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Objective:To investigate the effects of naringenin on oxidative stress and Tau protein phosphorylation of adrenal pheochromocytoma（PC12） cells injured by β-amyloid（Aβ）25-35 and its relationship with estrogen receptor（ER） and phosphatidylinositol -3 kinase/protein kinase B（PI3K/Akt） signaling pathway. Method:The PC12 cells were intervened with Aβ25-35 to prepare the injury model. The experiment was divided into blank group， model group， naringenin（400，40，4，0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）group， positive drugs estradiol（E2）（1 nmol·L-1）+Aβ25-35 group， naringenin（0.4，0.04，4×10-3，4×10-4，4×10-5 μmol·L-1）+Aβ25-35 group， E2+Aβ25-35+ER antagonist（ICI182780）（1 μmol·L-1） group， naringenin+Aβ25-35+ICI182780 group， E2+Aβ25-35+PI3K blocker（LY294002）（50 μmol·L-1） group， naringenin+Aβ25-35+LY294002 group. Methye thiazolye telrazlium（MTT）method was used to detect the cell proliferation index， 2'，7'-Dichlorodi -hydrofluorescein diacetate（DCFH-DA） was used as a fluorescent probe to detect the content of reactive osygen species（ROS）， the content of malondialdehyde（MDA） and the activity of superoxide dismutase（SOD） were measured by thiobarbituric acid（TBA） and oxidase methods， Western blot was used to detect the expression of phosphorylated Tau protein/total Tau protein（p-Tau/t-Tau）. Result:According to the results of MTT experiment， 0.4 μmol·L-1 was selected as the best effective concentration of naringenin， compared with the blank group， the cell proliferation index of model group decreased significantly （P<0.01）， compared with model group， the cell proliferation index of naringenin+Aβ25-35 group increased significantly （P<0.01）. In addition， compared with blank group， the content of ROS， MDA and the expression of p-Tau/t-Tau in the model group increased significantly （P<0.01）， and the activity of SOD decreased significantly （P<0.01）， compared with model group， the content of ROS， MDA and the expression of p-Tau/t-Tau in naringenin+Aβ25-35 group decreased significantly （P<0.01）， and the activity of SOD increased significantly （P<0.01）， compared with naringenin+Aβ25-35 group， the addition of ICI182780 and LY294002 significantly reversed the role of naringenin in the above indicators （P<0.01）. The effect of naringenin was similar to that of E2. Conclusion:Naringenin can improve the cell proliferation index and protect PC12 cells from Aβ25-35 injury， which may be achieved by activating ER and PI3K/Akt signaling pathway to reduce ROS， MDA content， p-Tau/t-Tau expression and promote SOD activity.
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OBJECTIVE@#To investigate the effects of stachydrine (STA) on apoptosis of Aβ-induced PC12 cells mimicking Alzheimer's disease and explore the mechanisms.@*METHODS@#The differential genes of STA were analyzed based on GSE85871 data, and the target genes of STA were identified using STITCH database. PC12 cells were treated with Aβ to establish a cell model of Alzheimer's disease, and the changes in cell viability and cell cycle in response to STA treatment were assessed using MTT assay and flow cytometry, respectively. RT-PCR and Western blotting were used to detect the relevant gene or protein expressions in the treated cells.@*RESULTS@#GSE85871 data showed 37 up-regulated genes and 48 down-regulated genes in cells following treatment with STA. Analysis of the data from the STITCH database indicated that RPS8 and EED were the target genes of STA. Treatment of PC12 cells with Aβ significantly lowered the cell viability ( < 0.05) and the expressions of RPS8 and EED at both the mRNA and protein levels ( < 0.05), and obviously inhibited the expression of apoptosis-related proteins Bcl-2 and p53 ( < 0.05). STA treatment of the cells significantly reversed the effect of Aβ and induced cell cycle arrest in G2/M phase, causing also significantly increases in the expression levels of RPS8, EED, Bcl-2 and p53 ( < 0.05).@*CONCLUSIONS@#STA plays an important role in inhibiting the apoptosis of PC12 cells induced by Aβ possibly by regulating RPS8 and EED expression to promote the expressions of Bcl-2 and p53.
