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Background Neurotransmitter secretion disorder induced by chronic manganese poisoning has always been one of the important causes of body injury, but the mechanism of neurotransmitter secretion disorder caused by manganese is not clear at present. Objective To investigate the effects of presynaptic membrane intracellular protein 13-1 (Munc13-1) and synapse fusion protein binding protein 18-1 (Munc18-1) on dopamine secretion dysfunction induced by manganese chloride (MnCl2) in human neuroblastoma (SH-SY5Y) cells. Methods A SH-SY5Y cell model induced by MnCl2 was established. Cell viability was measured by MTT assay. Four experimental groups were set up: control group and low-, medium-, and high-dose manganese groups (0, 100, 200, and 400 μmol·L−1 MnCl2). They were treated with corresponding doses of MnCl2 for 24 h. The secretion of dopamine was measured by enzyme-linked immunosorbent assay. The mRNA expression of Syntaxin-1 was detected by real-time quantitaive PCR. Total cell proteins were extracted, and the protein expression levels of Munc13-1, Munc18-1, and Syntaxin-1 were detected by Western blotting. The correlations of MnCl2 exposure and dopamine secretion with the protein expressions of Munc13-1 and Munc18-1 were also analyzed by Pearson correlation. Results Compared with the control group, the cell viability rate decreased gradually with the increase of manganese exposure concentration, and the difference between the medium- and the high-dose manganese groups was statistically significant (P<0.05). The concentration of dopamine in cell culture medium of all manganese exposure groups decreased with the increase of manganese concentration, and compared with the control group and the low-dose manganese group, the medium- and the high-dose manganese groups were statistically significant (P<0.05). The expression of Syntaxin-1 at mRNA or protein level did not change significantly among groups (P>0.05). Compared with the control group, the protein expression of Munc13-1 decreased and that of Munc18-1 increased with the increase of manganese concentration (P<0.05). Compared with the low-dose manganese group, the changes of Munc13-1 protein in the high-dose manganese group and Munc18-1 protein in the medium- and high-dose manganese groups had statistical significance (P<0.05). Compared with the medium-dose manganese group, the protein changes of Munc18-1 in the high-dose manganese group were statistically significant (P<0.05). The correlation analysis showed that MnCl2 dose was negatively correlated with Munc13-1 protein expression (r=−0.898, P<0.05), and positively correlated with Munc18-1 protein expression (r=0.678, P<0.05). Dopamine secretion was positively correlated with Munc13-1 protein expression (r=0.932, P<0.05), and negatively correlated with Munc18-1 protein expression (r=−0.817, P<0.05). Conclusion The inhibition of dopamine secretion in SH-SY5Y cells induced by manganese exposure is related to up-regulation of Munc18-1 and down-regulation of Munc13-1 expression levels, which may be one of the reasons for nerve injury caused by manganese.
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Background A large amount of iron deposition in the brain can cause neuronal damage by inducing oxidative stress, neuroinflammation, and abnormal mitochondrial function. In addition, iron deposition is also reported to be closely related to the pathogenesis of Alzheimer's disease (AD). The neurofibrillary tangles aggregated by tau hyperphosphorylation are one of the important pathological features of AD. Objective To investigate potential effect of exogenous trivalent iron ions on neuronal activity in human neuroblastoma (SH-SY5Y) cells and tau hyperphosphorylation and aggregation. Methods SH-SY5Y cells were treated with ferric chloride (FeCl3) at four concentrations (10, 100, 200, and 400 mg·L−1). Cell survival rate was then detected by CCK8 assay. Intracellular iron content was determined prussian blue (Perl's) by iron staining after 24 h exposure to FeCl3 at 10 or 200 mg·L−1. Transfection of tau-P301L plasmid was conducted to construct an AD-like cell model for tau overexpression. The differences in the expression of the phosphorylated tau (p-tau) protein in SH-SY5Y cells and SH-SY5Y cells with tau overexpression were detected by Western blotting