Korean red ginseng decreases 1-methyl-4-phenylpyridinium-induced mitophagy in SH-SY5Y cells / 中西医结合学报

OBJECTIVE@#Mitophagy is known to contribute towards progression of Parkinson's disease. Korean red ginseng (KRG) is a widely used medicinal herb in East Asia, and recent studies have reported that KRG prevents 1-methyl-4-phenylpyridinium ion (MPP@*METHODS@#SH-SY5Y cells were incubated with KRG for 24 h, and subsequently exposed to MPP@*RESULTS@#MPP@*CONCLUSION@#KRG effectively prevents MPP

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblasto-ma SH-SY5Y cells by inhibition of oxidative stress / 中国病理生理杂志

AIM:To investigate the role of zerumbone ( ZER) in 1-methyl-4-phenylpyridinium ( MPP+)-in-duced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS:Human neuroblastoma SH-SY5Y cells were cul-tured in vitro and the protective effect of ZER against MPP+-induced cytotoxicity was measured by CCK-8 assay. Flow cy-tometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease pro-tein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS:MMP+remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP+ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 ( P<0.05), and obvious increases in apoptosis ( P<0.05) and ROS level ( P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION:ZER protects SH-SY5Y cells against MPP+-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.

Dihydromyricetin inhibits PC12 cell injury induced by 1-methyl-4-phenylpyridinium by up-regulating autophagy activity / 中国药理学与毒理学杂志

OBJECTIVE To observe the effect of dihydromyricetin (DMY) on cell injury induced by 1-methyl-4-phenylpyridinium (MPP+) in PC12 cells and explore the possible mechanism. METHODS After being pretreated with DMY 5, 10, 20, 40 and 80 μmol · L-1 for 0.5 h, PC12 cells were incubated with DMEM culture medium including autophagy inhibitor 3-methyladenine (3-MA) 10 mmol · L-1 and MPP+500μmol·L-1 for 48 h. MTT Assay was used to test the viability of PC12 cells. The level of lactate dehy-dregenase (LDH) was measured colorimetrically. The ultrastructure of PC12 cells was observed under transmission electron microscope. Expressions of autophagy related protein, Beclin-1, microtubule-associated protein light chain 3 (LC3) and p62 were measured by Western blotting. RESULTS Compared with cell control group, the cell survival rate was significantly decreased, the level of LDH was signifi-cantly increased, the number of autophagosomes was obviously decreased, the percentage of autophagic vacuoles in the total cytoplasm area was significantly decreased (P<0.05), expression of Beclin-1 and LC3-Ⅱ and the ratio of LC3-Ⅱ to LC3-Ⅰ were significantly down-regulated (P<0.05) and the expres-sion of p62 was significantly up-regulated in MPP+group (P<0.05). Compared with MPP+group, the cell survival rate was significantly increased, while the level of LDH was significantly decreased in MPP++DMY 10-80 μmol · L-1 group (P<0.05). Compared with MPP+ group, the number of autophagosomes obviously increased, the percentage of autophagic vacuoles in the total cytoplasm area was significantly increased, the expression of Beclin-1 and LC3-Ⅱ and the ratio of LC3-Ⅱ to LC3-Ⅰwere significantly up-regulated and the expression of p62 was significantly down-regulated in MPP++DMY 20 μmol · L-1 group (P<0.05). Compared with MPP++DMY 20μmol · L-1 group, the cell survival rate was significantly decreased, but the level of LDH was significantly increased in MPP ++DMY+3-MA group (P<0.05). CONCLUSION DMY may inhibit the cell injury induced by MPP+in PC12 cells, and the mechanism may be related to the up-regulation of autophagy activity induced by DMY.

Protective effect of protocatechuic acid on midbrain dopaminergic neurons injured by 1-methyl-4-phenylpyridinium / 中草药

Objective: To investigate the effects of protocatechuic acid (PCA) on the midbrain dopaminergic neurons injured by 1-methyl-4-phenylpyridinium (MPP+). Methods: Midbrain neuron cells from KM mice pregnant 14 d were used in this experiment, and divided into control group, model group, low-, mid-, and high-dose (0.05, 0.1, and 0.5 mmol/L) groups. MTT method was used to determine the neuronal survival rate. The activity of lactate dehydrogenase (LDH) in culture, content of intracellular reactive oxygen species (ROS), activity of mitochondrial complex I, and mitochondrial membrane potential were further determined. Results: PCA can enhance the viability of dopaminergic neurons damaged by MPP+, reduce the release of LDH and the generation of ROS, increase the activity of the mitochondrial complex Ι, and prevent the reduction of mitochondrial membrane potential. Conclusion: PCA has the neroprotective effects against MPP+-induced damage of midbrain dopaminergic neurons.

