sirolimus and 3-n-butylphthalide

To explore the effects of modulating autophagy on neuroamyloidogenesis in an ischemic stroke model of cultured neuroblastoma 2a (N2a)/Amyloid precursor protein (APP)695 cells.. The ischemic stroke model of N2a/APP695 cells was made by 6h oxygen-glucose deprivation/12h reperfusion (OGDR). Drug administration of 3-methyladenine (3-MA), rapamycin or dl-3-n-butylphthalide (NBP) was started at the beginning of the OGDR and lasted until the end of reperfusion, in order to explore their effects on N2a/APP695 cells under OGDR conditions. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 12h. Cell viability and injury were investigated. The key proteins of nuclear factor kappa B (NF-κB) pathway and a key component of autophagy Beclin 1 were detected by Western blotting; immunofluorescence double-staining of amyloid-β (Aβ)1-42 with Beclin 1 was performed to investigate their cellular co-localization relationship; β-secretase and γ-secretase activity assay and Aβ1-42 enzyme-linked immunosorbent assay were performed to investigate the amyloidogenesis.. The results showed that, OGDR enhanced cell injury, autophagy activity, neuroinflammation and Aβ generation in N2a/APP695 cells; down-regulating autophagy by 3-MA and NBP increased cell viability, decreased lactate dehydrogenase (LDH) production, inhibited the activation of NF-κB pathway, suppressed β- and γ-secretase activities and Aβ generation; while up-regulating autophagy by rapamycin got the opposite results; immunofluorescence double-staining results showed elevated Aβ1-42(+) signal was co-localized with Beclin 1(+) signal.. Our data suggested that down-regulating autophagy may inhibit ischemia-induced neuroamyloidogenesis via suppressing the activation of NF-κB pathway. This study might help us to find a new therapeutic strategy to prevent brain ischemic damage and depress the risk of post-stroke dementia.

Topics: Adenine; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Autophagy; Benzofurans; Brain Ischemia; Cell Survival; Mice; Neuroblastoma; Sirolimus; Stroke; Tumor Cells, Cultured