after 24 h exposure to FeCl3 at 10 and 200 mg·L−1. After dilution with phosphate buffered saline (PBS), FeCl3, human tauR3, and FeCl3 + tauR3 were incubated at 37℃, and the fluorescence intensity reflecting tau aggregation level was measured by thioflavin T(ThT) method at 12, 24, 36, 48, 60, 72, 84, and 96 h, respectively. Meanwhile, after 96 h coincubation of FeCl3 and tauR3, the fibers formed by tau aggregation were observed under a transmission electron microscope (TEM). Results After 24 h of FeCl3 exposure, the cell survival rate of SH-SY5Y cells among all groups was statistically different (F=8.63, P<0.01). The cell survival rates in the 200 and 400 mg·L−1 groups were 80.1% and 68.7% of the control group, respectively (P<0.05). Compared with the control group, the nuclei of the 200 mg·L−1 FeCl3 group were mainly yellowish-brown after iron staining and the positive cell rate was up-regulated by 12.9% (P<0.01). After 24 h of FeCl3 exposure , the p-tau (Ser396) protein expression was statistically different among all groups (F=11.6, P<0.01). Compared with the control group, the p-tau protein expression level of SH-SY5Y cells in the 200 mg·L−1 group was up-regulated by 72.7% (P<0.01). After FeCl3-treated SH-SY5Y cells with tau overexpression for 24 h, the p-tau (Ser396) protein expression was statistically different among all groups (F=27.8, P<0.01). Compared with the tau group, the p-tau (Ser396) protein expression level of SH-SY5Y cells in the tau + 200 mg·L−1 group was up-regulated by 44.6% (P<0.05). Compared with the tauR3 group, the fluorescence intensities in the 84 and 96 h tauR3 + FeCl3 groups were up-regulated by 49.9% and 53.7% (P<0.01) respectively. After 96 h of coincubation, compared with the tauR3 group, FeCl3 + tauR3 aggravated tau aggregation and formed fiber deposition under TEM. Conclusion Exogenous trivalent iron ions may inhibit SH-SY5Y cell viability, promote the phosphorylation of tau in SH-SY5Y cells transfected with tau-P301L plasmid, and aggravate tauR3 aggregation and fiber production.
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[Abstract] Objective To study whether the regulation of mammalian target of rapamycin complex 2(mTORC2) / Akt signaling pathway has a protective effect on SH-SY5Y cell line damaged by 6-hydroxydopamine (6-OHDA), and to clarify its molecular mechanism. Methods SH-SY5Y cells treated with retinoic acid (RA) were given 6-OHDA, mTORC2 signaling pathway inhibitor PP242 and agonist A-443654 respectively. The changes of cell number in each group were investigated by immunofluorescent staining; The total protein was extracted and the expression level and interaction of key proteins in mTORC2 signaling pathway were determined by Western blotting and co-immunoprecipitation (CoIP); The apoptosis rate of cells in each group was detected by flow cytometry. At the same time, the co-culture Parkinson’ s disease (PD) model was made using SH-SY5Y cell line and Bv-2 cell line; MTT colorimetric method was used to detect the cell viability of each group; ELISA was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in cell culture supernatant. Results The number of tyrosine hydroxylase(TH) / proliferating cell nuclear antigen (PCNA) / hochest-, TH / 5-bronmo-2’ -deoxyuridine(BrdU) -labeled positive cells in 6-OHDA-lesioned PD cell model group was significantly lower than that in the normal group; The apoptosis rate was higher; The expression of Rictor, p-Akt and regulated in DNA damage and development 1(REDD1) was increased; There was an interaction between Rictor and p-Akt or REDD1; The cell viability was significantly reduced in the co-culture model; the content of TNF-α and IL-β increased in the cell culture supernatant. With further up-regulation of the abovementioned protein expressions, the cell survival, apoptosis and pro-inflammatory cytokine levels in A-443654 group were significantly ameliorated, while PP242 group showed the opposite changes. Conclusion A-443654 activates mTORC2 signaling pathway by p-Akt, which increases the expression of Rictor and REDD1 protein. These changes contribute to the amelioration in cell survival rate, apoptosis rate, and the proliferation and differentiation and decreasion of apoptosis rate of SH-SY5Y cells. These result improved 6-OHDA-induced cell damage and inhibited the release of pro-inflammatory cytokines.