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Animals , Rats , Alzheimer Disease , Amyloid beta-Peptides , Apoptosis , Cell Survival , PC12 Cells , Peptide FragmentsABSTRACT
Objective: To investigate the neuroprotective effect and mechanism of tetrahydroxy stilbene glucoside (TSG) on β-amyloid protein 25-35 (Aβ25-35)-induced neuron synapses damage. Method: Primary neurons were isolated and purified from cerebral cortex of suckling mouse. Then neurons were divided into control group, model group (incubation with Aβ25-35) and TSG groups (after incubation with Aβ25-35, add 6.25, 12.5, 25, 50, 100 μmol·L-1 TSG). Cell counting kit-8 (CCK-8) and Lactate dehydrogenase (LDH) methods were used to observe the viability of neuron, immunocytochemical staining was performed to determine the expressions of synapsin-1 (SYN-1), and the concentration of postsynaptic density-95 (PSD-95) and synaptophysin (SYP) were detected by enzyme-linked immunosorbent assay (ELISA) method. The level of cyclic adenosine monophosphate response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of CREB, Phosphorylated CREB (p-CREB) and BDNF proteins were determined by immunocytochemical staining or Western blot (WB). Result: Compared with normal group, the cell survival rate of model group was significantly reduced, LDH release was significantly increased (PPPPPPP-1,25 μmol·L-1 TSG can significantly enhance the expression of SYN-1(PPPPConclusion: TSG possesses the neuroprotective effect on Aβ25-35-induced neuron synapses, the mechanism may be associated with the activation of CREB/BDNF signaling pathway.
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Aim To investigate the protective effect of epigallocatechin(EGC) on SH-SY5Y cells induced by Aβ25-35 injury. Methods MTT and LDH assay were employed to investigate the effects of EGC on viability of SH-SY5Y cells. DCFH-DA fluorescent probe was used to detect the intracellular ROS levels, and the activity of SOD, GSH-Px and MDA in cells was measured to investigate the level of oxidative stress in cells. Western blot analysis was used to determine Nrf2, NQ01, HO-1, Prdx6 and Trxl levels in cells. Results SH-SY5Y cells were injured by Aβ25-35 treatment, and the cell viability was significantly reduced. 5, 10 and 20 μmol . L 1 of EGC alleviated Aβ25-35 induced SH-SY5Y cell injury and reduced LDH release. In addition, EGC could reduce the intracellular ROS level induced by Aβ25-35 and inhibit oxidative stress. At the same time, EGC could significantly enhance the expression of Nrf2, NQ01, HO-1, Prdx6 and Trxl after Aβ25-35 injury. Conclusions EGC could inhibit Aβ25-35 -induced SH-SY5Y cell injury, increase the cells antioxidant levels and reduce the neurotoxicity of Aβ25-35 by promoting nuclear translocation of Nrf2.
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OBJECTIVE: To study the chemical constituents from the rhizome of Drynaria fortunei and the protective effects of them on PC12 cells induced by Aβ25-35. METHODS: The compounds were isolated and purified by silica gel, Sephadex LH-20, ODS column chromatography, and their structures were identified on basis of spectroscopic METHODS:, such as MS and NMR. PC12 cells were treated with Aβ25-35 to establish the Alzheimer' s disease models. The compounds of different concentrations were added into culture medium to detect the protection. MTT assay was used to detect cell vitality and to observe the protective effects of compounds on PC12 cells induced by Aβ25-35. RESULTS: Nine compounds were isolated and identified as naringin(1), neoeriocitrin(2), 5,7-dihydroxychromone-7-neohesperidoside(3), (E)-4-O-β-D-glucopyranosyl caffeic acid(4), kaempferol(5), luteolin(6), protocatechoic acid(7), psoralen(8), and β-sitosterol(9). The cell experiments were performed on the compounds 1-8 and the RESULTS: showed they can promote the proliferation of PC12 cells. The cell vitality increase with concentration rising, and the difference is statistically significant (P<0.05). CONCLUSION: Compounds 1-8 play an important role in protecting Aβ25-35-induced injury in PC12 cells and they are the main active components of Drynaria fortunei in the protection of central nervous function.