Involvement of mitochondria-mediated pathway and Akt/GSK3β signaling in protective action of albi-florin against MPP+-induced PC12 cells apoptosis / 中国药学杂志

OBJECTIVE: To determine the neuro-protective effects of albiflorin against MPP+-induced PC12 cells death and related molecular mechanisms. METHODS: PC12 cells were treated with 4 mmol·L-1 MPP+ to establish apoptotic cell models. MTT method, DCFH-DA staining, JC-1 staining and Western Blot were used to determine the changes of cell viability, intracellular ROS concentration, mitochondrial membrane potential and the expression of Bel-2 and Bcl-X1, and the phosphoration of Akt/GSK3β in PC12 cells. RESULTS: Albiflorin showed significantly neuro-protective effects against MPP+-induced cell damage. Results showed that albiflorin enhanced the cell viability, reduced intracellular ROS level and caspase 3 activation, restored the mitochondrial membrane potential, increased the expression of Bcl-2 and Bcl-X1, and enhanced the phosphoration of Akt/GSK3β. CONCLUSION: Albiflorin shows neuro-protective effects against MPP+-induced PC12 cells damage via mitochondrial-dependent pathway and Akt/GSK3β signaling.

Inhibitory Effect of Rotundarpene on Parkinsonian Neurotoxin 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

BACKGROUND: The extract and hemiterpene glycosides of Ilex Rotunda Thunb exert antioxidant and anti-inflammatory effects. The effect of rotundarpene on apoptosis in neuronal cells caused by the 1-methyl-4-phenylpyridinium (MPP⁺) has not been reported previously. METHODS: Using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells, we investigated the effect of rotundarpene on MPP⁺-caused apoptosis in relation to the cell death process. RESULTS: MPP⁺-induced cell death was identified using the MTT and neutral red uptake tests. Apoptosis was induced by eliciting decreases in the cytosolic levels of Bid and Bcl-2 proteins, increases in the cytosolic levels of Bax and p53, disruption of the mitochondrial transmembrane potential, and the release of cytochrome c and the activation of caspase-8, -9, and -3 in differentiated PC12 cells and SH-SY5Y cells. Treatment with rotundarpene reduced the MPP⁺-induced changes in the levels of apoptosis-regulated proteins, formation of reactive oxygen species, depletion and oxidation of glutathione, and cell death in both PC12 and SH-SY5Y cells. CONCLUSIONS: Rotundarpene may reduce MPP⁺-induced apoptosis in neuronal cells by suppressing the activation of the mitochondria-mediated pathway and the caspase-8 and Bid pathways. Rotundarpene appears to act by inhibiting the production of reactive oxygen species and by the depletion and oxidation of glutathione.

Inhibitory Effect of 3,4,5-Tricaffeoylquinic Acid on Parkinsonian Toxin 1-Methyl-4-phenylpyridinium-induced Apoptosis

BACKGROUND: 1-Methyl-4-phenylpyridinium (MPP+) causes a neuronal cell injury that is similar to the findings observed in Parkinson's disease. Caffeoylquinic acid derivatives have demonstrated anti-oxidant and anti-inflammatory effects. Nevertheless, the effect of 3,4,5-tricaffeoylquinic acid (3,4,5-triCQA) on the neuronal cell death due to exposure of parkinsonian toxin MPP+ remains unclear. METHODS: Using differentiated PC12 cells, the preventive effect of 3,4,5-triCQA on the MPP+-induced cell death in relation to apoptotic process was examined. RESULTS: MPP+ induced a decrease in Bid, Bcl-2 and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9 and -3), cleavage of PARP-1, and an increase in the tumor suppressor p53 levels. 3,4,5-Tricaffeoylquinic acid attenuated the MPP+-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, depletion of GSH, nuclear damage and cell death. 3,4,5-Tricaffeoylquinic acid attenuated another parkinsonian neurotoxin rotenone-induced cell death. CONCLUSIONS: 3,4,5-Tricaffeoylquinic acid may attenuate the MPP+-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect seems to be ascribed to its inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