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Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aβand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aβ_(1-42)to establish AD cell model,and Aβimmunofluorescence detection showed that Aβdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aβ_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aβdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3Ⅱwas up-regulated after Aβinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3Ⅱ/LC3Ⅰamong groups was the same as the protein expression level of LC3Ⅱ，the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aβ_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.
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Humans , Amyloid beta-Peptides , Apoptosis , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Isoflavones/pharmacology , Lung Neoplasms , ProteomicsABSTRACT
Background: Proton pump inhibitors (PPIs) largely used a drug to treat gastroesophageal disease such as gastric ulcers. Moreover, in recent years, several studies suggest that PPIs have an important anti-cancer effect in monotherapy and or combination with chemotherapy. The aim of this study was to investigate whether esomeprazole and pantoprazole exhibit anti-cancer effect alone or could enhance chemosensitivity on the human neuroblastoma cell line SH-SY5Y to cisplatin.Methods: The human neuroblastoma SH-SY5Y cells were cultured and treated with different concentrations of esomeprazole, pantoprazole, and cisplatin alone. Also, these cells exposed to cisplatin+ esomeprazole and cisplatin + pantoprazole combinations, respectively and incubated 24 h. The antiproliferative activities of the (PPIs) alone or in a combination of cisplatin was evaluated using the XTT colorimetric assay.Results: According to experimental data, neither PPIs showed no cytotoxicity on the human neuroblastoma cell line SH-SY5Y at all concentrations. However, when combined with cisplatin separately, they were found to have significant antiproliferative effects on the human neuroblastoma SH-SY5Y cell lines when compared to cell lines treated with cisplatin alone (p<0.05).Conclusions: Taken together, the inhibition of V-ATPase via esomeprazole and pantoprazole might enhance the chemosensitivity of cisplatin on the human neuroblastoma cell line SH-SY5Y. However, further studies are needed to be able to utilize PPIs in human neuroblastoma cells.
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OBJECTIVE： To investigate the effects of tetrandrine （TET） on the proliferation and migration， invasion ability of neuroblastoma cells and its mechanism. METHODS： Human neuroblastoma cell lines IMR-32 and SH-SY5Y were chosen as objects. MTT assay was used to detect the effects of 0 （blank control）， 2.5， 5， 10， 15， 20 μmol/L TET on cell proliferation. The transmembrane number of normal control group （TET concentration of 0 μmol/L） and TET group （10 μmol/L for IMR-32 cells， 15 μmol/L for SH-SY5Y cells） were investigated by Transwell cell migration and invasion experiments. The protein levels of MMP-2， MMP-9， β-catenin， GSK3β and p-GSK3β in IMR-32 cells and SH-SY5Y cells were tested by Western blotting assay after treated with TET of above concentrations. RESULTS： TET with 5， 10， 15， 20 μmol/L can reduce the survival rate of IMR-32 cells and SH-SY5Y cells significantly （P＜0.05 or P＜0.01）. IC50 of which to IMR-32 cells and SH-SY5Y cells were 10.148 and 14.461 μmol/L， respectively. Results of migration and invasion experiments showed that compared with normal control group， the number of transmembrane cells was decreased significantly in TET group （P＜0.01）. Results of Western blotting assay showed that compared with normal control group， the protein expression levels of MMP-2，MMP-9，β-catenin and p-GSK3β were decreased significantly in TET group （P＜0.01）. CONCLUSIONS： TET shows significant inhibitory effects on the proliferation， migration and invasion ability of neuroblastoma cells， the mechanism of which may be associated with down-regulating the protein expression of MMP-2 and MMP-9 and inhibiting Wnt/β-catenin signaling pathway.