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Objective To observe the learning and memory ability of rats after injection of Aβ25-35 protein in different concentrations into the lateral ventricle assessed by Morris water maze test, and to explore the optimal concentration of Aβ25-35 in the preparation of AD model rats.Methods Male SD rats were randomly divided into sham operated group and model group.The rats of model group received Aβ25-35 injection in concentrations of 2 μg/μL, 4 μg/μL and 8 μg/μL, respectively.According to the Rat Brain Stereotaxic Atlas, 5 μL of aggregation of Aβ25-35 was injected into the right lateral ventricle to establish the AD rat model.7 days after successful modeling, Morris water maze was used to test thechanges of learning and memory ability of the rats.Results There was no significant difference in the average swimming speed between the two groups (P > 0.05).The escape latency time of rats in the model group was significantly increasedcompared with the sham group (P 0.05).The activity time and distance of target quadrant of the rats injected with different concentration of Aβ25-35in the model group were significantly reduced compared with the sham group (P 0.05).Compared with the sham-operated group, the number of platform-crossing of rats injected with different doses of Aβ25-35in the model group were significantly reduced (P 0.05).Conclusions The recommended dose and concentration of Aβ25-35 to be injected into the unilateral ventricle to establisha rat model of Alzheimer's disease is 4 μg/μL in a volume of 5 μL.
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Objective: To compare the neuroprotective effect of different elution fractions and compounds of Epimedium brevicormon on SH-SY5Y cells injury induced by Aβ25-35 so as to screen pharmacologically fraction and compounds. Methods: The isolation and purification of extract from E. brevicormon were performed on D101 macroporous resin column chromatography, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative liquid chromatography. Hoechst 33342/PI double staining method was used to screen neuroprotective fractions and components. Results: Compared to model group, all fractions and kaempferol-3-O-[α-L-rhamnopyranosyl (1→6)-β-D-galactoside]-7-O-α-L-rhamnopyranoside, icariin, epimedin A, and epimedin B from E. brevicormon could significantly increase the cells viability, except of 95% ethanol fraction. Conclusion: E. brevicormon show some protective effect against Aβ25-35 induced SH-SY5Y cells injury. The CH2CI2-MeOH (5:1) extract from E. brevicormon has the most pharmacologically activity, followed by 30% ethanol and 50% ethanol extracts from E. brevicormon.
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OBJECTIVE Lychee seed, a famous traditional Chinese medicine, recently were reported to improve the learning and memory abilities in mice. However, it is still unclear whether lychee seed saponins (LSS) can improve the cognitive function and associated mechanisms. METHODS In present studies, we established the Alzheimer disease (AD) model by injecting Aβ25-35 into the lateral ventricle of rats. Then the spatial learning and memory abilities of LSS- treated rats were evaluated with the Morris water maze, meanwhile the protein expressions of AKT, GSK3β and Tau in the hippo?campal neuron were analyzed by immunohistochemistry and Western blotting. RESULTS The results showed LSS can improve the cognitive functions of AD rats through shortening the escape latency, increasing the number across the platform, platform quadrant dwell time and the percentage of the total distance run platform quadrant. The protein expression of AKT was significantly up-regulated and that of GSK3β and Tau were decreased remarkably in the hippocampal CA1 area. CONCLUSION Our study is the first to show that LSS significantly improve the cognitive function and prevent hippocampal neuronal injury of the rats with AD by activation of the PI3K/AKT/GSK3β signaling pathway, suggesting LSS may be developed into the nutrient supplement for the treatment of AD.
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Objective To explore the protective effects of butylphthalide against Aβ25-35 induced dementia-like pathological rat model and reveal the mechanism.Methods Rats were divided into five groups:control group, model group and NBP groups (10,30,and 100 mg/kg).In model group and butylphthalide groups,Aβ25-35 was injected into the lateral ventricle,while the rats in intervention group were administered with butylphthalide (gastric infusion dose of 2.5 mL/kg).Learning and memory abilities of the rats were observed with water maze test. Mitochondrial function in brain tissue was observed by ATP assay,and the mitochondrial related enzyme activities were detected by the kit.Results Water maze test showed that learning and memory abilities of model group were poorer than those of control group.They were significantly improved in NBP 10 mg/kg group and 30 mg/kg group (P <0.05),but did not change significantly in 100 mg/kg group.Compared with control group,model group had significantly decreased ATP level (P < 0.05 ); cytochrome c oxidase, pyruvate dehydrogenase complex and ketoglutarate dehydrogenase activities were also significantly decreased (P < 0.05 ).Compared with model group, butylphthalide group had significantly improved activities of mitochondrial enzymes that improved mitochondrial function.Conclusion Butylphthalide can improve learning and memory abilities of rats with Aβ25-35 -induced dementia by improving mitochondrial function.
