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OBJECTIVE To study the neuroprotective effects of Shenzao jianna o oral liquid （SZJN）on Alzheimer ’s disease （AD）model mice and its mechanism. METHODS The mice were randomly divided into sham operation group ，model group ， Donepezil hydrochloride tablet group （0.65 mg/kg），SZJN low-dose ，medium-dose and high-dose groups （0.3，1.5 and 7.5 g/kg， calculated by crude drug quantity ），with 12 mice in each group ，half male and half female. Each group was given relevant medicine（intragastric administration of water at constant volume in sham operation group and model group ），twice a day ，for consecutive 28 d. On the 15th day of administration ，intracerebroventricular injection of β-amyloid 1-42（Aβ1-42）combined with intraperitoneal injection of scopolamine hydrobromide were used to induce AD model. Morris water maze was used to detect the learning and memory ability of mice. HE staining and Nissl staining were used to evaluate the pathological changes of brain tissue in mice. The levels of MDA and SOD in brain tissue of mice were detected. The phosphorylation level of cyclic adenosine monophosphate response element binding protein （CREB） and expression of brain-derived neurotrophic factor （BDNF） in hippocampal tissues were detected by Western blot. RESULTS Compared with sham operation group ，the escape latency of the model group was significantly prolonged ，and the number of crossing the platform and the percentage of residence time in the target quadrant were significantly reduced （P＜0.01）. The level of SOD in brain tissue ，the phosphorylation level of CREB and the expression level of BDNF in hippocampus decreased significantly （P＜0.01），while the level of MDA increased significantly （P＜ 0.01）. In hippocampal CA 1 area and cortical tissue ，nerve cells showed significantly decreased number ，the disordered arrangement and large gap ；the shape of nucleus was irregular and deeply stained ，and Nissl body was blurred ，loosely arranged and the number decreased. Compared with model group ，the escape latency of mice in each dose group of SZJN was significantly shortened ，and the times of crossing the platform and the percentage of residence time in the target quadrant were significantly jing- increased（P＜0.01）. Above indexes of brain tissue in mice were reversed sig nificantly in SZJN high-dose group （P＜0.01），and pathological damage of brain tiss ue was improved. CONCLUSIONS SZJN can significantly improve the learning and memory ability of AD model mice ，and alleviate the pathological injury and oxidative stress of brain tissue ，which may be related to the activation of CREB/BDNF signaling pathway.
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OBJECTIVE：To study the effects and mechanism of panaxadiol （PD） on Tau protein phosphorylation in the SH-SY5Y cells transfected with APP gene（APP-SH-SY5Y）. METHODS ：The target of PD and non-receptor tyrosine kinases Fyn was verified by molecular docking. SH-SY 5Y cells were cultured in vitro ，and the APP-SH-SY 5Y cell models and green fluorescent （GFP）-SH-SY5Y cell model （control cell ）was constructed. The expression of Aβ1-42 was detected so as to verify the success of APP-SH-SY5Y cell model. Taking GFP-SH-SY 5Y cells as control ，the effects of 5，10，20，30，40 μmol/L PD and 125，250， 500，1 000，2 000 nmol/L PP 2（Fyn inhibitor ，positive control ）on the survival rate of APP-SH-SY 5Y cells were detected by CCK-8 assay after treated for 24 h，so as to confirm the optimal concentration. The concentration of Ca 2 + ，the ratio fophosphorylated Tau protein （p-Tau）/Tau，phosphorylatedn Src（p-Src）/Fyn and phosphorylated glutamate receptor 2B（p-GluN2B）/ GluN2B were detected in APP-SH-SY 5Y cells after trated with the optimal concentration of PD and PP 2 for 24 h. RESULTS ：The results of molecular simulation docking showed that PD could target Fyn protein. Compared with GFP-SH-SY 5Y cells ，the protein expression of Aβ1-42 in APP-SH-SY 5Y cell were increased significantly （P＜0.01）. The optimal concentration of PD and PP 2 were 20 μmol/L and 500 nmol/L. The 20 μmol/L PD and 500 nmol/L PP 2 could increase the survival rate of the cells and reduced the concentration of Ca 2+，the ratio of p-Tau/Tau ，p-Src/Fyn，and p-GluN 2B/GluN2B. CONCLUSIONS：PD can reduce the the phosphorylation of Tau protein through inhibiting Fyn/GluN 2B signaling pathway.
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OBJECTIVE@#To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism.@*METHODS@#The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR.@*RESULTS@#The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all <0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all <0.01).@*CONCLUSIONS@#Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.
Subject(s)
Humans , Alzheimer Disease , Amyloid beta-Peptides , Amyloid beta-Protein Precursor , Genetics , Cell Line , Coumarins , Pharmacology , Gene Expression Regulation , Genetics , MicroRNAs , Genetics , MetabolismABSTRACT
Aim To investigate the neuroprotective effects of osthole(Ost)on the primary cultured cortical neurons transfected with APP595/596 gene and its underlying mechanism.Methods Neonatal mouse cortical neurons were transfected with APP595/596 gene to establish AD cell models for the further study.Then,the cell viability was detected by CCK-8 assay,and the leakage of lactate dehydrogenase(LDH)was assayed by LDH kit to evaluate the injury degree.Transferase-mediated nick end labeling(TUNEL)was used to evaluate the cell apoptosis.The expression of β-amyloid peptide(Aβ)and β-site APP cleaving enzyme 1(BACE1)was measured by immunofluorescence,while the miRNA-107 was measured by RT-PCR.Results Compared to model group,Ost could significantly improve the neurons viability,decrease the LDH release and prevent the apoptosis.Ost also inhibited the expression of Aβ and BACE1 at protein level,while enhanced the expression of miRNA-107 at gene level.Conclusion Ost plays a neuroprotective role in neurons transfected with APP595/596 gene in part through up-regulating miRNA-107.