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Ischemic cerebrovascular disease and cerebral ischemia/reperfusion injury threaten the health of human being. We studied the protective effect of Ginkgo biloba extract 50 (EGb50) on the mitochondrial function in SH-SY5Y cells after hypoxia/reoxygenation (H/R) injury and explored its mechanisms, so as to provide new ideas for studies on the treatment for ischemic cerebrovascular disease. We established the H/R injury model in SH-SY5Y cells after administrating EGb50. Subsequently, the mitochondrial membrane potential and the concentration of intracellular Ca²⁺ were measured by flow cytometer. The levels of optic atrophy1 (Opa1) and dynamin-like protein 1 (Drp1) were evaluated by immunofluorescence and western blot. The results showed that the mitochondrial membrane potential was decreased and the level of intracellular Ca²⁺ was increased after H/R injury. Moreover, the expression of mitochondrial fusion protein Opa1 was decreased, while the expression of mitochondrial fission protein Drp1 was increased. However, EGb50 significantly increased the mitochondrial membrane potential and suppressed the level of intracellular Ca²⁺. In addition, EGb50 increased the expression of Opa1 and decreased the expression of Drp1. The results demonstrated that EGb50 has a neuroprotective effect on SH-SY5Y cells after H/R injury, and could improve the energy metabolism and mitochondrial function. The underlying mechanisms may be associated with the regulation of mitochondrial fusion and fission, which provided data support for the treatment of ischemic cerebrovascular disease with EGb50.
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Humans , Cell Hypoxia , Membrane Potential, Mitochondrial , Mitochondria , Plant Extracts , Reperfusion InjuryABSTRACT
Objective To understand the role of NOD-like receptor pyrin domain containing 3 (NLRP3)-dependent activation of cysteinyl aspartate specific proteinase 1 (caspase-1) in mediating the neuroinflammatory response in mice with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced Parkinson's disease(PD) and to investigate the underlying mechanism. Methods Male C57BL/6 mice were randomly divided into two groups:experimental and control groups. MPTP solution (30 mg·kg-1·d-1) was given to mice through intraperitoneal injection to prepare the PD model and equal amount of saline was used to set up the control group. Changes in mouse behavior were observed through pole climbing and suspension experiment. Expression of inflammasome in the midbrain of mice was detected by Western blot. SH-SY5Y cells,a human neuroblastoma cell line,were cultured in vitro and randomly divided into five groups including control,nonsense,siRNA-NLRP3,MPP+(1-methyl-4-phenylpyridinium) and siRNA+MPP+groups. siRNA-NLRP3 was transfected into SH-SY5Y cells using Lipofectamine 2000 to silence the expression of NLRP3 gene. Then the transfected cells were incubated with MPP+for 48 h to observe the protective effect of NLRP3 on nerve cells. MTT assay and flow cytometry were performed to measure the viability and the apoptosis rate of SH-SY5Y cells,respectively. Western blot was used to detect the levels of NLRP3,B-cell lymphoma 2 (Bcl-2),Bcl-2-associated X protein (Bax) and cleaved caspase-3. Results Compared with the control group,the mice in the experimental group spent more time climbing from the top to the bottom of the pole (P<0. 05),got a lower score on suspension experiment (P<0. 05) and showed enhanced expression of NLRP3, IL-1β,Pro-IL-1β and caspase-1 in the substantia nigra (P<0. 05). Compared with the MPP+group,the siR-NA+MPP+group showed significantly inhibited expression of NLRP3, cleaved caspase-3 and Bax ( P<0. 05),enhanced cell viability (P<0. 05),suppressed cell apoptosis (P<0. 05) and promoted expression of Bcl-2 ( P<0. 05). Conclusion NLRP3-dependent activation of caspase-1 plays an important role in the nerve injury in PD. Inhibition of NLRP3 expression in vitro can significantly enhance the vitality and inhibit the apoptosis of SH-SY5Y cells,which may have a protective effect on nerve cells through regulating mito-chondrial apoptosis signaling pathway and provide a new drug target for the treatment of PD.
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Objective To explore the mechanism of SH?SY5Y mitochondrial dysfunction treated by manganese to find a new potential therapeutic target. Methods Transmission Electron Microscopy(TEM)to observe the morphology of mitochondria. Cell treated with 250μmol/L for periods of time(2 h, 4 h, 6 h)while mitochondrial membrane potential(MMP)and ROS can be detected by FCM and fluorescence microplate reader. Results After treating with MnCl2 in 6 h, TEM images showed early vacuoles, lamellar structures of SH?SY5Y cells. Then test the mitochondrial membrane potential and showed that MMP would be decreased gradually. Meanwhile, analysis showed that in comparison with control, treatment group had a higher ROS level respectively (P < 0.05). Conclusion MnCl2 can cause mitochondrial damage through a mechanism closely related to disrupt the MMP or generate abundant ROS.
