BACKGROUND:In studies concerning cell injury induced by cerebral ischemia-reperfusion,current experiments have primarily focused on altered protein levels.In addition,the apoptotic proteins Bax and Bcl-2 have been thoroughly studied with regard to initiating neuronal apoptosis. OBJECTIVE:To establish an in vitro model of oxygen-glucose deprivation and reintroduction in the rat hippocampus to simulate cerebral ischemia-reperfusion injury;to observe c-Jun N-terminal kinase 3(JNK3) mRNA expression in hippocampal neurons following Astragalus injection;and thus to determine changes in the signaling and downstream pathways of neuronal apoptosis at the cellular and molecular level. DESIGN,TIME AND SETTING:A randomized,controlled,cellular and molecular experiment was performed at the Department of Central Laboratory,Chengde Medical College from February to June 2008. MATERIALS:Astragalus injection,the main ingredient of astragaloside,was purchased from Chengdu Di’ao Jiuhong Pharmaceutical Manufactory,China.JNK3 mRNA probe and in situ hybridization kit were purchased from Tianjin Haoyang Biological Technology,China,and JNK3 RT-PCR primers were designed by Shanghai Bio-engineering,China. METHODS:Primary cultures of hippocampal neurons derived from Sprague Dawley rats,aged 1-2 days,were established.After 8 days,the hippocampal neurons were assigned to the following interventions:model group,Astragalus group,and vehicle control group,cells were subjected to oxygen-glucose reintroduction after oxygen-glucose deprivation for 30 minutes in sugar-free Earle’s solution and a hypoxia device,which contained high-purity nitrogen.The normal control group was subjected to primary culture techniques and was not treated using above-mentioned interventions. In addition,the Astragalus and vehicle control groups were treated with Astragalus injection(0.5 g/L raw drug) or sterile,deionized water at 2 hours prior to oxygen-glucose deprivation,respectively. MAIN OUTCOME MEASURES:JNK3 mRNA expression was measured by in situ hybridization and RT-PCR at 0,0.5,2,6,24,72,and 120 hours after oxygen-glucose reintroduction. RESULTS:Hippocampal neuronal morphology was normal in the normal control group. Hippocampal neurons exhibited apparent apoptosis-like pathological changes in the model,as well as the vehicle control,groups.The apoptosis-like pathological changes in the hippocampal neurons were less in the Astragalus group.Results from in situ hybridization and RT-PCR showed that JNK3 mRNA expression significantly increased in hippocampal neurons from model group,as well as the vehicle control group,compared with the normal control group(P<0.05).In addition,JNK3 mRNA expression significantly decreased in hippocampal neurons of the Astragalus group,compared with the model group and vehicle control group(P<0.05). CONCLUSION:Astragalus injection inhibited apoptosis-related JNK3 mRNA expression following oxygen-glucose deprivation and reintroduction,and accordingly played a role in inhibiting hippocampal neuronal apoptosis. BACKGROUND: In studies concerning cell injury induced by cerebral ischemia-reperfusion, current experiments have primarily focused on altered protein levels. In addition, the apoptotic proteins Bax and Bcl-2 have been thoroughly studied with regard to initiating neuronal apoptosis. OBJECTIVE: To establish an in vitro model of oxygen-glucose deprivation and reintroduction in the rat hippocampus to simulate cerebral ischemia-reperfusion injury; to observe c-Jun N-terminal kinase 3 (JNK3) mRNA expression in hippocampal neurons following Astragalus injection; and thus determine changes in the signaling and downstream pathways of neuronal apoptosis at the cellular and molecular level. DESIGN, TIME AND SETTING: A randomized, controlled, cellular and molecular experiment was performed at the Department of Central Laboratory, Chengde Medical College from February to June 2008. MATERIALS : Astragalus injection, the main ingredient of astragaloside, was purchased from Chengdu Di’ao Jiuhong Pharmaceutical Manu METHODS, Primary cultures of hippocampal neurons derived from Sprague Dawley rats, China, JNK3 mRNA probes and in situ hybridization kit were purchased from Tianjin Haoyang Biological Technology, China, and JNK3 RT-PCR primers were designed by Shanghai Bio-engineering, China. , aged 1-2 days, were established. After 8 days, the hippocampal neurons were assigned to the following interventions: model group, Astragalus group, and vehicle control group, cells were subjected to oxygen-glucose reintroduction after oxygen-glucose deprivation for 30 minutes in sugar-free Earle’s solution and a hypoxia device, which contained high-purity nitrogen. The normal control group was subjected to primary culture techniques and was not treated using above-mentioned interventions. In addition, the Astragalus and vehicle control groups were treated with Astragalus injection (0.5 g / L raw drug) or sterile, deionized water at 2 hours prior to oxygen-glucose deprivation, respectively. MAIN OUTCOME MEASURES: JNK3 mRNA expression was measured by in situ hybridization and RT-PCR at 0, 0.5, 2,6, 24,72, and 120 hours after oxygen-glucose reintroduction. RESULTS: Hippocampal neuronal morphology was normal in the normal control group. apoptosis-like pathological changes in the model, as well as the vehicle control, groups. apoptosis-like pathological changes in the hippocampal neurons were less in the Astragalus group. Results from in situ hybridization and RT-PCR showed that JNK3 mRNA expression increased in hippocampal neurons from model group, compared with the normal control group (P <0.05) .In addition, JNK3 mRNA expression was significantly decreased in hippocampal neurons of the Astragalus group, compared with the model group and vehicle control group (P <0.05). CONCLUSION: Astragalus injection regulated apoptosis-related JNK3 mRNA expression following oxygen-glucose deprivation and reintroduction, and advantageously played a role in inh ibiting hippocampal neuronal apoptosis.