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Objective: To investigate the protective effect of human lipoxin A4 (LXA4) on N2a cell damage induced by β-amyloid protein 25-35 (Aβ25-35) and the underlying mechanism. Methods: Aβ25-35 was used to treat N2a cells to establish Alzheimer's disease (AD) cell injury model. Meanwhile, LXA4 was added to the experimental group at different concentrations (50, 100 and 200 nmol·L-1 ). MTT assay was used to detect the activity of N2a cells. The apoptosis was detected by Hoechst 33258-PI staining, the expression of P62 and TRAF6 mRNA was detected by RT-PCR, and the expression of P62 and TRAF6 protein was detected by Western blot. Results: Compared with that of the model group, the cell survival rate of LXA4 protective group (50,100 and 200 nmol·L-1 ) increased (P <0. 01) and the apoptosis of N2a cells induced by Aβ25-35 was reduced by LXA4 (100 and 200 nmol·L-1 ) . Compared with that of the model group, the expression of P62-mRNA and protein-P62 of N2a cells treated with Aβ25-35 increased (P <0. 05 or P <0. 01) and the expression of TRAF6-mRNA and protein-TRAF6 of N2a cells treated with Aβ25-35 were reduced (P <0. 05 or P <0. 01). Conclusion: LXA4 has protective effect on N2a cell damage induced by Aβ25-35 , and its mechanism may be related to the up-regulation of P62 gene and down-regulation of TRAF6 gene.
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AIM:To explore the role of microRNA-181b (miR-181b) in ischemic injury and autophagy pro-tein 5 (Atg5) levels of mice .METHODS:Oxygen-glucose depletion (OGD) model in N2A cells to mimic ischemic in-jury in vitro was established .A middle cerebral artery occlusion ( MCAO) model to mimic ischemic injury in vivo was also induced in mice.The N2A cell apoptosis after OGD was assessed by in situ cell death detection kit.The Atg5 and caspase-9 expressions were determined by Western blotting .Luciferase reporter assay was performed to identify the direct binding of miR-181b with 3’-UTR of Atg5 mRNA.RESULTS:The alteration of miR-181b expression level by transfection with pre-miR-181b or anti-miR-181b significantly affected N2A cell apoptosis (P<0.05).Accordingly, the changes of miR-181b levels significantly altered the protein level of Atg 5 ( P<0.05 ) .Co-transfection of the luciferase reporters with pre-miR-181b or anti-miR-181b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3’-UTR of Atg5 mRNA (P<0.05).In addition, the miR-181b antagonist significantly reduced the cleaved caspase-9 levels in cerebral ischemic cortex of the mice after MCAO ( P<0.05 ) .CONCLUSION: Down-regulation of miR-181b plays an important role in ischemic injury of mice through regulating Atg 5 protein level.
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To study the prevention of dauricine (Dau) on bradykinin (BK) induced alteration of intracellular calcium homeostasis and tau phosphorylation, fluorescence spectrophotometer with dual excitation was utilized to measure the intracellular calcium concentration ([Ca2+]i), MTT to detect cell viability and immuncytochemistry to examine tau phosphorylation. The results showed (1) cells treated with BK 1 μmol/L induced a transit increase in [Ca2+]i in all the cell lines detected, among them, the sustained increase of [Ca2+]i level was only seen in PS1Δ9/APPswe cell at 2 h and 24 h after the treatment. Dau (3μmol/L or 6 μmol/L) prevented BK-induced transit and sustained elevation and fluctuation of [Ca2+]i;(2) BK treatment decreased the cell metabolism detected at 2 h in PS1Δ9/APPswe and Dau antagonized the effect; (3) BK induces Alzheimer-like tau hyperphosphorylation at tau-1 epitope and Dau partially antagonized this effect. In conclusion,Dau inhibits BK-induced disturbance in intracellular calcium homeostasis and tau hyperphosphorylation at tau-1 sites.