BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal dege...BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear. OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used. RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P 〈 0.01). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P 〈 0.01), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition (P 〈 0.01). By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION: γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.展开更多
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
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
Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/xanthine oxidase (XO)-induced injury to PC12 cells. Methods PC12 cells were damaged b...Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/xanthine oxidase (XO)-induced injury to PC12 cells. Methods PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isofavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. Results Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-β-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/XO-induced injury to PC12 cells. Their ECs0values of formononetin and calycosin were 0.05 μg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 μg/mL and 50 μg/mL. Formononetin and calycosin had no significant infuence on Bcl-2 or Bax protein contents. Conclusion Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.展开更多
Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctonus, Rhizoma chuanxiong, Seme...Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctonus, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na+-K+-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase I1. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.展开更多
Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease. Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake. As an...Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease. Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake. As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract, berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer's disease. The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity. Results demonstrated that berberine protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability. Lactate dehydrogenase release, reactive oxygen content, and malonyl dialdehyde levels were significantly decreased (P 〈 0.01). The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.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
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
BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription fact...BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE: To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING: A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS: Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract (0.185 g/kg/d) to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma (St. Louis, USA), and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf (1:2:2), by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS: PC12 cells were treated with DMEM culture media containing 10% blank serum (normal control group), tunicamycin (1 μg/mL; model group), and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin (1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups), for 2 hours. MAIN OUTCOME MEASURES: PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS: The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group (P 〈 0.05). In contrast, GRP78 mRNA and protein expressions were significantly decreased (P 〈 0.05). CONCLUSION: Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.展开更多
Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-...Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5-500 μg/mL) for 24 hours resulted in PCl2 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 pg/mL), and exposed to 125 pg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, lac- tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib- ited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.展开更多
In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesen...In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.展开更多
One of our previous studies showed that Yizhijiannao Granule,a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer’s disease.In the present study,we established a model of Alzheimer’s ...One of our previous studies showed that Yizhijiannao Granule,a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer’s disease.In the present study,we established a model of Alzheimer’s disease using beta-amyloid(25-35)in PC12 cells,and treated the cells with Yizhijiannao Granule and its four monomers,i.e.,icariin,catechin,Panax notoginseng saponins,and eleutheroside E.Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin,Panax notoginseng saponins,and icariin+Panax notoginseng saponins were protective against beta-amyloid(25-35)-induced injury in PC12 cells.Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25-35)-induced injury compared to icariin or Panax notoginseng saponins alone.The effects of icariin+Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule.The findings indicate that two of the effective monomers of Yizhijiannao Granule,icariin and Panax notoginseng saponins,can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid(25-35).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, bu...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 Fmol/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)%, respectivelyl 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
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
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 radica...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 SETrlNG: 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 pmol/L rotenone for 26 hours; rotenone group, treated with 2.5 pmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 pmol/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
BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative str...BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE: To investigate protective effects of EGB on peroxide (H2O2)-induced oxidative stress damage in PC12 cells. DESIGN: Observational contrast study. SETTING: Department ofPathophysiology, Guangdong Pharmacological College. MATERIALS: EGB was provided by Xi'an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium (MTT), trypsin and dimathyl sulfoxide (DMSO) by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase (LDH) kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS: The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ①Cell culture: PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1 × 10^7 L^-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention: PC12 cells (1 × 10^8L^-1) were plated in 96-well plates with the density of 200 μ L/hole and divided into three groups: normal control group (routinely adding media), H2O2 group (treating with media and H2O2 for 20 hours) and EGB group (adding media, 100μmol/L EGB and 100 μmol/L H2O2). ③ MTT assay: PC12 cells (1 × 10^8L^-1) were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT (100μL) and cultured for 4 hours. And then, 200 μ L DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly ④ Experimental evaluation: Absorbance (A) at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES: Results of MTT assay and LDH activity. RESULTS: ① Results of MTT assay: A value was lower in the H2O2 group than that in the normal control group (P 〈 0.01), while A value was higher in the EGB group than that in the H2O2 group (P 〈 0.01). ② LDH activity: LDH activity was higher in the H2O2 group than that in the normal control group (P 〈 0.01 ), while LDH activity was lower in the EGB group than that in the H2O2 group (P 〈 0.01). CONCLUSION: EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.展开更多
Nerve growth factor(NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol ...Nerve growth factor(NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate(cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF-and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1(Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.展开更多
文摘BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear. OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used. RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P 〈 0.01). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P 〈 0.01), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition (P 〈 0.01). By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION: γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.
