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.     

Protective effect of procyanidins on PC12 cells exposed to Aβ25-35

AIM: To investigate the protective effect of procyanidins on the PC12 cells exposed to Aβ_25-35 and the mechanisms.METHODS: Aβ_25-35 at 25 μmol/L was used to treat the PC12 cells for 48 h, and the PC12 cells were pretreated with procyanidins at 25, 50 and 100 mg/L for 24 h. The cell vitality was measured by MTT assay. The content of reactive oxygen species (ROS) was detected by DCFH-DA staining. The change of mitochondrial membrane potential was examined by JC-10 staining. The apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of activated caspase-3 was determined by Western blot.RESULTS: Under the exposure of the PC12 cells to Aβ_25-35, procyanidins increased the cell viability, reduced intracellular ROS level, prevented mitochondrial membrane potential decline, attenuated the caspase-3 activation and inhibited the apoptosis of PC12 cells (P<0.05 or P<0.01).CONCLUSION: Procyanidins have a significant protective effect on the PC12 cells exposed to Aβ_25-35. Its mechanism may be related to removing intracellular ROS induced by Aβ_25-35, relieving the damage to the mitochondrial membrane, and thereby inhibiting cell apoptosis.     

Curcumin reduces neuroinflammation stimulated by Aβ25-35 in primary rat microglial cells

AIM: To investigate the effects of curcumin (Cur) on the expression of High mobility group box 1 protein (HMGB1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) in amyloid-β (Aβ)-induced primary rat microglial cells. METHODS: Microglia were derived from the cerebral cortices of postnatal rat brains. The cells were identified by immunocytochemistry using mouse anti rat Iba-1 monoclonal antibody. A cell model using primary rat microglial cells incubated with Aβ_25-35 as an inflammation model of Alzheimer's disease (AD) was set up. The morphological characters of primary rat microglial cells were observed. The concentration of Aβ_25-35 and the treatment concentration of curcumin were selected by CCK-8 assay. Cultured primary rat microglial cells were divided into 5 groups:normal cell group, Aβ_25-35 group, Cur group, Aβ_25-35+Cur group and Aβ_25-35+DMSO group. The expression of HMGB1, NF-κB, and receptor for advanced glycation end products (RAGE) was detected by Western blot. The levels of HMGB1, IL-1β, and TNF-α in the culture supernatant were measured by ELISA. RESULTS: The purity of primary microglias determined by Iba-1 immunofluorescence was more than 95%. The protein levels of HMGB1, RAGE and NF-κB were significantly increased after Aβ_25-35 stimulation. After treatment with Cur, the protein levels of HMGB1, RAGE and NF-κB were significantly decreased (P<0.05). The levels of HMGB1, IL-1β and TNF-α in the supernatant were significantly increased after Aβ_25-35 stimulation. Cur significantly decreased the level of HMGB1, IL-1β and TNF-α in the supernatant. CONCLUSION: Curcumin significantly inhibits neuroinflammation stimulated by Aβ_25-35 in primary rat microglial cells.     

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.     

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.     

Eeffect of HMGB1 on expression of NF-κB in BV-2 cells stimulated with Aβ25-35

AIM: To investigate the effect of high mobility group box-1 protein (HMGB1) on the expression of nuclear factor-κB (NF-κB) in BV-2 cells stimulated with amyloid β-protein (Aβ)_25-35. METHODS: Cultured BV-2 cells in logarithmic growth phase were divided into 4 groups:normal cell group (without any treatment), model group (treated with Aβ_25-35 at 40 μmol/L), RNA interference (RNAi) group (conducted with HMGB1-siRNA followed by Aβ_25-35 stimulation) and solvent control group (treated with 0.1% DMSO). After treatment with Aβ_25-35 for 24 h, the protein levels of HMGB1 and NF-κB in BV-2 cells were determined by Western blot. RESULTS: Aβ_25-35 at 40 μmol/L was used to stimulate BV-2 cells. The GFP fluorescence-tagged HMGB1-siRNA (30 nmol/L) was used to transfect BV-2 cells and its transfection efficiency was about 80%~90%. The results of Western blot showed that the protein level of HMGB1 was significantly decreased after the interference of siRNA fragment (P<0.05). The protein levels of HMGB1 and nucleic NF-κB p65 were dramatically increased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). After RNA interference with HMGB1, the expression of HMGB1 and nucleic NF-κB p65 were significantly decreased in BV-2 cells stimulated with Aβ_25-35 (P<0.05). CONCLUSION: RNA interference with HMGB1 reduces the expression of nucleic NF-κB in BV-2 cells stimulated with Aβ_25-35.     