Neuroprotective Effect of Chebulagic Acid via Autophagy Induction in SH-SY5Y Cells

Autophagy is a series of catabolic process mediating the bulk degradation of intracellular proteins and organelles through formation of a double-membrane vesicle, known as an autophagosome, and fusing with lysosome. Autophagy plays an important role of death-survival decisions in neuronal cells, which may influence to several neurodegenerative disorders including Parkinson's disease. Chebulagic acid, the major constituent of Terminalia chebula and Phyllanthus emblica, is a benzopyran tannin compound with various kinds of beneficial effects. This study was performed to investigate the autophagy enhancing effect of chebulagic acid on human neuroblastoma SH-SY5Y cell lines. We determined the effect of chebulagic acid on expression levels of autophagosome marker proteins such as, DOR/TP53INP2, Golgi-associated ATPase Enhancer of 16 kDa (GATE 16) and Light chain 3 II (LC3 II), as well as those of its upstream pathway proteins, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Beclin-1. All of those proteins were modulated by chebulagic acid treatment in a way of enhancing the autophagy. Additionally in our study, chebulagic acid also showed a protective effect against 1-methyl-4-phenylpyridinium (MPP+) - induced cytotoxicity which mimics the pathological symptom of Parkinson's disease. This effect seems partially mediated by enhanced autophagy which increased the degradation of aggregated or misfolded proteins from cells. This study suggests that chebulagic acid is an attractive candidate as an autophagy-enhancing agent and therefore, it may provide a promising strategy to prevent or cure the diseases caused by accumulation of abnormal proteins including Parkinson's disease.

Sulforaphane protects PC12 cells against oxidative toxicity / 中华神经科杂志

Objective To investigate the effect of sulforaphane on 1-methyl-4-phenylpyridinium (MPP +)-induced cell viability loss in cultured PC12 cells and to explore the possible mechanism.Methods MPP + induced damage in PC12 cells was prepared as oxidative damage model.Sulforaphane (0.5,1.O,2.5,5.0 and 10.0 μmol/L) was added in each group cell growth medium.Subsequent experiments were divided into 4 groups:(A) normal control group,(B) sulforaphane group,(C) MPP+ injury group,(D)sulforaphane pretreatment + MPP+ injury group.Cell viability was detected by MTT assay,and the sulforaphane pretreatment PC12 cell viability was observed in different concentrations.Flow cytometry was used to detect changes in the rate of apoptosis in different packet PC12 cells,and protein expression levels of nuclear factor erythroid2-related factor 2 (Nrf2),heme oxygenase (HO-1) and human NAD (P) H dehydrogenase,quinone 1 (NQO1) were detected by Western blot when the PC12 cells were incubated with sulforaphane (2.5 μmol/L) and (or) MPP+ (500 μmol/L) for 24 h in vitro.Results Compared to control group (cell survival rate was 98.70％),the survival percents of PC12 cells were significantly decreased in MPP+-treated group (58.16％).A significant difference was showed between group A and C (F =21.83,P ＜ 0.05),and the cell survival rate in group D was significantly improved.Compared to control group,the rate of apoptosis in MPP+ injury group was increased,and the rate of apoptosis after pretreatment of the sulforaphane was significantly reduced.Compared to MPP+ injury group,the levels of Nrf2,HO-1 and NQO1 protein expression were significantly increased in sulforaphane pretreatment group.Conclusion Sulforaphane have a protective effect against MPP+-induced PC12 cell model damage,and the protective effect may be achieved by activating the Nrf2-antioxidant response element pathway.

Epigallocatechin gallate suppresses 1-methy1-4-phenyl-pyridinium-induced the damage of oxidative stress in PC12 cells by activating nuclear factor-related factor 2 / 中华神经科杂志

