Effect of progesterone on SH-SY5Y cells injured by ATP

AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca²⁺ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X₇ receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X₇ receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca²⁺ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca²⁺ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X₇ receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X₇ receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X₇ receptor expression, membrane pore formation, intracellular Ca²⁺ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; OGD group:the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.     

Protective effect of aliskiren on SH-SY5Y cells injured by oxygen-glucose deprivation and its possible mechanisms

AIM: To investigate the effects of aliskiren on the injury of SH-SY5Y cells induced by oxygen-glucose deprivation (OGD) and its possible mechanisms. METHODS: The SH-SY5Y cells were randomly divided into control group, OGD group and aliskiren (5.0, 10.0 and 20.0 μmol/L) groups. The cell viability was measured by CCK-8 assay. The levels of excitatory amino acid transporter 2 (EAAT2/GLT-1), EAAT3/EAAC1, EAAT4, endothelin-1 (ET-1) and S100 calcium-binding protein β subunit (S-100β) in the SH-SY5Y cells were detected by ELISA. The morphological changes of the cells were observed by Hoechst 33258 staining. Meanwhile, the content of lactic acid (LD) and activity of Na⁺-K⁺-ATPase were also analyzed. RESULTS: The viability of SH-SY5Y cells was not more than 60% after OGD injury for 4 h, so the appropriate time for OGD injury was 4 h. Compared with control group, the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells of OGD group were significantly decreased (P<0.05), but the protein levels of ET-1 and S-100β were significantly increased (P<0.05). Compared with OGD group, treatment with aliskiren dose-dependently increased the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells, but decreases in the levels of ET-1 and S-100β were observed (P<0.05). The results of Hochest 33258 staining showed that aliskiren significantly reduced the apoptosis of SH-SY5Y cells. Compared with control group, a significant increase in the content of LD (P<0.05) and a significant decrease in Na⁺-K⁺-ATPase activity (P<0.05) were found in the SH-SY5Y cells of OGD group. Compared with OGD group, aliskiren dose-dependently decreased the content of LD, but increased the Na⁺-K⁺-ATPase activity in the SH-SY5Y cells (P<0.05). CONCLUSION: Aliskiren has good neuroprotective effects on SH-SY5Y cells after OGD injury. The underlying mechanisms may be associated with the increases in the protein levels of GLT-1, EAAC1 and EAAT4, the enhancement of Na⁺-K⁺-ATPase activity, and the decreases in the levels of ET-1 and S-100β and the content of LD.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 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.     

Effect of salidroside on mitochondrial membrane potential during injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cells

AIM: To investigate the effects of salidroside on intracellular free calcium concentration [Ca²⁺]i, apoptosis, mitochondrial membrane potential (MMP) and activity during injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cells. METHODS: Mitochondrial activity was measured by methylthiazolyl tetrazolium test. MMP,[Ca²⁺]i and apoptosis were measured by flow cytometry. RESULTS: SH-SY5Y cells were cultured in a hypoxia/hypoglycemia condition for 2, 4, 6 and 12 h,[Ca²⁺]i and apoptosis rate significantly increased compared with control group (P<0.01). After hypoxia /hypoglycemia cultures, MMP and mitochondrial activity declined 29.17% (P<0.01) and 38.80% (P<0.01) at 2 h, 56.72% (P<0.01) and 63.58% (P<0.01) at 12 h, were lower than that in control group (P<0.01). Salidroside significantly decreased [Ca²⁺]i and apoptosis rate, and increased MMP and mitochondrial activity in hypoxia /hypoglycemia-treated SH-SY5Y cells. CONCLUSIONS: Salidroside might inhibit the decline in MMP and mitochondrial activity induced by hypoxia /hypoglycemia, and has an inhibitory effects on neuronal apoptosis. The mechanism might be related to inhibiting intracellular calcium overload.     