Inhibitory effect of basic fibroblast growth factor on apoptosis induced by β-amyloid in PC12 cells

AIM:To clarify the effect of bFGF on the neurotoxity of Aβ_25-35 in PC12 cells and its potential application in the treatment of Alzheimer's disease. METHODS:Giema's, PI stainning, DNA agarose gel electrophoresis, Western blot and FCM were used to detect the morphological and biochemical changes of cultured PC12 cells treated with Aβ_25-35 and bFGF+ Aβ_25-35, and the expression of apoptosis-related gene bcl-2, bax. RESULTS:Morphological and biochemical characteristics of apoptosis, such as internuclear DNA fragmentation, compaction of nuclear chromatin, membrane blobbing, formation of apoptotic bodies, were observed in PC12 cells treated with Aβ_25-35. However, in PC12 cells treated with bFGF+ Aβ_25-35 , the above changes were significantly reversed, the expression of Bcl-2 was up-regulated while that of Bax was down-regulated. CONCLUSION:bFGF can inhibit the neurotoxity of Aβ_25-35 to neurons by regulating the expression of the apoptosis-related gene Bcl-2 and Bax.     

Role of P2Y1 receptor in activation of astrocytes induced by Aβ25-35

AIM: To study the role of P2Y₁ receptor in the activation of astrocytes induced by Aβ_25-35.METHODS: Astrocytes were isolated and cultured from newborn Wistar rats and divided into control group, Aβ_25-35 group, MRS2179(P2Y₁receptor inhibitor)+Aβ_25-35 group and MRS2179 group by treating the cells with the corresponding reagents. The expression levels of GFAP and P2Y₁ were determined by the methods of immunohistochemistry, immunofluorescence and Western blotting.RESULTS: No significant change of the astrocyte numbers in all groups was observed. Compared with the control cells, the fluorescence intensity of GFAP significantly increased in Aβ_25-35 group and decreased in both MRS2179+Aβ_25-35 group and MRS2179 group. The expression level of GFAP determined by Western blotting and immunofluorescence showed the similar trend of change in each group. Compared with control group, the expression of P2Y₁ in Aβ_25-35 group was significantly increased (P<0.05), and no significant change between MRS2179+Aβ_25-35 group and MRS2179 group was found (P>0.05).CONCLUSION: Aβ_25-35 activates astrocytes by activation of P2Y₁ receptor.     

Effects of Aβ1-42-induced microglial cells on survival of neural stem cells in vitro

AIM: To explore the effects of β-amyloid protein 1-42 (Aβ_1-42)-induced microglia on the survival of cultured neural stem cells (NSCs) in vitro . METHODS: Using the Transwell chambers to build a coculture system of NSCs and microglia, we detected the proliferation, differentiation and apoptosis of the NSCs with the microglia before and after induction by Aβ_1-42. RESULTS: Compared with non-intervention group, the proliferation rate of NSCs in Aβ_1-42 intervention coculture group decreased, as well as the positive expression rates of microtubule-associated protein 2 (MAP-2) and choline acetyltransferase. CONCLUSION: The inflammation mediated by Aβ_1-42 inhib their the proliferation of NSCs and induces their apoptosis. Inflammation also significantly reduces the ratio of NSCs differentiating to neurons, especially to cholinergic neurons.     