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Objective To explore the protective effects of butylphthalide against Aβ25-35 induced dementia-like pathological rat model and reveal the mechanism.Methods Rats were divided into five groups:control group, model group and NBP groups (10,30,and 100 mg/kg).In model group and butylphthalide groups,Aβ25-35 was injected into the lateral ventricle,while the rats in intervention group were administered with butylphthalide (gastric infusion dose of 2.5 mL/kg).Learning and memory abilities of the rats were observed with water maze test. Mitochondrial function in brain tissue was observed by ATP assay,and the mitochondrial related enzyme activities were detected by the kit.Results Water maze test showed that learning and memory abilities of model group were poorer than those of control group.They were significantly improved in NBP 10 mg/kg group and 30 mg/kg group (P <0.05),but did not change significantly in 100 mg/kg group.Compared with control group,model group had significantly decreased ATP level (P < 0.05 ); cytochrome c oxidase, pyruvate dehydrogenase complex and ketoglutarate dehydrogenase activities were also significantly decreased (P < 0.05 ).Compared with model group, butylphthalide group had significantly improved activities of mitochondrial enzymes that improved mitochondrial function.Conclusion Butylphthalide can improve learning and memory abilities of rats with Aβ25-35 -induced dementia by improving mitochondrial function.
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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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Alzheimer's disease (AD) is one of the major neurodegenerative disorders of the elderly, which is characterized by the accumulation and deposition of amyloid-beta (Aβ) peptide in human brains. Oxidative stress and neuroinflammation induced by Aβ in brain are increasingly considered to be responsible for the pathogenesis of AD. The present study aimed to determine the protective effects of walnut peptides against the neurotoxicity induced by Aβ25-35 in vivo. Briefly, the AD model was induced by injecting Aβ25-35 into bilateral hippocampi of mice. The animals were treated with distilled water or walnut peptides (200, 400 and 800 mg/kg, p.o.) for five consecutive weeks. Spatial learning and memory abilities of mice were investigated by Morris water maze test and step-down avoidance test. To further explore the underlying mechanisms of the neuroprotectivity of walnut peptides, the activities of superoxide dismutase (SOD), glutathione (GSH), acetylcholine esterase (AChE), and the content of malondialdehyde (MDA) as well as the level of nitric oxide (NO) in the hippocampus of mice were measured by spectrophotometric method. In addition, the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-6 in the samples were determined using ELISA. The hippocampal expressions of inducible nitric oxide synthase (iNOS) and nuclear factor κB (NF-κB) were evaluated by Western blot analysis. The results showed that walnut peptides supplementation effectively ameliorated the cognitive deficits and memory impairment of mice. Meanwhile, our study also revealed effective restoration of levels of antioxidant enzymes as well as inflammatory mediators with supplementation of walnut peptides (400 or 800 mg/kg). All the above findings suggested that walnut peptides may have a protective effect on AD by reducing inflammatory responses and modulating antioxidant system.