Effect of MG132 on Aβ generation in SH-SY5Y cells

AIM: To observe the influences of different concentrations of MG132 on apoptosis and beta-amyloid protein(Aβ) generation in SH-SY5Y cells, and to explore the underlying mechanism.METHODS: SHSY-5Y cells were incubated with MG132 for 24 h. The final concentrations of MG132 were 2.5, 5 and 10 μmol/L. The cell viability was determined by MTT assay. The cell apoptosis was assessed by flow cytometry. The levels of Aβ were measured by ELISA. The relative protein levels were detected by Western blot.RESULTS: In the SH-SY5Y cells, MG132 reduced the cell viability, induced the cell apoptosis, increased the level of Aβ, and increased the expression of the related proteins for Aβ generation in a concentration-dependent manner.CONCLUSION: MG132 induces apoptosis and increases the levels of Aβ_1-42 and Aβ_1-40 by regulating the proteins related to Aβ generation in the SH-SY5Y cells.     

High concentration of extracellular ATP causes autophagy and apoptosis of SH-SY5Y cells

AIM：To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5Y cell injury. METHODS：Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile, apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blotting. RESULTS：Compared with control group, the survival rate of SH-SY5Y cells was significantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h). The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment, trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment, which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2~3 h with ATP treatment, and returned to control level at 6 h. Compared with control group, the apoptotic rate and the expression level of caspase-3 were enhanced synchronously. The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION：High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5Y cells. The increased autophagy shows up, followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP, apoptosis is the main process in the cells.     

Overexpression of α-synuclein in SH-SY5Y cells in vitro leads to oxidative stress

AIM: To observe the influence of overexpression of α-synuclein on the cultured SH-SY5Y cells. METHODS: The plasmid of α-synuclein-pcDNA3 was transfected into SH-SY5Y cells with LipofectAMINE. The expression of α-synuclein was determined by anti-α-synuclein immunocytochemistry. The intracellular reactive oxygen species was determined with 2', 7'-dichlorofluorescein diacetate (DCF-DA) by using a FACSCAN flow cytometer and fluorescent microscope. The intracellular content of reduced GSH was detected with glutathione assay kit by spectrophotometer. RESULTS: The α-synuclein was expressed in cultured SH-SY5Y cells transfected with the plasmid of α-synuclein-pcDNA3. The DCF loading analysis and the intracellular level of reduced GSH suggested that the transfected cells were under oxidative stress. CONCLUSION: Overexpression and accumulation of α-synuclein in SH-SY5Y cells increase intracellular reactive oxygen species levels, it is suggested therefore that the α-synuclein does play an important role in the pathogenesis of Parkinson's disease.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Effect of autophagy in SH-SY5Y cells injured by Aβ25-35

AIM: To explore whether the damage of neurons induced by amyloid β-protein (Aβ) is related to the regulation of autophagy and its mechanism based on Akt/mTOR pathway. METHODS: SH-SY5Y cells were incubated with Aβ_25-35 (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L) for 24 h, and the cell viability was measured by MTT assay. The protein levels of LC3-I, LC3-II, Akt, p-Akt, mTOR and p-mTOR in the SH-SY5Y cells were determined by Western blot. After the SH-5Y5Y cells were incubated with autophagy inducer rapamycin (Rapa) or autophagy inhibitor 3-methyladenine (3-MA) combined with Aβ_25-35 for 24 h, the cell viability and related protein expression were detected by the same methods above mentioned. RESULTS: Each concentration of Aβ_25-35 damaged SH-SY5Y cells and decreased the viability of SH-SY5Y cells. Aβ_25-35 increased the expression of autophagy marker protein LC3-II, increased the level of LC3-II/LC3-I, and down-regulated the phosphorylation level of Akt and mTOR proteins (P<0.05). When combined with autophagy inducer Rapa, the cell viability was not significantly affected, the expression of LC3-II protein was increased, LC3-II/LC3-I was increased significantly, and p-mTOR/mTOR level was decreased (P<0.05). When combined with autophagy inhibitor 3-MA, the protein expression of LC3-II and the level of LC3-II/LC3-I showed a downward trend, while the level of p-Akt/Akt was decreased (P<0.05). CONCLUSION: Aβ_25-35 may induce SH-SY5Y cell autophagy and injury by down-regulating phosphorylation levels of Akt and mTOR proteins.     