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.
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 by the Natural Science Foundation of China(No.NSFC.30670415).
文摘Objective To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/xanthine oxidase (XO)-induced injury to PC12 cells. Methods PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isofavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. Results Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-β-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/XO-induced injury to PC12 cells. Their ECs0values of formononetin and calycosin were 0.05 μg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 μg/mL and 50 μg/mL. Formononetin and calycosin had no significant infuence on Bcl-2 or Bax protein contents. Conclusion Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.
基金supported by the National Natural Science Foundation of China, No. 30973979the Science-Technology Foundation for Excellent Young Scholar of Anhui Province China, No. 10040606Y17Kanion Innovation Fund of Traditional Chinese Medicine, No. KYCX2010007
文摘Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctonus, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na+-K+-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase I1. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.
基金the National Natural Science Foundation of China,No.30772768
文摘Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease. Oxidative damage could be prevented by augmenting the endogenous defense capacity against oxidative stress by antioxidant intake. As an effective alkaloid component of Chinese herbal medicine Rhizoma coptidis extract, berberine exhibits antioxidative properties and ameliorates memory impairment in a rat model of Alzheimer's disease. The present study investigated the protective effects of berberine on H2O2-induced PC12 cell toxicity. Results demonstrated that berberine protects PC12 cells from H2O2-induced apoptosis and increases PC12 cell viability. Lactate dehydrogenase release, reactive oxygen content, and malonyl dialdehyde levels were significantly decreased (P 〈 0.01). The protective effects of berberine on H2O2-induced PC12 cell toxicity were achieved via the antioxidative effects of berberine.
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.
基金Supported by:Scientific and Technological Foundation of the National Administration of Traditional Chinese Medicine of China,No.02-03LP41the Scientific and Technological Key Project of Guangdong Province,No. 2006B35630007
文摘BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE: To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING: A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS: Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract (0.185 g/kg/d) to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma (St. Louis, USA), and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf (1:2:2), by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS: PC12 cells were treated with DMEM culture media containing 10% blank serum (normal control group), tunicamycin (1 μg/mL; model group), and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin (1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups), for 2 hours. MAIN OUTCOME MEASURES: PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS: The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group (P 〈 0.05). In contrast, GRP78 mRNA and protein expressions were significantly decreased (P 〈 0.05). CONCLUSION: Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.
基金supported by the National Natural Science Foundation of China,No.31201951 and 31272613the Scientific and Technological Innovation Talent Scientific Research Foundation for the Returned Overseas Chinese Scholars by State Education Ministry and Heilongjiang Province in China,No.2012RFLXN005 and LC201018+1 种基金the Youth Science and Technology Foundation of Liaoning Medical University in China,No.Y2012Z023the Science and Technology Department of Liaoning Provincial Foundation Programs,No.2011214001
文摘Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate (62.5-500 μg/mL) for 24 hours resulted in PCl2 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin (25, 50 and 100 pg/mL), and exposed to 125 pg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, lac- tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib- ited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.
基金supported by the Science and Technology Development Foundation of Beijing Science and Technology Commission, No. Z101107052210004
文摘In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.
