline and apoptosis with oxidative stress and mitochondrial damage in differentiated rat pheochromocytoma cells(PC12)for 24 h incubation.Compared with Al(10 mmol/L)treatment alone,ALA(50μmol/L)pretreatment for 24 h significantly enhanced cell viability by 28.40%,and hindered cell apoptosis by 12.35%,together with recovering redox state balance and alleviating mitochondrial damage.It was measured that ALA treatment upregulated Bcl-2 expression and down-regulated Bax level,accompanied with an expression decline of caspase-3 and caspase-9.Meanwhile,ALA pretreatment was proved to increase protein kinase A(PKA)expression and to promote phosphorylation of cAMP response element-binding protein(p-CREB),resulting in elevation on the level of brain-derived neurotrophic factor(BDNF).The above results showed that ALA attenuated Al toxicity in PC12 cells by mediating the PKA-CREBBDNF signaling pathway.展开更多
Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The pu...Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The put- pose of this study was to investigate the role of baicalein in modulating dopamine （DA） metabolism in PC12 cells and to explore possible mechanisms of its actions. Methods The intracellular content and extracellular release of DA in both rotenone-treated and untreated PC12 cells were examined. Second, PC12 cells were first pretreated with baicalein （ 10 μmol · L^-1 ） for 10 rain, and then incubated with or without ionomycin （5 μmol · L^-1 ） for 10 rain to test whether short-term exposure to baicalein affected calcium-dependent or spontaneous DA release. Third, the intracellular and extracellular contents of DA and its related metabolites were examined. After treatment with baica- lein for 24 h, the The tyrosine hydroxylase （TH）, monoamine oxidase B （MAOB）, catechol-O-methyltransferase （COMT） and dopamine transporter （DAT） were detected by immunoblot analysis. Results The results showed that baicalein prevented rotenone-induced cytotoxicity and significantly increased the DA content in both rotenone- treated and untreated PC12 cells. Furthermore, it had no effect on ionomycin-induced or spontaneous DA release after short-term exposure but significantly increased DA content in a time- and dose-dependent manner after treat- ment for 6 h. Baicalein also significantly decreased the intracellular and extracellular homovanillic acid （HVA） content but increased the intracellular 3,4-dihydroxy phenylacetic acid （DOPAC） content. Finally, baicalein sig- nificantly decreased the expression of COMT and DAT, but it had no effect on the expression of TH and MAOB. Conclusion These data suggest that bacalein has the ability to increase DA content and modulate DA metabolism by inhibiting the expression of COMT and DAT. Our study provides evidence that baicalein may be a potential anti- PD drug that merits further study.展开更多
Objective To investigate the protective effect of Xanthoceraside on various injured PC12 cells models and to indicate the mechanism of Xanthoceraside therapying dementia.Methods Four injured PC12 models induced by glu...Objective To investigate the protective effect of Xanthoceraside on various injured PC12 cells models and to indicate the mechanism of Xanthoceraside therapying dementia.Methods Four injured PC12 models induced by glutamate,hydrosulfurous sodium,sodium nitroprusside and potassium chloride accordingly were used to assay the effect of Xanthoceraside on PC12 cells by using morphological examination and MTT assay.In addition,in the model of glutamate injury,the Lactate dehydrogenase(LDH)and the lipid peroxidation products malondialdehyde(MDA)were measured by a spectrophotometric method,reactive oxygen species(ROS)generation were measured with flow cytometry.Results It was found that Xanthoceraside could obviously increase the viability of PC12 cells injured by four injury models.Xanthoceraside could also decerase the levels of LDH release,MDA production,and ROS generation induced by glutamate.Conclusions These data indicate that Xanthoceraside may provide a useful therapeutic strategy for the treatment of progressive neurodegenerative diseases such as Alzheimer's disease(AD).展开更多
OBJECTIVE Mu-Xiang-You-Fang(MXYF)is a classic prescription of Hui medicine,composed of five herbs,which has been used to treat ischemic stroke for many years.However,the potential pharmacological mecha⁃nisms of MXYF r...OBJECTIVE Mu-Xiang-You-Fang(MXYF)is a classic prescription of Hui medicine,composed of five herbs,which has been used to treat ischemic stroke for many years.However,the potential pharmacological mecha⁃nisms of MXYF remain unclear.The present research is to investigate the neuroprotective effect of MXYF and its role in modulating autophagy via AMPK/mTOR signaling pathway in the PC12 oxygen-glucose deprivation and reperfusion(OGD/R)injury model.METHODS MXYF was extracted by supercritical CO2 fluid extraction apparatus.PC12 OGD/R injury model was established by oxygen-glucose deprivation for 2 h and reperfusion for 24 h.The effects of MXYF on the viability and cytotoxicity of PC12 