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Aim To study the apoptotic inducing effects of deguelin on SH-SY5Y cells.Methods SH-SY5Y cells were treated with 0,0.625,1.25,2.5,5,10 and 20 μmol·L-1 deguelin for different time(24,48,72 h);cell viability was detected by CCK-8 assay.SH-SY5Y cells were treated with 0,8,20,50 μmol·L-1 deguelin for 24 h;light microscope and AO/EB double stained method were employed for observing the morphology and apoptotic morphology of treated cells.Apoptotic rate of treated cells was determined by flow cytometry.Cells were stained by DCFH-DA,and the whole reactive oxygen species(ROS)was determined by flow cytometry.Spectrophotometry was employed to determine the activation degree of caspase-3.Results Deguelin inhibited cell growth in a time-and dose-dependent manner,and the IC50 value of deguelin was(26.07±2.18),(18.33±0.94),(12.5±1.49)μmol·L-1 when treated with 24,48,72 h respectively.After treated with 8,20,50 μmol·L-1 deguelin for 24 h,cell apoptotic rate,ROS and activation rate of caspase-3 increased markedly(P<0.05),all of which performed a dose related effect.Conclusion Deguelin can inhibit SH-SY5Y cell proliferation and induce cell apoptosis,and the mechanism may be concerned with the elevated ROS and activated caspase-3.
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OBJECTIVE: To explore the effect of 1,2-dichloroethane(1,2-DCE) induced apoptosis on the expression of related proteins in human neuroblastoma cells(SH-SY5 Y cells). METHODS: SH-SY5 Y cells were cultured in complete medium with 1,2-DCE at final concentrations of 0,10,20,30,40,50,60,70 and 80 mmol/L. After being cultured for24 hours,the apoptosis of SH-SY5 Y cells was tested by flow cytometry using annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. Western blot was used to detect the protein expression of P53,B cell lymphoma/leukmia-2(BCL-2)and BCL-2 associated X protein(BAX). RESULTS: At 1,2-DCE concentrations of 0-80 mmol/L,the total apoptosis rate of SH-SY5 Y cells increased with 1,2-DCE concentrations in a dose-dependent manner(P < 0. 01). At 1,2-DCE concentrations of 30-80 mmol/L,the early apoptosis rate and total apoptosis rate of SH-SY5 Y cells increased significantly than the control group(P < 0. 05). Compared with the other groups,the protein expression of P53 was the lowest when the1,2-DCE concentration was 20 mmol/L(P < 0. 05),and the protein expression of BCL-2 and the BCL-2/BAX ratio were the lowest when the 1,2-DCE concentration was 70 mmol/L(P < 0. 05). There is no dose-response relationship in the1,2-DCE concentrations and the protein expression levels of P53,BCL-2 and BAX,and BCL-2/BAX ratio. Linear multiple regression analysis revealed that the total apoptosis rate of SH-SY5 Y cells treated with 1,2-DCE was associated with the protein expression of P53 and BCL-2,and BCL-2/BAX ratio(P < 0. 05). CONCLUSION: 1,2-DCE could inhibit the apoptosis of SH-SY5 Y cells. The mechanisms may be related to the changes of P53 and BCL-2 protein expression,and BCL-2/BAX relative amount.