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.     

CDK4-pRB-E2F1 pathway may mediate Aβ1-40-induced apoptosis in rat cortical neurons

AIM:To study the possible molecular mechanism of beta-amyloid peptide_1-40-induced apoptosis in rat cortical neurons.METHODS:40 mg/L beta-amyloid peptide_1-40 (Aβ_1-40) was used to induce apoptosis in cultured rat cortical neurons. The level of CDK4, phosphorylated pRB were detected by flow cytometry and immunoblotting; RT-PCR was used to examine the mRNA expression of E2F1 while fluorescent spectrofluorometer was used to measure caspase-3 activity. All of the above study was designed to observe whether the level of CDK4, phosphorylated pRB and E2F1 mRNA expression could be affected by Aβ_1-40.RESULTS:(1)The level of CDK4, phosphorylated pRB increased markedly 2-4 hours after treatment with Aβ_1-40, and caspase-3 activity elevated remarkably 12-24 hours after treatment with Aβ_1-40; (2) E2F1 mRNA expression was upregulated 3 hours after incubation with Aβ_1-40.CONCLUSION:Aβ_1-40 may induce apoptosis in rat cortical neurons in a manner dependent on CDK4-pRB-E2F1 pathway.     

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.     

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.     

Overexpression of neuroglobin attenuates Aβ-induced apoptosis in brains of double transgenic AD mice

AIM: To observe the effects of neuroglobin(NGB) overexpression on the apoptosis induced by Aβ in the brains of double transgenic AD(APPswe/PS1dE9) mice and to explore its potential mechanisms.METHODS: Twenty-four 13-month-old double transgenic AD mice were randomly divided into 3 groups:intracerebroventricular injection with normal saline(NS) group, intracerebroventricular injection with pcDNA3.1 and NS group, and intracerebroventricular injection with pcDNA3.1 and pNGB group. Immunohistochemistry was used to detect the expression of Aβ_1-42 in the brains. TUNEL staining was used for analyzing the apoptosis, and the protein levels of cleaved caspase-3, caspase-9, PI3K, Akt and p-Akt were determined by Western blot.RESULTS: After intracerebroventricular injection with pNGB, the areas of Aβ_1-42 in the hippocampus and cortex were decreased compared with NS group and pcDNA3.1+NS group(P<0.01). The TUNEL-positive staining cells in the pNGB group were less than those in NS group and pcDNA3.1 group(P<0.01). NGB overexpression attenuated the protein levels of cleaved caspase-3 and caspase-9(P<0.01), but induced the production of PI3K and p-Akt(P<0.01).CONCLUSION: Overexpression of pNGB significantly inhibits the generation of Aβ and attenuates the apoptosis induced by Aβ, indicating that NGB overexpression activates PI3K/Akt pathway and inhibits the production of cleaved caspase-3 and caspase-9, which were tightly related with apoptosis.     

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.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

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.     

Chrysophanol ameliorates spatial memory ability of rats by inhibiting Aβ1-42-induced oxidative stress