文摘One of our previous studies showed that Yizhijiannao Granule,a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer’s disease.In the present study,we established a model of Alzheimer’s disease using beta-amyloid(25-35)in PC12 cells,and treated the cells with Yizhijiannao Granule and its four monomers,i.e.,icariin,catechin,Panax notoginseng saponins,and eleutheroside E.Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin,Panax notoginseng saponins,and icariin+Panax notoginseng saponins were protective against beta-amyloid(25-35)-induced injury in PC12 cells.Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25-35)-induced injury compared to icariin or Panax notoginseng saponins alone.The effects of icariin+Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule.The findings indicate that two of the effective monomers of Yizhijiannao Granule,icariin and Panax notoginseng saponins,can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid(25-35).
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 Fmol/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)%, respectivelyl 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 SETrlNG: 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 pmol/L rotenone for 26 hours; rotenone group, treated with 2.5 pmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100, 200, and 300 pmol/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Initial Funding for Doctor from Guangdong Science and Technology Bureau, No. 06300709Initial Funding for Doctor from Guangdong Pharmaceutical College, No. 43543096
文摘BACKGROUND: Extracts of ginkgo biloba leaves (EGB) and its metabolites have been reported to enhance brain function and nerve behavior. It has also been hypothesized that they can protect neurons from oxidative stress. OBJECTIVE: To investigate protective effects of EGB on peroxide (H2O2)-induced oxidative stress damage in PC12 cells. DESIGN: Observational contrast study. SETTING: Department ofPathophysiology, Guangdong Pharmacological College. MATERIALS: EGB was provided by Xi'an Fujie Biotechnological Development Company; 1640 culture medium, methylthiazolyl tetrazolium (MTT), trypsin and dimathyl sulfoxide (DMSO) by Sigma Company; PC12 cell strain by Cell Center of Medical College of Zhongshan University; calf serum by Hangzhou Sijiqing Bioengineering Company; lactate dehydrogenase (LDH) kit by Nanjing Jiancheng Bioengineering Research Institute. METHODS: The experiment was carried out in Department of Cell Biology of Guangdong Pharmacological College from June to December 2005. ①Cell culture: PC12 cells were cultured in 1640 medium containing 200 g/L fetal calf serum. The cells were diluted to 1 × 10^7 L^-1 and washed every two days. Those cells were used to experiment until they grew in logarithm on solid wall. ② Grouping and intervention: PC12 cells (1 × 10^8L^-1) were plated in 96-well plates with the density of 200 μ L/hole and divided into three groups: normal control group (routinely adding media), H2O2 group (treating with media and H2O2 for 20 hours) and EGB group (adding media, 100μmol/L EGB and 100 μmol/L H2O2). ③ MTT assay: PC12 cells (1 × 10^8L^-1) were plated in 96-well plates and divided into three groups with 8 holes for each group. Under sterile condition, cells were added with 5 g/L MTT (100μL) and cultured for 4 hours. And then, 200 μ L DMSO fluid was added and shaken for 30 minutes until blue crystal products formed were dissolved soundly ④ Experimental evaluation: Absorbance (A) at 630 nm was measured and LDH activity was measured at the same time. MAIN OUTCOME MEASURES: Results of MTT assay and LDH activity. RESULTS: ① Results of MTT assay: A value was lower in the H2O2 group than that in the normal control group (P 〈 0.01), while A value was higher in the EGB group than that in the H2O2 group (P 〈 0.01). ② LDH activity: LDH activity was higher in the H2O2 group than that in the normal control group (P 〈 0.01 ), while LDH activity was lower in the EGB group than that in the H2O2 group (P 〈 0.01). CONCLUSION: EGB can inhibit H2O2-induced oxidative stress damage in PC12 cells possibly by preventing damage to the cell membrane.
基金supported partly by the National Natural Science Foundation of China,No.30873057,81171245a grant from the Key Basic Project of Shanghai Municipal Science and Technology Commission of China,No.08JC1413600,11JC1406600
文摘Nerve growth factor(NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate(cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF-and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1(Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.