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Objective To investigate the effects of extract of Ginkgo biloba leaves EGb761 on 1-methy-l 4-phenylpyridium (MPP+)-induced injury in human neuroblastoma SH-SY5Y cells; To discuss its mechanism of action.Methods Cell culture method was used and SH-SY5Y cell damage model was induced with different concentrations of MPP+ to build Parkinson’s disease model in vitro. The experiment was divided into control group, MPP+ model group, low-, medium-, and high-dose EGb761 groups. The survival rate was determined by MTT assay, and the apoptotic rate was detected by flow cytometry according to AnnexinV apoptosis detection kit. The cell morphology was observed by inverted microscope. NO content in SH-SY5Y cells was detected by Nitric acid reduction method.Results Compared with the control group, the survival rate of SH-SY5Y cells decreased and the apoptotic rate and NO content increased in the model group (P<0.05); Compared with the model group, the survival rate of SH-SY5Y cells increased and the apoptotic rate and NO content decreased in the low-, medium- and high-dose EGb761 groups (P<0.05).Conclusion EGb761 can protect MPP+-induced SH-SY5Y cell from damage by the inhibition of the content of NO free radical.
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Objective: To identify neuroprotective extracts with the protective effects on Aβ25-35-induced SH-SY5Y cell injury via high content screening (HCS). Methods: Hoechst 33342/PI double staining method was used to screen neuroprotective extracts from 60 Chinese materia medica (CMM) extracts. Further more, the effects of neuroprotective extracts on Aβ25-35-induced changes in the levels of Caspase-3/7 were detected. Results: The results showed that 17 extracts had obviously neuroprotective effects. Among the 17 extracts, 8 of them inhibited Aβ25-35-induced up-regulation of Caspase-3/7. Conclusion: HCS is an efficient method to screen neuroprotective extracts with the protective effects of Aβ25-35-induced SH-SY5Y cell injury. The neuroprotective extracts have potential medicinal value in Alzheimer's disease.
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OBJECTIVE:To investigate the inhibitory effect of astragalus polysaccharide(APS)on the proliferation of human neuroblastoma SH-SY5Y cells. METHODS:After the cells were cultured with 0(blank control),25,50 and 100 mg/ml APS for 6,12 and 24 h,MTT method was used to determine cell viability and calculate inhibition rate. Following cell cultured with 0 (blank control),25,50 and 100 mg/ml APS for 24 h,Hoechst 33258 fluorescent staining was performed,and then cell nucleus morphology was observed under the fluorescence microscope;flow cytometer was used to detect the distribution of cell cycles and apoptosis;western blot was employed to determine the expression of extracellular regulated protein kinases (ERK) 1/2 protein in cells. Enzyme linked immunosorbent assay (ELISA) was conducted to determine the contents of interleukin 2 (IL-2),IL-6 and IL-12 in the cells. RESULTS:Compared to the blank control,those cultured with 100 mg/ml APS for 6 h,50 and 100 mg/ml APS for 12 h and 25,50 and 100 mg/ml APS for 24 h demonstrated higher inhibition rate. After the cells were cultured with 50 and 100 mg/ml APS for 24 h,those in G0/G1 phase increased and those in G2/M and S phases decreased,and the contents of IL-2 and IL-6 increased. After cells were cultured with 25,50 and 100 mg/ml APS for 24 h,the apoptosis rate was higher,densely hyperchromat-ic fragments in cell nuclei and apoptotic bodies appeared,the phosphorylation level of ERK1/2 protein in the cells was lower,and the content of IL-12 was higher. There was statistically significance (P<0.01 or P<0.05). CONCLUSIONS:APS can inhibit the proliferation of SH-SY5Y cells by arresting cell cycle and inducing cell apoptosis through a mechanism which may be correlated to the decrease in the phosphorylation of ERK1/2 and increase in cytokine.
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Objective To discuss the effect of bupivacaine on SH‐SY5Y cells in high sugar environment and observe the ROS and apoptosis .Methods 1 mmol/L bupivicaine was added to medium with different concentrations of glucose for SH‐SY5Y cells , and the quantity of ROS in the cells and the situation of apoptosis were detected by flow cytometry ,and Western blot was uesd to detect the change of the related protein ,GRP78 .Results The contents of intarcellular ROS in different groups ,which had different concentration(7 .80 ,11 .10 ,13 .30 mmol/L) of sugar medium ,were high than those in groups with concentrations 5 .56 ,6 .10 ,7 .00 mmol/L of sugar medium ,and it showed statistical significance (P<0 .05) .Cell apoptosis rates among different groups had statisti‐cal significance(P<0 .05) .Expression of GRP78 in group of 5 .56 ,6 .10 and 7 .80 mmol/L were (1 .02 ± 0 .12) ,(0 .97 ± 0 .06) and (0 .49 ± 0 .04) ,respectively ,and they all were higher than that in group of 7 .00 mmol/L (0 .46 ± 0 .06) .The groups of 11 .10 mmol/L (0 .22 ± 0 .03) and 13 .30 mmol/L (0 .15 ± 0 .07) were lower than the other groups in the expression of GRP78 ,and all the difference shows statistical significance (P<0 .05) .Conclusion Increasing and apoptosis of intracellular ROS are associated with strengthened endoplasmic reticulum stress by bupivacaine .With the function degrading of GRP78 ,endoplasmic reticulum stress (ERS) may strengthen further .
