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Objective To investigate the synergetic neuroprotection of glial cell line-derived neurotrophic factor (GDNF) and 17-Estradiol (17 β-E2) on dopaminergic (DA) neurons and the possible mechanism underlining it.Methods We selected the cultured brain slices of substantianigra from the male SD rats which were born 2 to 3 days to study.Being induced by addition of 200μ mol/ L 6-hydroxydopamine (6-OHDA) for 1h to the brain slices after cultured 7 days, the cultured brain slices— which were divided into seven groups: solvent control group, GDNF group, 17 β-E2 group, GDNF plus 17 β-E2 group, Wortmannin (selective inhibitor of PI3-K) plus GDNF group, Wortmannin plus 17β-E2 group, Wortmannin plus GDNF and 17 β-E2 group— and the normal control group were studied together.We tested the tyrosine hydroxylase (TH), Akt, ER and ER in DA neurons using Immunofluorescence method and determined TH, p-Akt, Bcl-2, Bad, Procaspase-3 using Western Blot assay.Results In our study, we found that all the DA neurons are TH positive (+), as well as Akt+, ER α+ and ER β+.The levels ofTH, Bcl-2, Procaspase-3 in GDNF plus 17 β-E2 group were higher than both in the 17 β-E2 and GDNF groups, whereas the expression of Bad was lower; the levels ofp-Ak t in GDNF, 17 β-E2, and GDNF plus 17 β-E2 groups were higher than the solvent control group; The levels of TH, Bcl-2, Procaspase-3 were decreased in the Wortmannin groups compared with groups without treating with Wortmannin, while the level of Bad was increased.Conclusion The results of our study showed that GDNF and 17 β-E2 had a synergetic neuroprotective effect on the DA neurons and PI3-K/Akt signal pathway might played a major role during this procedure.