Effects of DARPP-32 on the drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of DARPP-32 on the drug sensitivity of human neuroblastoma cell line SH-SY5Y. METHODS: The plasmid containing cDNA of DARPP-32 gene and the small interfering RNA eukaryotic expression vector specific to human DARPP-32 gene were constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting analysis. MTT assay and flow cytometry assay were used to investigate the effects of DARPP-32 on the drug sensitivity and the drug accumulation in cell models. The expressions of Bcl-2，Bax, P-gp and MRP were analyzed by RT-PCR and Western blotting. RESULTS: The stable clones with increased or decreased DARPP-32 expression were successfully established. Up-regulation of DARPP-32 significantly enhanced the drug sensitivity and the drug accumulation of SH-SY5Y cells due to down-regulation of P-gp and Bcl-2 expressions. Down-regulation of DARPP-32 significantly reduced the drug sensitivity of SH-SY5Y cells and decreased drug accumulation in SH-SY5Y cells. CONCLUSION: DARPP-32 might mediate the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents.     

Effects of survivin inhibitor YM155 on mitochondrial apoptotic pathway of retinoblastoma Y79 cells

AIM: To investigate the effects of survivin inhibitor YM155 {4,9-dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide} on the apoptosis, mitochondrial membrane potential (Δψ_m) and cytochrome C (Cyt C) of retinoblastoma Y79 cells, and to analyze the mitochondrial mechanisms of apoptosis.METHODS: Y79 cells were cultured in vitro and treated with YM155 at concentrations of 0, 0.5, 1, 2, 4 and 8 nmol/L. The cells in control group were treated without YM155. The proliferation of Y79 cells were measured by CCK-8 assay and bromodeoxyuridine (BrdU) labeling assay. Y79 cells were randomly divided into 4 groups:control group (with equal volume of RPMI-1640 nutrient medium), positive control group (10 nmol/L topotecan), low-dose (1 nmol/L) YM155 group and high-dose (2 nmol/L) YM155 group. The effects of YM155 on the apoptosis, the changes of Δψ_m, the mitochondrial distribution and the protein level of Cyt C in the Y79 cells were evaluated by flow cytometry with Annexin V-FITC/PI staining, JC-1 staining, immunofluorescence analysis and Western blot, respectively. RESULTS: Compared with control group, YM155 significantly inhibited the proliferation of Y79 cells and induced apoptosis (P<0.05). YM155 significantly reduced Δψ_m of the Y79 cells, promoted Cyt C which released from mitochondria to the cytosol and reduced the protein level of Cyt C in the mitochondria (P<0.05). CONCLUSION: YM155 inhibits Y79 cell proliferation and induces apoptosis, and the possible mechanisms may be involved in the mitochondrium-mediated apoptotic pathway.     

Protective effect of NAD(P)H:quinone oxidoreductase 1 on dopaminergic cells

AIM: To determine the influence of NAD(P)H:quinone oxidoreductase 1 (NQO1) on dopamine-induced toxicity in dopaminergic cells.METHODS: MTT assay was used to determine the toxic curve of dopamine in SH-SY5Y cells. Lipofection was applied to transfect SH-SY5Y cells with an NQO1 expression plasmid. The endogenous and transfected NQO1 expression was detected by immunofluorescence staining and Western blotting. The content of cellular quinone protein was measured by nitroblue tetrazolium (NBT) method. RESULTS: Dopamine reduced SH-SY5Y cell proliferation in a dose-dependent manner, which was correlated with an increase in the content of quinone protein. Increased expression of NQO1 by transient transfection or by phase II enzyme inducer sulforaphane treatment alleviated dopamine-induced toxicity and reduced the content of cellular quinone protein. CONCLUSION: Increased NQO1 expression protects SH-SY5Y cells against cytotoxicity caused by dopamine.     