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Objective To explore the possible mechanism of SchA ,which decreases MPP+induce SH-SY5Y cell damage .Meth-ods Cultured cells were divided into 5 groups ,one as control group ,cultured by free-blood serum media;the other 4 groups were treated with different concentrations of SchA(1 ,3 ,5 μmol/L) and MPP+ (1 mmol/L) for 48 h named model group ,1 ,3 ,5 μmol/L SchA group respetivly .The content of nitric oxide(NO) were measured by NO kit ;The expression levels of total Akt and p-Akt proteins were detected by Western blot .Results Compared with the control group ,the content of NO in group significantly in-creased after MPP+stimulating(P0 .05) .The expression levels of p-Akt in model group significantly lowered ,while SchA(1、3、5 μmol/L) significantly increased the expression levels of p-Akt in comparision with cells in model group .Conclusion Decreasing MPP+ induced SH-SY5Y cell damage of SchA may be related to the content of NO and p-Akt expression .
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Parkinson's disease is a multifactorial disorder with several genes linked to the familial types of the disease. ATP13A2 is one of those genes and encode for a transmembrane protein localized in lysosomes and late endosomes. Previous studies suggested the roles of this protein in lysosomal functions and cellular ion homeostasis. Here, we set out to investigate the role of ATP13A2 in lysosomal function and in metabolism of alpha-synuclein, another PD-linked protein whose accumulation is implicated in the pathogenesis. We generated non-sense mutations in both copies of ATP13A2 gene in SH-SY5Y human neuroblastoma cells. We examined lysosomal function of ATP13A2-/- cells by measuring the accumulation of lysosomal substrate proteins, such as p62 and polyubiquitinated proteins, induction of acidic compartments, and degradation of ectopically introduced dextran. None of these measures were altered by ATP13A2 deficiency. The steady-state levels of alpha-synuclein in cells or secretion of this protein were unaltered either in ATP13A2-/- compared to the normal cells. Therefore, the proposed roles of ATP13A2 in lysosomal functions may not be generalized and may depend on the cellular context. The ATP13A2-/- cells generated in the current study may provide a useful control for studies on the roles of PD genes in lysosomal functions.
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Humans , alpha-Synuclein , Dextrans , Endosomes , Homeostasis , Lysosomes , Metabolism , Neuroblastoma , Parkinson Disease , PolyubiquitinABSTRACT
Parkinson's disease is known to exhibit progressive degeneration of the dopaminergic neurons in the substantia nigra via inhibition of glutathione metabolism. It is well known that 6-Hydroxydopamine (6-OHDA) induces Parkinson's disease-like symptoms, while resveratrol (3,5,4'-trihydroxystilbene) has been shown to have anti-inflammatory and antioxidant effects. In the present study, we investigated the neuroprotective effects of resveratrol, a phytoalexin found in grapes and various plants, on 6-OHDA-induced cell damage to the SH-SY5Y human neuroblastoma cell line. Resveratrol (5 and 10 microM) inhibited 6-OHDA (60 microM)-induced cytotoxicity in SH-SY5Y cells and induced a reduction of the number of apoptotic nuclei caused by 6-OHDA treatment. Additionally, the total apoptotic rate of cells treated with both resveratrol (10 microM) and 6-OHDA (60 microM) was less than that of 6-OHDA treated cells. Resveratrol also dose-dependently (1, 5 and 10 microM) scavenged reactive oxygen species (ROS) induced by 6-OHDA in SH-SY5Y cells and prevented depletion of glutathione in response to the 6-OHDA-induced cytotoxicity in the glutathione assay. Overall, these results indicate that resveratrol exerts a neuroprotective effect against 6-OHDA-induced cytotoxicity of SH-SY5Y cells by scavenging ROS and preserving glutathione.