cells were determined through cell counting kit(CCK-8)assay.Colorimetric method was performed to determine the LDH leakage rate.The calcium concentration was determined by chemical fluorescence method and the mitochondrial membrane potential was determined through flow cytometry.Monodansylcadaverine(MDC)staining was conducted to detect autophagosome formation.The expression of LC3,Beclin1,p62,p-AMPK,ULK1,p-mTOR and p-p70s6k proteins were determined by immunofluorescence and Western blotting analyses.RESULTS MXYF(1,2 and 4 mg·L^-1)could significantly increase the cell viability and mitochondrial membrane potential,while decreased the release of lactate dehydrogenase(LDH)and calcium concentration in PC12 cells.Mechanistic studies showed that MXYF reduced the LC3-II/LC3-I ratio and inhibited the expression of beclin1,p-AMPK and ULK1.In comparison,the expres⁃sion of p-mTOR,p-p70s6k and p62 were significantly enhanced.CONCLUSION MXYF inhibits autophagy after OGD/Rinduced PC12 cell injury through AMPK-mTOR pathway,thus MXYF might have therapeutic potential for treating the ischemic stroke.展开更多
Nitric oxide (NO) regulates a wide range of physiological processes. Recent studies show that NO can regulate the release of catecholamines (CA) from the adrenal medulla. In the current study, the PC12 cell line was u...Nitric oxide (NO) regulates a wide range of physiological processes. Recent studies show that NO can regulate the release of catecholamines (CA) from the adrenal medulla. In the current study, the PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine Nmethyltransferase (PNMT). Treatment of PC12 cells with the NO donor, sodium nitroprusside (SNP) for 6 hours significantly increased TH, DBH and PNMT mRNA levels. In addition, NO potentiates the regulation of gene expression of all three CA biosynthetic enzymes by glucocorticoids and cholinergic agonists. The signaling pathways involved in NO regulation of CA biosynthetic enzymes were investigated with the use of specific kinase activators and inhibitors, with results supporting a contributing role of PKA, PKC and PKG in SNP-mediated induction for all three CA genes (p < 0.01). In addition, inhibitors of transcription and translation inhibited SNP-mediated induction of all three genes (p < 0.001) suggesting that both transcriptional and translational mechanisms may be involved in CA gene regulation by NO. Results from this study show that in addition to regulating CA biosynthetic enzymes, NO can also potentiate cholinergic and glucocorticoid activation of CA genes.展开更多
BACKGROUND:Electromagnetic radiation can influence dopamine(DA) synthesis in brain tissues or cells,but electromagnetic frequencies,intensities,and radiation time can produce different effects.In addition,the signal p...BACKGROUND:Electromagnetic radiation can influence dopamine(DA) synthesis in brain tissues or cells,but electromagnetic frequencies,intensities,and radiation time can produce different effects.In addition,the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial.OBJECTIVE:To determine tyrosine hydroxylase(TH) expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field(LF-PEF) stimulation,and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors.DESIGN,TIME AND SETTING:A parallel,controlled,cell experiment was performed at the Laboratory of Cell Biology,School of Life Science,East China Normal University,between January and October 2006.MATERIALS:PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,China.Nerve growth factor was purchased from PeproTech,USA.The protein kinase A inhibitor,H-89,and mitogen-activated protein kinase kinase inhibitor,U0126,were purchased from Sigma,USA.METHODS:(1) Following routine culture in Dulbecco's modified eagle medium,primary PC12 cells were stimulated under LF-PEF(pulse frequency 50 Hz,pulse width 20 μs,peak field strength 1 V/m) for 5,10,15,20,and 30 minutes.(2) Inhibitors(H-89 or U0126,1 μmol/L) were added 30 minutes before LF-PEF stimulation for 10 minutes.MAIN OUTCOME MEASURES:(1) TH expression was determined by Western blot in PC12 cells at 0.5,1,2,3,and 4 days after LF-PEF stimulation.Similarly,DA was measured by high-performance liquid chromatography in media at 2,3,4,or 5 days after LF-PEF.(2) TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEF.RESULTS:(1) Short-term LF-PEF stimulation(5 and 10 minutes) increased TH expression and media DA levels after short-term culture(2 days)(P<0.01),but both parameters decreased with longer culture(3-4 days)(P<0.01).Long-term LF-PEF stimulation(15,20,or 30 minutes) decreased TH and DA synthesis,followed by a rapid increase(P<0.01).(2) H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes,while the inhibition rate of U0126 was 53.2%.CONCLUSION:Short-term LF-PEF first promotes then inhibits,while long-term LF-PEF first inhibits then promotes,TH and DA synthesis.LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.展开更多
基金supported by the National Natural Science Foundation of China,No.81973501the Natural Science Foundation of Shandong Province,No.ZR2019MH009(both to YLG).
