Acetylpuerarin increases cell viability and reduces apoptosis in rat hippocampal neurons following oxygen-glucose deprivation/reperfusion

The effects of acetylpuerarin treatment following oxygen-glucose deprivation/reperfusion (OGD/R) were examined in rat hippocampal neurons in vitro and compared with the effects of acetylpuerarin in normoxic cells to confirm acetylpuerarin's potential neuroprotective effects, including apoptosis inhibition. Wistar rat embryo hippocampal cells (day 18, E18) cultured for 8 days were subjected to 3 h OGD treatment, followed by reperfusion for 12, 24 or 36 h. For each time interval, a group of cells was left untreated (OGD/R-only groups) and treated with 0.1, 0.4 and 1.6 mu M acetylpuerarin (OGD/R+acetylpuerarin). Neuron viability, apoptosis and caspase-8 and -3 activities were assessed by the reduction of 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), 4 ',6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) and spectrophotometric assays, respectively. Fas-ligand (Fas-L), Fas-associated death domain (FADD) and tumor necrosis factor-a (TNF-alpha) were determined by western blot analysis. Compared with control cells, OCD/R+ acetylpuerarin cells treated with 0.1, 0.4 and 1.6 mu M doses showed a concentration-dependent increase in hippocampal cell survival and viability by 69.93 +/- 2.28%, 81.49 +/- 2.13% and 85.28 +/- 2.38% at 12 h, 68.59 +/- 3.02%, 77.85 +/- 2.84% and 85.64 +/- 4.39% at 24 h and 69.70 +/- 1.70%, 77.21 +/- 3.21% and 83.90 +/- 2.12% at 36 h (P<0.05). Furthermore, OCD/R+ acetylpuerarin cells exhibited a dose-dependent decrease in caspase-8 and -3 activation, TUNEL and DAPI-positive neurons and Fas-L, FADD and TNF-alpha a expression. In conclusion, acetylpuerarin protects against OGD/R-induced neuronal apoptosis predominantly in the first 24 h following ischemia, which may be useful in mediating neuronal apoptosis in ischemic stroke patients.