Objective To investigate the relationship between the neuroprotective effect of epigallocatechin gallate ( EGCG ) for PC12 cells induced by 1-methyl-4-phenyl-pyridinium ( MPP+ ) and activating nuclear factor-related factor 2 ( NRF2 ).Methods Well differentiated PC12 cells treated with MPP+ were used as the in vitro cell models,and PC12 cells were pretreated with different concentrations of EGCG.MTT assay was used to investigate the cell viability.Western blot was used to observe the expression of NRF2 in cells and distribution in the nucleus and the cytoplasm.Real-time PCR was used to observe the antioxidant enzymes,HO-1 and NQO1 mRNA expression.Results Pretreatment of PC12 cells with different concentrations of EG CG for an hour could restore cell viability.Western blot showed that expression of NRF2 in cells treated with MPP+ for 24 hours was increased 148％ +5％ compared with the control group (t =6.102,P ＜0.01 ).The level of NRF2 in EGCG pretreated group was 188％ + 6％ compared with the control group(t =11.172,P ＜0.01 ).Moreover the NRF2 protein level in the nuclear was also increased.Western blot showed that the NRF2 protein level in the nuclear was 258％ +2％ compared with the control group (t =21.995,P ＜ 0.01 ).Further research found U 120,an inhibitor of ERK,could inhibit the effect of EGCG.The levels of NRF2 in both samples were 148％ ± 15％ and 158％ ± 1％ compared with their respective control groups(t =6.118,8.058,both P ＜0.01 ).In accordance with the NRF2 data,real-time PCR indicated that the levels of HO-1 and NQO1 mRNA expression increased obviously in the group pretreated with EGCG.Likewise,U120 could also inhibit HO-1 and NQO1 mRNA expression induced by EGCG.Conclusions EGCG can repair oxidative damage to PC12 cells induced by MPP+.The protective effect may be related through the ways to activate ERK-NRF2 and induce downstream of antioxidant enzyme expression,such as HO-1 and NQO1.

Role of Casein Kinase 2 in Parkinsonian Toxin 1-Methyl-4-phenylpyridinium-induced Cell Death

BACKGROUND: Protein casein kinase 2 is involved in signal transduction, cell growth, and apoptosis. However, it has not been elucidated whether parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+)-induced neuronal cell death is mediated by a casein-kinase-2-mediated pathway. METHODS: We monitored apoptosis-related protein activation, changes in the level of casein kinase 2, nuclear damage, and apoptosis in differentiated PC12 cells exposed to MPP+ in combination with casein kinase 2 inhibitor. RESULTS: Casein kinase 2 inhibitors [4,5,6,7-tetrabromobenzotriazole (TBB), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, and apigenin] reduced MPP+- and rotenone-induced cell death in differentiated PC12 cells. TBB inhibited the MPP+-induced activation of apoptosis-related proteins (decreases in Bid and Bcl-2 levels, increase in Bax levels, cytochrome c release, and caspase-3 activation), increase in casein kinase 2 levels, and nuclear damage. CONCLUSIONS: Administering casein kinase 2 inhibitor TBB at concentrations that do not induce toxic effects may reduce MPP+-induced cell death in differentiated PC12 cells by suppressing the apoptosis-related protein activation that leads to cytochrome c release and subsequent activation of caspase-3. The results suggest that MPP+-induced cell death process is mediated by a casein kinase 2 pathway.

Promoting Effect of Hydrogen Peroxide on 1-Methyl-4-phenylpyridinium-induced Mitochondrial Dysfunction and Cell Death in PC12 Cells

The promoting effect of hydrogen peroxide (H2O2) against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MPP+ resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Addition of H2O2 enhanced the MPP+-induced nuclear damage and cell death. Catalase, Carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the cytotoxic effect of MPP+ in the presence of H2O2. Addition of H2O2 promoted the change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to MPP+ in PC12 cells. The results show that the H2O2 treatment promotes the cytotoxicity of MPP+ against PC12 cells. H2O2 may enhance the MPP+-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that H2O2 as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by neurotoxins.

Group Ⅱ and Ⅲ metabotropic glutamate receptors agonists reverse 1-methyl-4-phenylpyridinium -induced glutamate uptake inhibition / 中国临床药理学与治疗学

AIM: To study the effects of group Ⅱ and Ⅲ metabotropic glutamate receptors (mGluRs) agonists on 1-methyl-4-phenylpyridinium (MPP+) -induced glutamate uptake inhibition. METHODS: The glutamate uptake into astrocytes was measured by using radio-ligand binding assay method,and the viability of astrocytes was investigated by MTT method. RESULTS: It was shown that MPP+(150, 200 ?mol?L -1 ) inhibited glutamate uptake into astrocytes,but produced no effect on the viability of astrocytes,and the inhibition rates were 58.3 % and 70.1 %,respectively. Group Ⅱ mGluRs agonist (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) ( 0.1 ,1,10, 100 ?mol?L -1 ) and Group Ⅲ mGluRs agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) (1,10, 100 ?mol?L -1 ) significantly reversed MPP+-induced glutamate uptake inhibition. CONCLUSION: MPP+ directly inhibits the function of glutamate transporters,and group Ⅱ and Ⅲ mGluRs agonists produce neuroprotective effects by enhancing the activity of glutamate transporters.