文摘Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.
基金supported by the National Natural Science Foundation of China[grant number.3187010926]the National Science Foundation of Changchun Normal University[grant number.2021018]。
文摘Alzheimer’s disease is one of the most debilitating neurodegenerative diseases affecting the aging population. One of the neuropathological hallmarks of AD is an aggregation of the tau protein into neurofibrillary tangles(NFTs) in neurons and glial cells of the brain. Tau is a microtubuleassociated protein expressed mainly in neurons of the central nervous system[1].
文摘BACKGROUND: Insulin receptor (IR) expression in the substantia nigra of patients with Parkinson disease (PD) is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease.OBJECTIVE: To observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis of PC12.DESIGN: Controlled observation.SETTINGS: Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology, Huashan Hospital Affiliated to Fudan University.MATERIALS: PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Science. MPP+, MTT, HOECHST 33258 (Invitrogen Life Technologies), reverse transcription-polymerase chain reaction (RT-PCR) reagent (Takara Shuzo Co., Ltd.), flow cytometer (Bacton Dickionson, San Jose, CA), enzyme labelling instrument (Bio-Tek, Winooski, VT) and PCR circulation instrument (Takara Shuzo Co., Ltd) were used in this study.METHODS: This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. ① Cell culture and experimental grouping: PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP+ group and insulin group.② Detection of relative survival rate of cells: The relative survival rate of cells at different MPP+ final concentrations (0, 50, 100, 200, 300, 1 000 μmol/L) and at different culture time (0, 4, 8, 12, 18, 24 hours) in the 300 μmol/L MPP+ group and different concentrations of insulin (0, 15, 50, 100 nmol/L) in the insulin group was detected with MTT method according to the method from Hansen et al. ③ Observation of cell apoptosis: After stained by HOECHST 33258, the apoptotic cells were observed under the fluorescence miscroscope with the method from Chen et al. ④ Dection of apoptotic rate of cells: Apoptotic rate of cells was detected with flow cytometry according to the method from Zhang et al. ⑤ The expression of tyrosine hydroxylase (TH) mRNA in PC12 cells was detected with RT-PCR methods according to the modified method from Peng et al.MAIN OUTCOME MEASURES: Comparison of relative survival rate, apoptosis rate, the expression of IR mRNA and TH mRNA and cell apoptosis.RESULTS: ① After 12-hour incubation of 100, 200, 300 and 1 000 μmol/L MPP+, the relative survival rate of PC12 cells was (72.88±2.91)%, (60.64±0.81)%, (54.56±0.76)% and (16.89±2.83)%, respectively, which was significantly lower than that of blank control group (100%, P < 0.05); After 12, 18 and 24-hour incubation, the relative survival rate of PC12 cells was (54.56±0.76)%, (42.43±0.16)% and (23.56±0.17)% respectively, which was significantly lower than that of blank control group (100%, P < 0.05); When 15, 50 and 100 nmol/L insulin was pre-added to cells, the relative survival rate was (70.10±0.16)%, (78.01±2.43)% and (83.55±1.43)%, respectively, which was significantly higher than MPP+ alone [(54.56±0.76)%, P < 0.05]. ② Appototic bodies were rarely seen in the blank control group, but densely gathered in the MPP+ group and were significantly decreased in the insulin group. ③ Apoptosis rate of PC12 cells in the MPP+ group was significantly higher than that in the blank control group [(36.56±0.89)% vs. (2.34±0.23)%, P < 0.05], and that in the 15, 50, 100 nmol/L insulin group [(30.01±0.04)%, (24.23±0.37)%, (20.01±1.01)%, respectively] was significantly lower than that in MPP+ group (P < 0.05). ④ The TH mRNA expression in PC12 cells in MPP+ group was significantly lower than that in blank control group; The expression of TH mRNA in insulin group was gradually increased in an insulin dose-dependent manner. There were no significant changes in the expression of IR mRNA under different experimental conditions.CONCLUSION: Insulin can resist MPP+-induced apoptosis of PC12 cells, lessen the damage of PC12 cells, but does not change the gene expression of target cell insulin receptor.
基金the Natural Science Foundation of Shandong Province, No. Y2004C04
文摘BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN: Randomized controlled study. SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University. MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate. The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items: At 1, 2 and 3 hours after reoxygenation, absorbance (A value) of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAIN OUTCOME MEASURES: Comparisons between quantity and activity of PC12 cells and mitochondrial membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS: ① Effect of phycocyanin on quantity and activity of PC12 cells: A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation (0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P < 0.05). A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture (P < 0.05). With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation (P < 0.05). ② Effect of phycocyanin on mitochondrial membrane potential of PC12 cells: Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation (1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P < 0.05); but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation (P < 0.05). With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation (P < 0.05). CONCLUSION: Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.
