Objective    To investigate the effects of deproteinized brain tissue extracts from hypoxia-preconditioned mice (DHP) on the tolerance of differentiated PC12 cells (DPC12) to hypoxia and its relationship with adenosine (ADO).     Methods    The mouse model of acute repetitive hypoxia was reproduced and deproteinized brain tissue extracts w

ere prepared by means of perchloric acid. High performance liquid chromatogram (HPLC) was used to determine ADO contents in the extracts. DHP was added into the culture of 4-day DPC12 cells

and deproteinized brain tissue extracts of normal mice (DN) were used as control. Besides

ADO at final concentrations of 1.0

10.0

100.0  mol/L was used as positive controls. Then DPC12 cells were cultured in hypoxia (2%O

2

). After 24-

48-

72-hour hypoxia exposure

colorimetric method of tetrazolium salt MTT assays (A

570

) and lactate dehydrogenase (LDH) release assays were conducted. Furthermore

apoptotic percentages at late stage (72-hour hypoxia) were detected with Hoechst 33258 stained fluorescence microscopy; Then

adenosine A

1

A

2A

receptor antagonists

DPCPX and SCH 58261

were respectively added into DHP groups and 10.0  μmol/L adenosine positive control groups

with DN group as control; MTT

LDH assays were performed after 24-

48-

72-hour hypoxia.     Results    ADO contents in DHP significantly increased; MTT value of DHP group was significantly higher than that of DN group

and LDH release percentage was significantly lower than that of DN group after 24-hour hypoxia. As the period of hypoxia prolonged

DHP gradually lost its protective effect. After 72-hour hypoxia

MTT value

LDH release percentage and apoptotic percentage at late stage of DHP group showed no significant differences from those of DN group. SCH 58261 but not DPCPX blocked the protective effect of DHP.     Conclusion    The protective effects of DHP on DPC12 cells at early stage were related to ADO increase in DHP and the effects might be mediated via A

2A

receptor.