Subject(s)
Humans , Antioxidants , Apoptosis , Cell Line , Dopaminergic Neurons , Glutathione , Metabolism , Neuroblastoma , Neuroprotective Agents , Oxidopamine , Parkinson Disease , Reactive Oxygen Species , Substantia Nigra , VitisABSTRACT
Objective: To investigate the neuroprotective effects of paeoniflorin (PF) against cell death induced by hydrogen peroxide (H2O2) in human neuroblastoma cells and its mechanisms. Methods: H2O2 was used to induce SH-SY5Y cell damages, and the cell survival rate was detected by CCK-8 assay; the cell morphologic changes were observed by inverted optical microscope; the apoptosis was tested using Hoechst 33258 staining; flow cytometer (FCM) and propidium iodide staining were used to analyze the apoptosis and cell cycle alteration; reactive oxygen species (ROS) production was determined by 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence; lactic dehydrogenase (LDH) release was detected by reaction of diaphorase and INT; 8-OHdG production was determined by enzyme-linked immunosorbent assay (ELISA); caspase-3 activity was determined by caspase-3 catalyzing the specific substrate. Results: Compared with control group, after the treatment with H2O2 (200 μmol/L) for 24 h, the viability and proliferation index of CH-SY5Y cells were significantly decreased (P < 0.01, 0.05), the apoptosis rate and content of 8-OHdG were increased (P < 0.01), the LDH resease and ROS production were increased (P < 0.01); the activity of caspase 3 was increased (P < 0.01). Compared with H2O2 injury group, PF (20-40 μmol/L) significantly ameliorated the changes in SH-SY5Y cells induced by H2O2 in concentration-related manner (P < 0.05). PF (10 μmol/L) did not significantly change the above indexes except the cell viability, ROS, and caspase-3 activity induced by H2O2 (P < 0.05). Conclusion: PF has the significant protective effect against the H2O2-induced cell injury, which may be related to eliminatinging ROS, alleviating DNA oxidative damage, regulation of cell cycle, and inhibition of apoptosis of caspase pathway activation.
ABSTRACT
AIM: To examine the effects of the APP17-mer peptide against 2Aβ25-35-induced apoptosis and gain some insight into the neuroprotective mechanism of the APP17-mer peptide. METHODS: Protective effects of APP17-mer peptide against Aβ25-35- induced apoptosis in SH-SY5Y cell was proved by cell morphology, LM-PCR DNA ladder assay and FCM assay. The antiapoptotic mechanism of APP17-mer peptide was investigated using the MTT assay to measure mitochondrial energy redox state, using the fluorescent probe DCF-DA, Rhodamine 123 to measure relative levels of cellular peroxides and mitochondrial membrane potential and using Western blot for AIF and NF-kB to detect the expression of AIF and NF-KB. RESULTS: Damage of cell morphology was ameliorated by pretreating with APP17-mer peptide. The apoptotic rate of the SH-SY5Y cells exposed to Aβ25-35 in the presence of APP17-mer peptide decreased from 63.75% to 28.25%. Exposure of SH-SY5Y to Aβ25-35 for 48 h resulted in an increase in DCF-DA fluorescence, a decrease in Rhodamine 123 fluorescence and MTT reduction, the results were weakened by pre-incubating with APP17-me.r peptide for 30 minutes. Treatment of cells with APP17-mer peptide resulted in a significant attenuation in the expression of AIF and a strong increase in the expression of NF-KB. CONCLUSION: APP17-mer is protective against cell apoptosis induced by Aβ25-35 by provoking and sustaining upregulation of a key antiapoptotic transcription factor NF-KB, by suppressing oxyradical production and by preserving mitochondrial function and inhibiting the release of apoptotic protein from mitochondria.