文摘Autophagy has been shown to have a protective effect against brain damage.Ligustilide(LIG)is a bioactive substance isolated from Ligusticum chuanxiong,a traditional Chinese medicine.LIG has a neuroprotective effect;however,it is unclear whether this neuroprotective effect involves autophagy.In this study,PC12 cells were treated with 1×10^-5–1×10^-9 M LIG for 0,3,12 or 24 hours,and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay.Treatment with 1×10^-6 M LIG for 3 hours had the greatest effect on cell proliferation,and was therefore used for subsequent experiments.PC12 cells were pre-treated with 1×10^-6 M LIG for 3 hours,cultured in 95%N2/5%CO2 in Dulbecco’s modified Eagle’s medium without glucose or serum for 4 hours,and then cultured normally for 16 hours,to simulate oxygen-glucose deprivation/reoxygenation(OGD/R).Cell proliferation was assessed with the MTS assay.Apoptosis was detected by flow cytometry.The expression levels of apoptosis-related proteins,Bcl-2 and Bax,autophagy-related proteins,Beclin 1 and microtubule-associated protein l light chain 3B(LC3-II),and liver kinase B1(LKB1)-5′-adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway-related proteins were assessed by western blot assay.Immunofluorescence staining was used to detect LC3-II expression.Autophagosome formation was observed by electron microscopy.LIG significantly decreased apoptosis,increased Bcl-2,Beclin 1 and LC3-II expression,decreased Bax expression,increased LC3-II immunoreactivity and the number of autophagosomes,and activated the LKB1-AMPK-mTOR signaling pathway in PC12 cells exposed to OGD/R.The addition of the autophagy inhibitor 3-methyladenine or dorsomorphin before OGD/R attenuated the activation of the LKB1-AMPK-mTOR signaling pathway in cells treated with LIG.Taken together,our findings show that LIG promotes autophagy and protects PC12 cells from apoptosis induced by OGD/R via the LKB1-AMPK-mTOR signaling pathway.
基金supported by the National Natural Science Foundation of China,No.81671882,81471832the Natural Science Foundation of Guangdong Province of China,No.2016A030311039+1 种基金the Science and Technology Foundation of Guangdong Province of China,No.2015A020212012,2017A020224012the Science and Technology Foundation of Guangzhou City of China,No.201707010373(all to XL)
文摘Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.
基金This study was supported by grants from the National Natural Science Foundation of China(No.81801175 and No.81970722)the Fundamental Research Funds for the Central Universities(No.WK9110000044 and No.WK9110000036)+2 种基金China Scholarship Council(No.201706270155)the China Postdoctoral Science Foundation(No.2019M662179)the Anhui Province Postdoctoral Science Foundation(No.2019B324).
文摘Summary:Dexmedetomidine(DEX),a potent and highly selective agonist for a2-adrenergic receptors(a2AR),exerts neuroprotective effects by reducing apoptosis through decreased neuronal Ca^2+influx.However,the exact action mechanism of DEX and its effects on oxygen-glucose deprivation-reoxygenation(OGD/R)injury in vitro are unknown.We demonstrate that DEX pretreatment reduced OGD/R injury in PC12 cells,as evidenced by decreased oxidative stress,autophagy,and neuronal apoptosis.Specifically,DEX pretreatment decreased the expression levels of stromal interaction molecule 1(STIM1)and calcium release-activated calcium channel protein 1(Orail),and reduced the concentration of intracellular calcium pools.In addition,variations in cytosolic calcium concentration altered apoptosis rate of PC12 cells after exposure to hypoxic conditions,which were modulated through STIM 1/Orail signaling.Moreover,DEX pretreatment decreased the expression levels of Beclin-1 and microtubule-associated protein 1A/1B-light chain 3(LC3),hallmark markers of autophagy,and the formation of autophagosomes.In conclusion,these results suggested that DEX exerts neuroprotective effects against oxidative stress,autophagy,and neuronal apoptosis afier OGD/R injury via modulation of Caf-STIM1/Orai1 signaling.Our results offer insights into the molecular mechanisms of DEX in protecting against neuronal ischemia-reperfusion injury.
文摘Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the usable ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene is a novel compound that behaves as a free radical scavenger with excellent biology consistent. In the present study, we have synthesized and characterized a novel cystine C60 derivative for the first time, and investigated the effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by MTT, PI/Hoechst 33342 staining and flow cytometry analysis. These results suggested that cystine C60 derivative has the potential to prevent oxidative stress-induced cell death and has no evident toxicity.