Effects of RUNX3 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of RUNX3 gene on the growth and drug sensitivity of SH-SY5Y cells.METHODS: The siRNA plasmid of RUNX3 was constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting. The growth curve, cell cycle distribution, drug sensitivity assay and accumulation of adriamycin in cells were detected by MTT assay and flow cytometry. The expressions of cyclin D1, CDK4, CDK6, p21, p27, Bcl-2, Bax, P-gp and MRP were analyzed by Western blotting. RESULTS: mU6pro-RUNX3 siRNA was successfully constructed and transfected into SH-SY5Y cells. Down-regulation of RUNX3 significantly promoted the cellular proliferation, inhibit the drug sensitivity and intracellular adriamycin accumulation of cells, compared with that in the controls (P<0.05). The expressions of P-gp, Bcl-2 and cyclin D1 in transfected cells were increased, while p21 decreased.CONCLUSION: RUNX3 might play important roles in the development of neuroblastoma.     

Glycogen synthase kinase-3 beta overexpression enhances tau protein phosphorylation and attenuates tubulin acetylation in SH-SY5Y cells

AIM: To construct GSK3β-overexpressed SH-SY5Y cells and to observe the effects of GSK3β-overexpression on tau protein phosphorylation and tubulin acetylation in SH-SY5Y engineered cells. METHODS: The cDNA of GSK3β construct was subcloned into mammalian expression vector pBudCE4.1. The integrity of the GSK3β construct was confirmed by sequence analysis. GSK3β was transiently transfected into SH-SY5Y cells using Lipofectamine2000. Western blotting was used to measured protein levels of GSK3β and phosphorylating GSK3β, as well as, the total tau and phosphorylated tau protein and acetylated tubulin. RESULTS: 36 h after transfection, the levels of GSK3β and phosphorylating GSK3β in SH-SY5Y cells were significantly increased compared with non-transfection group and vector group. After 48 h, the levels of phosphorylated tau protein (Ser199/202, Thr231 and Thr205 residues) but not total tau protein were markedly increased in GSK3β-overexpressed SH-SY5Y cells. In addition, the level of acetylated tubulin was lower than that in non- transfection group and vector group. CONCLUSION: The over-expression of GSK3β in SH-SY5Y cells results in robust increases in tau protein phosphorylation at Ser199/202, Thr231 and Thr205 residues, and decreases in tubulin acetylation.     

Panax notoginseng saponins suppress oxygen-glucose deprivation/reoxygenation-induced pyroptosis of SH-SY5Y cells

AIM To investigate the effect of Panax notoginseng saponins (PNS) on pyroptosis of SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS The OGD/R was conducted to induce ischemia/reperfusion injury in SH-SY5Y cells. The effects of PNS on the viability (detected by CCK-8 assay) and membrane permeability [indicated by lactate dehydrogenase (LDH) leakage and propidium iodide (PI) staining positive cell proportion] of OGD/R-induced SH-SY5Y cells were observed. The protein levels of gasdermin D (GSDMD), GSDMD N-terminal fragment (GSDMD-N), caspase-1 and caspase-4, and the release of interleukin-1β (IL-1β) and IL-18 in the cells were also determined. RESULTS After exposure to OGD/R, the viability of SH-SY5Y cells dramatically decreased (P<0.01), while the LDH leakage, the PI staining positive cell proportion, the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4, and the release of IL-1β and IL-18 were significantly increased (P<0.01). However, PNS treatment enhanced the viability of SH-SY5Y cells inhibited by OGD/R (P<0.01), but reduced the leakage of LDH and the percentage of PI staining positive cells (P<0.05 or P<0.01). Moreover, PNS reversed the increases in the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4 and the release of IL-1β and IL-18 in OGD/R-induced SH-SY5Y cells (P<0.05 or P<0.01). CONCLUSION Treatment with PNS alleviates OGD/R-induced injury in SH-SY5Y cells. Its mechanism may be related to inhibition of SH-SY5Y cell pyroptosis induced by OGD/R.     