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Animals , Female , Male , Mice , Acetylcholinesterase , Metabolism , Alzheimer Disease , Drug Therapy , Amyloid beta-Peptides , Toxicity , Glutathione , Metabolism , Hippocampus , Metabolism , Interleukins , Metabolism , Juglans , Chemistry , Malondialdehyde , Metabolism , Maze Learning , Memory Disorders , Drug Therapy , NF-kappa B , Metabolism , Neuroprotective Agents , Pharmacology , Therapeutic Uses , Nitric Oxide , Metabolism , Peptide Fragments , Toxicity , Peptides , Pharmacology , Therapeutic Uses , Plant Extracts , Pharmacology , Therapeutic Uses , Superoxide Dismutase , Metabolism , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
Objective To observe the effects of moxibustion on learning and memory ability and nerve cell ultrastructure in CA1 region of hippocampus of rats with Alzheimer's disease (AD);To discuss its mechanism of action to AD.Methods A total of 40 male SD mice was randomly divided into normal group, model group, moxibustion group and sham-operation group. Stereotactic injection agglutinated Aβ25-35 into bilateral hippocampus of rats in model group and moxibustion group to prepare AD models. Rats in moxibustion group were treated by moxibustion at the points which were located above 2-3 cm away from Shenshu, Zusanli and Baihui. Rats in normal group received no treatment. The learning and memory ability was detected by Morris water maze test. Morphological changes of rat nerve cell ultra structure in hippocampal CA1 region were observed by transmission electron microscope.Results In model group, the average escape latency of five days and the last three days was significantly lengthened, and the times across the platform position significantly decreased. Transmission electron microscope indicated nerve cell structures were damaged and displayed unclearly;mitochondrion swelled, even mitochondrial crista was abnormal and deformed, endoplasmic reticulum of inter organelle expanded and the deposition of lipofuscin increased. After the treatment by moxibustion, the average escape latency of five days and the last three days was shortened obviously, and the times across the platform position significantly increased. In moxibustion group, the edema of nerve cells significantly decreased;dilation of endoplasmic reticulum and mitochondria swelling significantly improved;golgi bodies, mitochondria and ribosomes obviously increased in comparison with the model group. Conclusion Moxibustion can improve the learning and memory impairment caused by Aβ by promoting repairing of nerve cells in brain.
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Aim To explore the effect of N-acetyl-L-cysteine ( NAC ) on β amyloid peptide 25 - 35 ( Aβ25-35 )-induced the increase of calpain activity and its possible mechanism. Methods The activity of cal-pain was induced by 20μmol·L-1 Aβ 25-35 in primary cortical neuron. Neurons were incubated in the absent or present Aβ25-35 , or pre-incubated NAC ( 10 mmol ·L-1 ) , then co-incubated with Aβ25-35 . The meas-urement of calpain activity, H2 O2 level and mitochon-drial membrane potential was performed on a micro-plate fluorometer. The ATP level was detected using a luciferin/luciferase based ATP assay kit. Results In Aβ25-35 treated group, the activity of calpain and H2 O2 was obviously higher than that in control group. How-ever, in neurons pre-incubated in NAC and then co-in-cubated in Aβ25-35 , the calpain activity and H2 O2 level were significantly decreased compared with that in Aβ25-35 group. Upon Aβ25-35 exposure for 12 h, corti-cal neurons showed a significant decrease in mitochon-drial membrane potential and ATP level when com-pared to the control group. Pre-treatment with NAC showed an increase in mitochondrial membrane poten-tial and ATP level as compared to neurons treated with Aβ25-35 alone for 12h. Conclusion This result sug-gests that NAC can attenuate calpain activity induced by Aβ25-35 through protecting mitochondria.
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Aim To investigate the effect of genistein ( GEN) against Aβ( 25 -35 )-induced PC12 cells in regulation of Fas pathway through the activation of JNK. Methods Aβ( 25 -35 )-induced PC12 cells model was established. MTT and fluorescence activated cell sorting to analyze cell viability and apoptotic rate. Fluorescence quantitative PCR was used to detect Fas apoptotic pathways related gene Fas, FasL, caspase-3 and caspase-8 mRNA relative expression. Spectropho-tometry was used to detect caspase-3 and caspase-8 en-zyme activity. Western blot was adopted to detect JNK and p-JNK protein expression level changes. Results GEN attenuated Aβ( 25-35 )-induced upregulation of Fas and FasL, caspase-3 and caspase-8 mRNA lev-el, caspase-3 and caspase-8 enzyme activity, and sig-nificantly reduced Aβ(25-35) induced JNK phospho-rylation level. Conclusion GEN can protect PC12 cells from Aβ(25-35)-induced apoptosis via reducing Aβ( 25 -35 )-induced phosphorylation of JNK activa-tion, and then inhibit the JNK dependent Fas apoptotic pathway.
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Aim To determine TAT-Tcntf penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by β-amyloid peptide 25-35(Aβ_(25-35) ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by Aβ_(25-35).And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusion sTAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after Aβ25-35 exposure.
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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.