基金supported in part by the Japan Society for the Promotion of Science(JSPS)Grant-in-Aid for Scientific Research,No.17K11813(to LX)Tai Shan Industrial Experts Program of China No.TSCY20170233(to FL)。
文摘As an aging-associated degenerative disease,Alzheimer’s disease is characterized by the deposition of amyloid beta(Aβ),oxidative stress,inflammation,dysfunction and loss of cholinergic neurons.Colla Corii Asini(CCA)is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging.CCA might delay aging-induced degenerative changes in neurons.In the present study,we evaluated antioxidant activity,cytoprotective effects,and Aβremovability of enzyme-digested Colla Corii Asini(CCAD).Oxygen radical absorbance capacity(ORAC)activity assay showed that,as compared to gelatins from the skin of porcine,bovine and cold water fish,CCA exhibited the highest ORAC activity.The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage.Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008,2013,2017 and gelatin from cold water fish skin,CCA manufactured in 2008 presented the smoothest surface structure.We further tested the protective effects of CCAD(manufactured in 2008)and enzyme-digested gelatin from cold water fish skin(FGD)on hydrogen peroxide(H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells.Presto blue assay showed that both FGD and CCAD at 0.5 mg/m L increased cell viability in H2O2-treated neuronal-like PC12 cells.The protection of CCAD was significantly superior to that of FGD.Acetylcholinesterase(Ach E)assay showed that both FGD and CCAD inhibited Ach E activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1%and 74.5%of that in non-treated cells,respectively.The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine.Although CCAD inhibited Ach E activity in neuronal-like PC12 cells,CCAD prevented H2O2-induced abnormal deterioration of Ach E.ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ1–42-treated neuronal-like PC12 cells,suggesting that CCAD can enhance Aβclearance.Our results suggest that CCA might be useful for preventing and treating Alzheimer’s disease.
基金financially supported by the National Natural Science Foundation of China,No.81574038(to ZZW)the Natural Science Foundation of Guangdong Province of China,No.2017A030313842(to LHD)+1 种基金the Science and Technology Foundation of Guangdong Province of China,No.2017A050506007(to YHL)the Technology Research Foundation of Basic Research Project of Shenzhen City of China,No.JCYJ20170412161254416(to ZZW)
文摘Alpinia oxyphylla,a traditional herb,is widely used for its neuroprotective,antioxidant and memory-improving effects.However,the neuroprotective mechanisms of action of its active ingredients are unclear.In this study,we investigated the neuroprotective effects of various organic extracts of Alpinia oxyphylla on PC12 cells exposed to hydrogen peroxide-induced oxidative injury in vitro.Alpinia oxyphylla was extracted three times with 95%ethanol(representing extracts 1–3).The third 95%ethanol extract was dried and resuspended in water,and then extracted successively with petroleum ether,ethyl acetate and n-butanol(representing extracts 4–6).The cell counting kit-8 assay and microscopy were used to evaluate cell viability and observe the morphology of PC12 cells.The protective effect of the three ethanol extracts(at tested concentrations of 50,100 and 200μg/mL)against cytotoxicity to PC12 cells increased in a concentration-dependent manner.The ethyl acetate,petroleum ether and n-butanol extracts(each tested at 100,150 and 200μg/mL)had neuroprotective effects as well.The optimum effective concentration ranged from 50–200μg/mL,and the protective effect of the ethyl acetate extract was comparatively robust.These results demonstrate that organic extracts of Alpinia oxyphylla protect PC12 cells against apoptosis induced by hydrogen peroxide.Our findings should help identify the bioactive neuroprotective components in Alpinia oxyphylla.
基金the Guangdong Province Science and Technology Program Funded Projects,No.2005B33801003
文摘BACKGROUND:Previous studies have shown that rifampicin exhibits neuroprotective effects,but the precise mechanisms remain unclear.Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger.OBJECTIVE:To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells.DESIGN,TIME AND SETTING:A repeated measure,cell-based study was performed at the Department of Neurology,Second Affiliated Hospital,Sun Yat-sen University,China between December 2007 and November 2008.MATERIALS:PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School,Sun Yat-sen University,China.Rotenone and rifampicin were purchased from Sigma,USA.METHODS:PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco's modified Eagle's medium/Nutrient Mix F12(DMEM/F12) supplemented with 10% fetal bovine serum.The cells were assigned to six groups according to various treatment conditions:control,cultured with normal media;rifampicin group,treated with 300 μmol/L rotenone for 26 hours;rotenone group,treated with 2.5 μmol/L rotenone for 24 hours;rifampicin pretreatment groups,pretreated with 100,200,and 300 μmol/L rifampicin for 2 hours,respectively,followed by 2.5 μmol/L rotenone for 24 hours.MAIN OUTCOME MEASURES:Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry,respectively,using rhodamine123 staining.Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2',7'-dichlorofluorescin-diacetate staining,and intracellular reduced glutathione was measured with a microplate reader.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry.RESULTS:Increased apoptosis in rotenone-induced,differentiated,PC12 cells was accompanied by the loss of mitochondrial transmembrane potential,the formation of reactive oxygen species,and reduced glutathione depletion(P<0.01).Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin(P<0.05 or P<0.01).CONCLUSION:Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.
