Abstract
Oxidative glutamate toxicity is involved in diverse neurological disorders including epilepsy and ischemic stroke. Our present work aimed to assess protective effects of huperzine A (HupA) against oxidative glutamate toxicity in a mouse-derived hippocampal HT22 cells and explore its potential mechanisms. Cell survival and cell injury were analyzed by MTT method and LDH release assay, respectively. The production of ROS was measured by detection kits. Protein expressions of BDNF, phosphor-TrkB (p-TrkB), TrkB, phosphor-Akt (p-Akt), Akt, phosphor-mTOR (p-mTOR), mTOR, phosphor-p70s6 (p-p70s6) kinase, p70s6 kinase, Bcl-2, Bax, and β-actin were assayed via Western blot analysis. Enzyme-linked immunosorbent assay was employed to measure the contents of nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Our findings illustrated 10 μM HupA for 24 h significantly protected HT22 from cellular damage and suppressed the generation of ROS. Additionally, after treating with LY294002 or wortmannin [the selective inhibitors of phosphatidylinositol 3 kinase (PI3K)], HupA dramatically prevented the down-regulations of p-Akt, p-mTOR, and p-p70s6 kinase in HT22 cells under oxidative toxicity. Furthermore, it was observed that the protein levels of BDNF and p-TrkB were evidently enhanced after co-treatment with HupA and glutamate in HT22 cells. The elevations of p-Akt and p-mTOR were abrogated under toxic conditions after blockade of TrkB by TrkB IgG. Cellular apoptosis was significantly suppressed (decreased caspase-3 activity and enhanced Bcl-2 protein level) after HupA treatment. It was concluded that HupA attenuated oxidative glutamate toxicity in murine hippocampal HT22 cells via activating BDNF/TrkB-dependent PI3K/Akt/mTOR signaling pathway.
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This project was partly funded by the National Natural Science Foundation of China (No. 81302750).
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10571_2015_276_MOESM1_ESM.tiff
Supplementary material 1 S-Fig. 1 HupA attenuated neuronal impairment in human neuroblastoma SH-SY5Y cells under glutamate stimulation. SH-SY5Y cells was subjected to different concentrations of glutamate (2, 4, 6, 8 and 10 mM) for 24 h and cell viability was measured by MTT method (a). Nearly a 30% reduction of cell viability was found after glutamate exposure at the concentration of 10 mM for 24 h. HupA by the concentrations of 0-10 μM did not have any significant toxic effects on the cell viability of SH-SY5Y cells (b). It was also noted that 10 μM of HupA treatment for 24 h evidently promoted the recovery of SH-SY5Y cells after glutamate exposure (c). Results were expressed as mean ± standard deviation (SD) (n = 8). ** p<0.01 versus control group; ## p<0.01 versus glutamate-treated group. (TIFF 306 kb)
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Mao, XY., Zhou, HH., Li, X. et al. Huperzine A Alleviates Oxidative Glutamate Toxicity in Hippocampal HT22 Cells via Activating BDNF/TrkB-Dependent PI3K/Akt/mTOR Signaling Pathway. Cell Mol Neurobiol 36, 915–925 (2016). https://doi.org/10.1007/s10571-015-0276-5
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DOI: https://doi.org/10.1007/s10571-015-0276-5