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.     

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.     

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.     

Effect of antioxidants TA9901 on the fibril formation of Aβ1-40 injected into cerebral cortex of rat brain

AIM: To clarify if TA9901, a natural antioxidants, could inhibit the formation of β-amyloid (Aβ) fibril when Aβ_1-40 were injected into cerebral cortex of rat brain, and explore the mechanism of action of TA9901 on Alzheimer disesse. METHODS: Twelve Wistar rats (250-300 g) were randomly divided into four groups (n=3). (1) control group; (2) TA9901 treatment group (ip 100 mg·kg^-1 ·d^-1); (3) Vitamin E (VE) treatment group (ip 100 mg·kg^-1 ·d^-1); (4) PBS group. 5 μL 0.2% Aβ_1-40 was immediately injected into the right side of the deep cerebral cortex of control, TA9901 and VE group rats. The animals were sacrificed at the seventh day after the injection. The sections of the rat brain that contained the injected field were examined with transmission electron microscopy and Congo red staining with polarized microscopy. RESULTS: Many depositions of high electron density were observed by electron microscopy in the field where Aβ_1-40was injected. They are intimately intermingled with macrophages and astrocytes. In the field, abou10nm fibrillar structures were observed that appeared similar to the fibrils seen in senile plaque (SP) of the brain of Alzheimer disease (AD). The fields in control and VE group contained richer Aβ fibrils than that in TA9901 group. After the sections stained with Congo red, A1-40aggregation demonstrated intense birefringence under, indication the formation of amyloid fibrils. In TA9901 group, there was a weak birefringence. CONCLUSIONS: TA9901 can inhibit the fibril formation of Aβ that was injected into deep cerebral cortex of rat brain, this indicates primarily that TA9901 may be a potential therapeutic drug to interfere with the progression of amyloidgenesis in AD.     

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.     

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.     

Generation of induced pluripotent stem cells and neural cells from urine-derived cells of Alzheimer disease patients

AIM: In this study, we aim to obtain the induced pluripotent stem cells (iPSCs) from the patients with sporadic Alzheimer disease (AD). METHODS: Three typical Alzheimer's patients were chosen, and the epithelial cells were isolated from their urine. We reprogrammed these cells into induced pluripotent stem cells by transfection of 4 factors (Oct4, Sox2, Klf4 and SV40LT) with the technique of electro-transfection. After getting these iPSCs, we continue to differentiate them into neural cells by a specific method—dual inhibition of Smad signaling. RESULTS: The primary cells from 3 AD patients were successfully reprogrammed to iPSCs, and these patients-derived iPSCs were differentiated into neural cells. There was no significant difference, during iPSCs reprogramming and neural differentiation, between cells from AD patients and normal people. CONCLUSION: The urine cells from AD patients were able to transfer to iPSCs, functional neurons and neurogliocytes.     

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.     

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.     

Anti-inflammatory effect of huperzine A on protection of rat neural stem cells in vitro

AIM：To explore the anti-inflammatory effect of huperzine A (HupA) and its neuroprotective effect on rat neural stem cells (NSCs). METHODS：The microglia and NSCs were isolated from neonatal rat hippocampal tissues and co-cultured in a Transwell system. The cells were divided into 3 groups：control group, amyloid beta-peptide (Aβ) group and HupA group. The microglia layer in Aβ group was treated with Aβ1-42 (10 μmol/L), while that in HupA group was pretreated with HupA (1 μmol/L) before Aβ1-42 stimulation. The culture supernatant levels of inflammatory mediators, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 1α (MIP-1α), were detected by LiquiChip technique. The apoptosis of NSCs was determined by flow cytometry and Western blotting. RESULTS：The microglia secreted a large number of inflammatory mediators with the stimulation of Aβ. In Aβ group, the levels of IL-6, TNF-α and MIP-1α were significantly higher than those in control group at 72 h (P<0.01), and the apoptotic rate of NSCs was 25.46% (P<0.01). In HupA group, the concentrations of IL-6, TNF-α and MIP-1α decreased significantly as compared with Aβ group (P<0.01), and the apoptotic rate of NSCs was only 8.05% (P<0.01). The Bcl-2/Bax ratio in HupA group was higher than that in Aβ group (P<0.05). CONCLUSION：Huperzine A reduces the secretion of cytokines and chemokines, and attenuates microglia-mediated neuroinflammation, thus protecting NSCs against inflammation-induced apoptosis.     

Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro

AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS:Embryonic body induced by retinoic acid and retinal müller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and müller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.     

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.     

Over-expression of Hsp75 in neural stem cells reduced Aβ-mediated neurotoxicity

AIM: To investigate the effect of heat shock protein 75 (Hsp75) over-expression on Aβ-induced neurotoxicity in the neural stem cells and to explore its mechanism. METHODS: An adenovirus-mediated Hsp75 over-expression vector was used in vitro. The mouse neural stem cell C17.2 was cultured in vitro and divided into control group, Aβ group, negative adenovirus vector transfection group and Hsp75 over-expression adenovirus vector transfection group. The transfection and cellular immune identification were detected by fluorescence microscopy. The cell morphology was observed under inverted phase-contrast microscope. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Hsp75 over-expression and cleaved caspase-3 protein level were measured by Western blot. RESULTS: Observation by fluorescence microscopy indicated that C17.2 cells were successfully transfected and Hsp75 gene was effectively expressed in the neural stem cells after transfection. In addition, the morphology and viability of the cells did not change and these cells did not differentiate after transfection. As compared with control group, the cell viability in Aβ group and negative adenovirus vector transfection group was significantly decreased (P < 0.05), and the cell apoptotic rate and cleaved caspase-3 level (P < 0.05) were increased. As compared with Aβ group and negative adenovirus vector transfection group, Hsp75 over-expression significantly increased the cell viability, and decreased the cell apoptosis and cleaved caspase-3 level (P < 0.05). CONCLUSION: Hsp75 over-expression protects the neural stem cells against Aβ-induced injury. The mechanism may be related to inhibiting caspase-3 pathway-dependent apoptosis.     

Effects of gypenosides on cell cycle-related protein expression and calcium homeostasis in hippocampal neurons of rats treated with β-amyloid protein fragmant 1-40

AIM：To observe the effects of gypenosides on the expression of cell cycle-related protein and calcium homeostasis in the hippocampal neurons in the animal model of Alzheimer’s disease (AD) induced by Aβ₁-40.METHODS：The animal model of AD was established through injection of Aβ₁-40 into hippocampus in rats.The ability of learning and memory were verified by the performance of Y-shape maze task.With the method of immunohistochemical staining coupled integral absorbance analysis,the expression of cell cycle related protein in hippocampus was determined in rats.The contents of cytoplasm Ca2+ were determined using Fura-2/AM-fluorescence method.The effects of gypenosides on above indices were studied.RESULTS：Compared with the controls,the learning and memory ability of Aβ₁-40-injected rats were lower,the expression of cyclin A and cyclin B₁ protein in rat hippocampus neurons had hoisted,the contents of calcium in hippocampus were increased significantly.Gypenosides,however,improved all the indexes in varying degrees.CONCLUSION：These results imply that gypenosides improves the learning and memory ability of the rats treated with Aβ₁-40,reverses the expression of cell cycle-related protein and maintains the calcium homeostasis in hippocampus neurons.     

Effect of VEGF on proliferation and differentiation of neural stem cells from the hippocampal gyrus of rat in vitro

AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.     

Effects of TGF-β1 and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells(HSPC)

AIM:To study the effect of TGF-β₁ and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells (HSPC). METHODS:CD₃₄⁺cells were purified from fresh umbilical cord blood by immunomagnetic beads, and mononuclear cells were purified from bone marrow by Ficoll-hypaque. The effects of TGF-β₁ and /or TNF-α antisense PS-ODNS on ex vivo expansion of CD₃₄⁺ cells、CFU-GEMM、CFU-GM、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems.RESULTS:TGF-β₁ antisense PS-ODNS cooperated with cytokines increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E, which was as 4, 2.6, 2.7, 1.8, 2.1 times as that of the control (the cytokines combination), respectively. TNF-α antisense PS-ODNS cooperated with cytokines respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 4, 2.9, 2.6, 1.7, 1.8 times as that of the control. The above two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5.3, 2.1, 2.7, 1.9, 1.8 times as that of the control.CONCLUSION:Inhibition of endogenous TGF-β₁ and TNF-α by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.     