基金funded by Egyptian Cultural and Educational Bureau in London,Egyptian mission sector and ministry of higher education in Egypt(grant No.GAM2649)。
文摘Dental pulp stem cells(DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium(DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor(NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2(MAP-2) and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and βIII-tubulin were analysed by quantitative polymerase chain reaction(qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors(NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and βIII-tubulin immunostaining. Furthermore, DPSCsCM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls;however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.
基金financially supported by the Ministry of Small and Medium Enterprise and Startups (MSS),Korea,under the Social Economy Innovation Growth Project (R&D)(Project number P0013037)
文摘Objective:To explore the possible neuroprotective activities of Humulus japonicus extract against Parkinson's disease(PD)in a cellular model.Methods:PD was modeled in PC12 cells using 6-hydroxydopamine(6-OHDA).The cell activity,intracellular levels of reactive oxygen species(ROS),anti-oxidative and anti-apoptotic effects,and other related indicators and related signaling pathways were evaluated to elucidate the neuroprotective effects of Humulus japonicus extract.Results:Humulus japonicus extract exhibited anti-oxidative and anti-apoptotic effects in 6-OHDA-stimulated PC12 cells.It also reduced oxidative stress-induced ROS accumulation;upregulated antioxidant enzymes,such as glutathione,catalase,heme oxidase-1,and 8-oxguanine glycosylase 1;promoted cell survival by decreasing BAX and increasing Bcl-2 and sirtuin 1 expression via the MAPK and/or Nrf2 signaling pathways.Conclusions:Humulus japonicus extract has antioxidative and anti-apoptotic effects and could be developed as a promising candidate for preventing and treating oxidative stress-related neurodegenerative diseases.
基金supported by the Taishan Industry Leading Talent Project, Guangdong Provincial Key R&D Program (2020B020226005)the Specific Fund Program for Basic and Applied Basic Research of Guangdong Province (2019A1515011952)+2 种基金the Fundamental Research Funds for the Central Universities (No. x2skD2192510)the Natural Science Foundation of Guangdong for Basic and Applied Basic Research (2020A1515010659)Special Support Project of Guangxi Province for Innovation driven Development (Guangdong Huapeptides Biotechnology Co., Ltd., AA17204075)。
文摘In our previous study, defatted walnut meal hydrolysate(DWMH) could attenuate D-galactose-induced acute memory deficits in vivo, and six potent active peptides including WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG and ADIYTEEAGR were identified. The aim of this study was to investigate the possible mechanism underlying their neuroprotective effects on glutamate-induced apoptosis in PC12 cells and their digestive stability. Results showed that all these peptides could attenuate the reduction of cell viability caused by glutamate in PC12 cells, especially WSREEQEREE and ADIYTEEAGR. The addition of Arg residue in WSREEQEREE and ADIYTEEAGR might be the potential reason for their stronger protective effects. Additionally, these two peptides possibly protected PC12 cells against glutamate-induced apoptosis via activating intracellular antioxidant defence(superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px)) through Kelch-like ECH-associated protein 1(Keap1) inhibition, inhibiting ROS production, Ca;influx and mitochondrial membrane potential(MMP) collapse as well as regulating the expression of apoptosis-related proteins(Bax and Bcl-2). This might be due to the presence of Trp, Tyr and Arg in these two peptides. However, encapsulation of WSREEQEREE and ADIYTEEAGR should be considered based on their digestive sensibility during in vitro gastrointestinal digestion.