AIM: To investigate whether chrysophanol alleviates amyloid β-protein (Aβ)-induced cognitive dysfunction and the underlying antioxidative mechanism.METHODS:Adult male Wistar rats (230~250 g) were randomly divided into control group, Aβ_1-42 group, chrysophanol group, and Aβ_1-42+chrysophanol (1, 10 and 100 mg/kg) groups. Aβ_1-42 was delivered by intracerebroventricular injection under the guidence of a brain stereotaxic apparatus. Y-maze test, open-field test and Morris water maze test were performed 1 week after Aβ_1-42 injection to evaluate the ability of rat spacial learning and memory. Chrysophanol was intraperitoneally injected once daily for 5 consecutive days. After the behavioral tests, the animals were sacrificed immediately by decapitation, and the hippocampus were collected. The malondialdehyde (MDA) content and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in the hippocampus were measured.RESULTS:Multiple (7 consecutive days, once daily) but not single (once a day) chrysophanol treatment at 1, 10 and 100 mg/kg effectively prevented Aβ_1-42-induced cognitive function deficits in a dose-dependent manner as shown by Y-maze test and Morris water maze test. Moreover, the Aβ_1-42-induced increase in MDA content and decrease in the activity of antioxidant enzymes (SOD, GSH-Px and CAT) in the hippocampus of the rats were also attenuated by multiple chrysophanol treatment.CONCLUSION:Repeated chrysophanol treatment attenuates Aβ_1-42-induced cognitive deficits and synaptic plasticity dysfunction, and the mechanisms underlying the neuroprotective effects are likely due to its antioxidant activity.     

Cytotoxicity of lidocaine on SH-SY5Y cells

AIM: To observe the effects of lidocaine on SH-SY5Y cells and to investigate the neural cytotoxicity of lidocaine. METHODS: Cultured SH-SY5Y cells in vitro were divided into 4 groups: in group C (control group), SH-SY5Y cells were cultured under normal conditions; in group L1, SH-SY5Y cells were cultured with 0.5% lidocaine for 10 min; in group L2, SH-SY5Y cells were cultured with 1% lidocaine for 10 min; in group L3, SH-SY5Y cells were cultured with 2% lidocaine for 10 min. Cell morphology, cell viability and apoptosis were detected to evaluate the effects of lidocaine on the cells. RESULTS: SH-SY5Y cells in group C showed dendritic protrusions, enlarged nervous process and dense network. However, SH-SY5Y cells in the groups of L1, L2 and L3 showed the disappearance of dendritic protrusions, cell shrinkage, cell size reduction and round shape. Compared with SH-SY5Y cells in group C, the cell viability significantly decreased in the groups of L1, L2 and L3 with the increase in the concentration of lidocaine. Apoptotic rate of SH-SY5Y cells in group C was between 5.9% and 6.3%, and it increased with the increases in the concentration of lidocaine. CONCLUSION: Lidocaine at concentrations of 0.5%, 1%, 2% can damage SH-SY5Y cells, and this injury become obviously serious with the increase in the concentrations of lidocaine, indicating that we should use the minimum effective concentration of lidocaine in clinic to prevent the harmful effect of the drug.     

Effect of tenuigenin on tau protein phosphorylation at Ser396 site in neurons of AD rats induced by Aβ1-40

AIM:To investigate the effect of tenuigenin(TEN) on hyperphosphorylation of tau protein in neurons of amyloid β-peptide_1-40(Aβ_1-40) -induced Alzheimer disease(AD) rats. METHODS:Aβ_1-40 was injected into hippocampus CA1 region of the rats to establish AD model. TEN at different doses(18.5 mg/kg, 37.0 mg/kg and 74.0 mg/kg) was intragastrically administered. The protein expression of protein kinase A(PKA),protein phosphatase 2A(PP2A), total tau and p-tau(Ser³⁹⁶) in the neurons was observed by the method of immunohistochemistry. The protein content of total tau and p-tau(Ser³⁹⁶), and the expression level of PKA and PP-2A were detected by Western blotting analysis. RESULTS:In Aβ_1-40 group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were significantly increased compared with those in sham operation group. Meanwhile, the expression of PP2A in Aβ_1-40 group was lower than that in sham operation group. In TEN treatment group, the level of total tau, the phosphorylation of tau protein and the expression of PKA were markedly decreased, and the expression of PP2A was increased as compared with Aβ_1-40 group. CONCLUSION:TEN may protect the neurons from the toxic effect of Aβ_1-40 and reduce the hyperphosphorylation of tau(Ser³⁹⁶) in the neurons of AD rats by activating the expression of PP2A and inhibiting the expression of PKA.