Astragalus injection inhibits c-Jun N terminal kinase mRNA expression following oxygen-glucose deprivation and reintroduction in rat hippocampal neurons

摘要

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.