基金supported by the National Natural Science Foundation of China (31972041, 32101955)the China Agriculture Research System of MOF and MARA (CARS-12)+1 种基金the Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAASASTIP-2016-OCRI)the Key Scientific Research Projects of Hubei Province (2020BCA086)
文摘Aluminum has been associated with neurodegenerative diseases.ALA(α-linolenic acid),an essential dietary component for human health,possesses prominent biological activities.Herein,we aim to explore the neuroprotective effects of ALA on aluminum toxicity and reveal the underlying mechanism.Results show that aluminum chloride(denoted as Al)enabled cell viability decline and apoptosis with oxidative stress and mitochondrial damage in differentiated rat pheochromocytoma cells(PC12)for 24 h incubation.Compared with Al(10 mmol/L)treatment alone,ALA(50μmol/L)pretreatment for 24 h significantly enhanced cell viability by 28.40%,and hindered cell apoptosis by 12.35%,together with recovering redox state balance and alleviating mitochondrial damage.It was measured that ALA treatment upregulated Bcl-2 expression and down-regulated Bax level,accompanied with an expression decline of caspase-3 and caspase-9.Meanwhile,ALA pretreatment was proved to increase protein kinase A(PKA)expression and to promote phosphorylation of cAMP response element-binding protein(p-CREB),resulting in elevation on the level of brain-derived neurotrophic factor(BDNF).The above results showed that ALA attenuated Al toxicity in PC12 cells by mediating the PKA-CREBBDNF signaling pathway.
文摘Aim Baicalein is the major flavonoid obtained from the Scutellaria root. Our previous studies have dem- onstrated that baicalein has a clear positive effect on recovery in an experimental model of Parkinsonism. The put- pose of this study was to investigate the role of baicalein in modulating dopamine （DA） metabolism in PC12 cells and to explore possible mechanisms of its actions. Methods The intracellular content and extracellular release of DA in both rotenone-treated and untreated PC12 cells were examined. Second, PC12 cells were first pretreated with baicalein （ 10 μmol · L^-1 ） for 10 rain, and then incubated with or without ionomycin （5 μmol · L^-1 ） for 10 rain to test whether short-term exposure to baicalein affected calcium-dependent or spontaneous DA release. Third, the intracellular and extracellular contents of DA and its related metabolites were examined. After treatment with baica- lein for 24 h, the The tyrosine hydroxylase （TH）, monoamine oxidase B （MAOB）, catechol-O-methyltransferase （COMT） and dopamine transporter （DAT） were detected by immunoblot analysis. Results The results showed that baicalein prevented rotenone-induced cytotoxicity and significantly increased the DA content in both rotenone- treated and untreated PC12 cells. Furthermore, it had no effect on ionomycin-induced or spontaneous DA release after short-term exposure but significantly increased DA content in a time- and dose-dependent manner after treat- ment for 6 h. Baicalein also significantly decreased the intracellular and extracellular homovanillic acid （HVA） content but increased the intracellular 3,4-dihydroxy phenylacetic acid （DOPAC） content. Finally, baicalein sig- nificantly decreased the expression of COMT and DAT, but it had no effect on the expression of TH and MAOB. Conclusion These data suggest that bacalein has the ability to increase DA content and modulate DA metabolism by inhibiting the expression of COMT and DAT. Our study provides evidence that baicalein may be a potential anti- PD drug that merits further study.
文摘Objective To investigate the protective effect of Xanthoceraside on various injured PC12 cells models and to indicate the mechanism of Xanthoceraside therapying dementia.Methods Four injured PC12 models induced by glutamate,hydrosulfurous sodium,sodium nitroprusside and potassium chloride accordingly were used to assay the effect of Xanthoceraside on PC12 cells by using morphological examination and MTT assay.In addition,in the model of glutamate injury,the Lactate dehydrogenase(LDH)and the lipid peroxidation products malondialdehyde(MDA)were measured by a spectrophotometric method,reactive oxygen species(ROS)generation were measured with flow cytometry.Results It was found that Xanthoceraside could obviously increase the viability of PC12 cells injured by four injury models.Xanthoceraside could also decerase the levels of LDH release,MDA production,and ROS generation induced by glutamate.Conclusions These data indicate that Xanthoceraside may provide a useful therapeutic strategy for the treatment of progressive neurodegenerative diseases such as Alzheimer's disease(AD).