Overexpression of 14-3-3 protein alleviates the toxicity of MPP~+ to PC12 cell / 中华神经科杂志

Objective To investigate the protective effects of the 14-3-3 protein overexpression on the injury of PC12 cell induced by MPP~+ and its mechanisms.Methods For expression in mammalial cells, pcDNA3.1(+)-14-3-3 plasmid was constructed and transfeeted into PC12 cell with Lipofectamine~(TM)2000. The overexpression of transfected 14-3-3 gene in PC12 cell was determined by immunofluorescence and Western blotting.The effects of 14-3-3 overexpressing on the cells viability,apoptotie ratio and the activity of superoxide dismutase(SOD)as well as glutathione peroxidase(GSH-Px)of PC 12 cell treated with MPP~+ were measured by MTT assay,flow cytometry analysis and microplate reader respectively.Results The expression of 14-3-3 protein in transfection group(1.19?0.06)increased evidently compared with control group(0.75?0.05).And the antioxidant enzyme activity assession,MTT assay and flow cytometry analysis shows that the overexpression of 14-3-3 protein elevates the activity of SOD(transfection group:(9.13? 0.41)U/mg protein,MPP~+ group:(6.45?0.52)U/mg protein)and GSH-Px(transfection group: (89.66?3.42)?mol/mg,protein MPP~+ group:(82.73?4.15)?mol/mg protein),increases the cell viability(transfection group:0.78?0.06,MPP~+ group:0.54?0.07),and inhibits cell apoptosis (transfeetion group:11.87%?3.26%,MPP~+ group:36.30%?2.39%)of PC12 induced by MPP~. Conclusion The overexpression of 14-3-3 protein could elevate the activity of antioxidant enzymes SOD and GSH-Px,reduce oxidant stress,alleviate MPP~+ toxicity,and thus inhibit the apoptosis of PC12 cell induced by MPP~+.

Protection of Semen Cuscuta extracts from apoptosis PC12 cell of induced by 1-methyl-4-phenylpyridinium / 中成药

AIM: To establish an apoptotic model induced by 1-methyl-4-phenylpyridinium ion(MPP~+) in PC12 cell and assess the effect of Semen Cuscuta extracts on protecting PC12 cell from apoptosis induced by MPP~+. METHODS: The cell viability was analyzed by MTT method,the morphological changes were observed by Hoechst 33258 staining assay,and the apoptosis rate was measured by flow cytometry analysis(FCM).(RESULTS:) Treatment of 0.15 mM MPP~+ for 48 h induced apoptosis in PC12 cells;concentration of 50 mg/L Semen Cuscuta extracts could protect cells from apoptosis induced by MPP~+. CONCLUSION: MPP~+-induced apoptosis in PC12 cell is greatly inhibited by Semen Cuscuta extracts.

Antagonistic action of epigallocatechin-3-gallate on apoptosis in rat PC12 cells induced by 1-methyl-4-phenylpyridinium ion / 中国药理学通报

Aim To investigate the antagonistic action of EGCG on apoptosis of rat PC12 cell induced by MPP+.Methods PC12 cells were cultured and the apoptosis induced by MPP+(900 ?mol?L-1)was observed.The cells were randomly divided into 6 groups:blank group without any treatment,MPP+ control group,vitamin E group and EGCG groups(10,50,100 ?mol?L-1).After treatment of drugs,cell viability,leakage of LDH,morphological changes of mitochondria and apoptosis were detected by MTT,Hoechst 33342 staining,transmission electron microscope and flow cytometry,respectively.Results After treatment of cultured PC12 cells with MPP+,cell viability was decreased,leakage of LDH and apoptotic rate were increased,and mitochondria swelling,vacuole and cristae breakage were observed.Vitamin E and EGCG en-hanced cell viability,reduced the leakage of LDH and apoptotic rate,and decreased the damage degree of mitochondria.Conclusions EGCG possesses the ability of inhibiting rat PC12 cell apoptosis induced by MPP+,and its protective action may relate to its function of keeping mitochondria integrality.