Prevention and treatment of AD by over-expression of neuroglobin via activating PI3K/Akt signaling pathway

AIM: To study the neuroprotective roles of neuroglobin (NGB) over-expression in the SH-SY5Y cells transfected with pAPPswe.METHODS: The plasmid pEGFP-NGB was successfully constructed and transfected into the SH-SY5Y cells, which were pretreated with pAPPswe. MTT assay was applied to detect the effect of NGB over-expression on the cell survival rates. JC-1 staining was used to detect the level of mitochondrial transmembrane potential. The cell apoptosis was analyzed by flow cytometry. The effects of NGB over-expression on the protein level of p-Akt, Akt and caspase-3/9 were determined by Western blotting. The generation of Aβ42 in the cells was measured by ELISA.RESULTS: The cell survival rate was remarkably increased after transfection with NGB compared with control group and empty plasmid group (P<0.05). The over-expression of NGB significantly inhibited the decrease in mitochondrial membrane potential induced by pAPPswe. The over-expression of NGB inhibited the apoptosis of the cells. Furthermore, over-expression of NGB not only inhibited the expression of caspase-3 and caspase-9, but also induced the production of p-Akt, which was prevented by LY294002, an inhibitor of PI3K/Akt. The generation of Aβ42 was inhibited in the cells with the over-expression of NGB. CONCLUSION: Over-expression of NGB significantly inhibits the SH-SY5Y cell injuries induced by pAPPswe and inhibits the expression of caspase-3/9, which is tightly related with cell apoptosis. Furthermore, the neuroprotective roles of NGB may be via activating PI3K/Akt signaling pathway.     

Role of divalent metal transporter 1 in degeneration of dopaminergic neuron

AIM: To observe the expression of divalent metal transporter 1(DMT1) in SH-SY5Y cells with lactacystin-induced injury, and to investigate the possible role of DMT1 in the degeneration of dopaminergic neuron in Parkinson disease(PD). METHODS: An in vitro model of cell injury was established in SH-SY5Y cells induced by lactacystin. The protein expression of DMT1 was detected by immunofluorescence and Western blotting. Under the environment of high iron level, the cellular oxidative stress was observed by fluorescent probe DCFH-DA. The level of α-synuclein was determined by immunohistochemistry and Western blotting. RESULTS: The cell viability rate was reduced by lactacystin in a concentration-dependent pattern. Compared with the control, the protein level of DMT1 was obviously increased in lactacystin-treated cells. The arrangements of the changes from high to low in decreased cell viability, increased intracellular oxidative stress and increased aggregation of α-synuclein(43-55 kD) were Fe²⁺ treatment group > lactacystin treatment group > control group(P<0.05). CONCLUSION: Lactacystin up-regulates the protein expression of DMT1. By this way, the increased function of DMT1-mediated iron uptake may play an important role in iron or iron-catalyzed oxidative reactions to enhance α-synuclein aggregation, leading to the degeneration of neurons in PD.     

Protective effect of osthole on SH-SY5Y cells transfected with APP595/596 gene

AIM: To explore the protective effect of osthole on the SH-SY5Y cells transfected with APP595/596 gene, and to investigate the molecular mechanism. METHODS: The SH-SY5Y cells were transfected with APP595/596 gene in vitro for establishing a cell model to study the pathogenic role of amyloid β-protein (Aβ). The cell viability was detected by CCK-8 assay. The release of lactate dehydrogenase (LDH) was determined by the colour reaction of diaphorase-INT. The cell apoptotic rate was analyzed by flow cytometry. The expression of β-site APP cleaving enzyme 1(BACE1) at mRNA and protein levels was detected by RT-PCR and Western blot. The expression of Aβ was measured by the technique of immunofluorescence cytochemistry and Western blot. RESULTS: Treatment with osthole inhibited the LDH release, and increased the viability of the cells. The percentage of apoptotic cells was also significantly decreased. Osthole also inhibited the expression of BACE1 at mRNA and protein levels and the protein expression of Aβ. CONCLUSION: Osthole has protective effect on SH-SY5Y cells transfected with APP595/596 gene. The mechanism may be association with inhibiting the mRNA and protein expression of BACE1.