Comparison of 2 methods for inducing iPSC to differentiate into neural stem cells

AIM: To select an efficient way of promoting induced pluripotent stem cells (iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods. METHODS: The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100% initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density. For comparison and identification of the 2 methods, the growth state was observed under microscope, and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry. The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis. RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax6, nestin, Sox1 and Sox2. The growth state was better and the cells differentiated into neurons and astrocytes normally. CONCLUSION: The method A was superior to method B, and we recommend the method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density as the method for differentiating NSC.     

Effect of antioxidants on secondary conformational transition of aged amyloid β-peptide1-40 by FT-IR quantitative study

AIM: To investigate the mechanism that antioxidants TA9901, inhibit the formation of amyloid-β-protein(Aβ) fibril.METHODS: Fourier-transform infrared spectroscopy was used to study the secondary structure changes on aging Aβin vitro.RESULTS: Aβ aged alone for 30 min, the content of β-pleated sheet and β-turn were 43.17% and 32.9% respectively. Aβ aged alone for 7 days, the content of β-pleated sheet increased abuot 10% and produced a shift of random coil toward β-pleated sheet. TA9901 induced a significant decrease of the content of β-turn (23.5%) and β-pleated sheet (26.4%). VE mainly decreased the β-pleated sheet content (30.8%). The combination of TA9901 and VE promoted transition of β-turn (16.7%) toward α-helix and random coil. CONCLUSIONS: Both of TA9901 and VE can effectively diminish the β-structural content. TA9901 showed more intensitive inhibition than VE. The effect of TA9901 on the secondary structure of aged Aβ was associated with the mechanism that TA9901 inhibited Aβ aggregation and fibril formation.     

Effect of autophagic inhibitor 3-methyladenine on apoptosis of hippo-campal neurons induced by Aβ(25-35)

AIM: To investigate the influence of autophagy on the apoptosis of hippocampal neurons in the rat model of Alzheimer disease.METHODS: Sprague-Dawley rats were divided into model group, autophagic inhibitior 3-methyladenine (3-MA) pretreatment group and control group.In model group, the rats were anesthetized and placed in a stereotaxic apparatus.Hippocampus CA1 area microinjection was performed and Aβ(25-35) was applied to establish the model of AD.3-MA in 0.9% saline was administered by the same way prior to Aβ(25-35) infusion.The learning and memory ability of the rats was observed by Morris water maze.The ultrastructure of the hippocampal neurons, the formation of autophagic vesicles, beclin-1 expression and cell apoptosis were detected after behavioral experiment.RESULTS: Compared with model group, the learning and memory ability of the rats in 3-MA group significantly impaired (P<0.05) and the apoptotic rate of the hippocampal neurons significantly increased (P<0.05).Moreover, the expression of beclin-1 was declined.In model group, hippocampal neurons showed double membrane wrapped in the autophagic vacuoles, and the neuronal damages were significantly milder than that in 3-MA group.CONCLUSION: Decrease in the levels of neuronal autophagy increases the neuronal apoptosis, indicating that increasing neuronal autophagy may have therapeutic potential for AD.     

Effect of rhizoma acori graminei on the secondary structure of amyloid beta-protein 25-35

AIM: To study the effect and the mechanism of rhizoma acori graminei’s water extract and its naphtha on the secondary structure of amyloid beta-protein 25-35 (abeta 25-35). METHODS:Abeta 25-35 protein was cultured in PBS for 30 min, 24 h, 7 d in 37 ℃, then the change of secondary structure in abeta 25-35 was measured by circular dichroism (CD). Under the same condition, abeta 25-35 protein was cultured with different concentrations of RAG’s water extract and its naphtha instead of PBS, its secondary structure was also observed with CD. RESULTS:Obvious changes were observed between the CD graphs: when cultured in PBS for 30 min, the secondary structure of abeta 25-35 protein all were α-helix; but after 24 h, its secondary structure change, only 10.10% α-helix and 18.40% β-sheet was observed. When cultured for 7 days, its secondary structure mainly were β-sheet (27.00%) and random (56.40%). After cultured with RAG’s water extract and its naphtha, α-helix structure increased evidently, and the result showed that the best effect was when it is cultured with 100% RAG’s water extract and 100% naphtha for 7 days, its secondary structure all converted to α-helix. CONCLUSION: Definite concentration of RAG’s water extract and its naphtha effectively prevented abeta 25-35 protein’s secondary structure change from α-helix to β-sheet in definite times.