基金National Natural Science Foundation of China(8166070081260679)Ningxia College FirstClass Discipline Construction Project(Chinese Medicine)Funded Project(NXYLXK2017A06)
文摘OBJECTIVE Mu-Xiang-You-Fang(MXYF)is a classic prescription of Hui medicine,composed of five herbs,which has been used to treat ischemic stroke for many years.However,the potential pharmacological mecha⁃nisms of MXYF remain unclear.The present research is to investigate the neuroprotective effect of MXYF and its role in modulating autophagy via AMPK/mTOR signaling pathway in the PC12 oxygen-glucose deprivation and reperfusion(OGD/R)injury model.METHODS MXYF was extracted by supercritical CO2 fluid extraction apparatus.PC12 OGD/R injury model was established by oxygen-glucose deprivation for 2 h and reperfusion for 24 h.The effects of MXYF on the viability and cytotoxicity of PC12 cells were determined through cell counting kit(CCK-8)assay.Colorimetric method was performed to determine the LDH leakage rate.The calcium concentration was determined by chemical fluorescence method and the mitochondrial membrane potential was determined through flow cytometry.Monodansylcadaverine(MDC)staining was conducted to detect autophagosome formation.The expression of LC3,Beclin1,p62,p-AMPK,ULK1,p-mTOR and p-p70s6k proteins were determined by immunofluorescence and Western blotting analyses.RESULTS MXYF(1,2 and 4 mg·L^-1)could significantly increase the cell viability and mitochondrial membrane potential,while decreased the release of lactate dehydrogenase(LDH)and calcium concentration in PC12 cells.Mechanistic studies showed that MXYF reduced the LC3-II/LC3-I ratio and inhibited the expression of beclin1,p-AMPK and ULK1.In comparison,the expres⁃sion of p-mTOR,p-p70s6k and p62 were significantly enhanced.CONCLUSION MXYF inhibits autophagy after OGD/Rinduced PC12 cell injury through AMPK-mTOR pathway,thus MXYF might have therapeutic potential for treating the ischemic stroke.
文摘Nitric oxide (NO) regulates a wide range of physiological processes. Recent studies show that NO can regulate the release of catecholamines (CA) from the adrenal medulla. In the current study, the PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine Nmethyltransferase (PNMT). Treatment of PC12 cells with the NO donor, sodium nitroprusside (SNP) for 6 hours significantly increased TH, DBH and PNMT mRNA levels. In addition, NO potentiates the regulation of gene expression of all three CA biosynthetic enzymes by glucocorticoids and cholinergic agonists. The signaling pathways involved in NO regulation of CA biosynthetic enzymes were investigated with the use of specific kinase activators and inhibitors, with results supporting a contributing role of PKA, PKC and PKG in SNP-mediated induction for all three CA genes (p < 0.01). In addition, inhibitors of transcription and translation inhibited SNP-mediated induction of all three genes (p < 0.001) suggesting that both transcriptional and translational mechanisms may be involved in CA gene regulation by NO. Results from this study show that in addition to regulating CA biosynthetic enzymes, NO can also potentiate cholinergic and glucocorticoid activation of CA genes.
基金the National Natural Science Foundation of China,No.50677022
文摘BACKGROUND:Electromagnetic radiation can influence dopamine(DA) synthesis in brain tissues or cells,but electromagnetic frequencies,intensities,and radiation time can produce different effects.In addition,the signal pathway by which electromagnetic radiation influences DA synthesis remains controversial.OBJECTIVE:To determine tyrosine hydroxylase(TH) expression in PC12 cells and DA levels in cell culture media after different periods of low-frequency pulsed electric field(LF-PEF) stimulation,and to determine how LF-PEF signaling stimulates TH synthesis using inhibitors.DESIGN,TIME AND SETTING:A parallel,controlled,cell experiment was performed at the Laboratory of Cell Biology,School of Life Science,East China Normal University,between January and October 2006.MATERIALS:PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,China.Nerve growth factor was purchased from PeproTech,USA.The protein kinase A inhibitor,H-89,and mitogen-activated protein kinase kinase inhibitor,U0126,were purchased from Sigma,USA.METHODS:(1) Following routine culture in Dulbecco's modified eagle medium,primary PC12 cells were stimulated under LF-PEF(pulse frequency 50 Hz,pulse width 20 μs,peak field strength 1 V/m) for 5,10,15,20,and 30 minutes.(2) Inhibitors(H-89 or U0126,1 μmol/L) were added 30 minutes before LF-PEF stimulation for 10 minutes.MAIN OUTCOME MEASURES:(1) TH expression was determined by Western blot in PC12 cells at 0.5,1,2,3,and 4 days after LF-PEF stimulation.Similarly,DA was measured by high-performance liquid chromatography in media at 2,3,4,or 5 days after LF-PEF.(2) TH expression was detected 1 day after H-89 or U0126 treatment and LF-PEF.RESULTS:(1) Short-term LF-PEF stimulation(5 and 10 minutes) increased TH expression and media DA levels after short-term culture(2 days)(P<0.01),but both parameters decreased with longer culture(3-4 days)(P<0.01).Long-term LF-PEF stimulation(15,20,or 30 minutes) decreased TH and DA synthesis,followed by a rapid increase(P<0.01).(2) H89 could completely inhibit TH expression in PC12 cells stimulated by LF-PEF for 10 minutes,while the inhibition rate of U0126 was 53.2%.CONCLUSION:Short-term LF-PEF first promotes then inhibits,while long-term LF-PEF first inhibits then promotes,TH and DA synthesis.LF-PEF stimulation regulates TH expression primarily by activating protein kinase A to regulate DA synthesis.
