Aim To investigate the effect of astragalus injection on the expression of caspase-3 interrelated by apoptosis after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats.Method The hippocampal neurons cultured for eight days were divided into four groups : normal control group( normal ) , hypoxia/hypoglycemia and reoxygenation group( model ).astragalus injection solution group( solution ), astragalus injection group( astragalus ).All groups except normal group were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 minutes.Methods of Immunohistochemistry and Western blot were used to measure the expression of caspase-3 protein.In situ hubridization was used to measure the expression of caspase-3 mRNA after hypoxia/hypoglycemia and reoxygenation 0 h,0.5 h.2 h,6 h,24 h,48 h,72 h,120 h.Results The results of immunohistochemistry were similar to those of in situ hybridization.Compared with normal group, the numbers, the percentage of caspase-3 protein positive neuronal cells at every time point increased obvious in the model group except 0 h and 0.5 h( P ＜0.05 ).and the peak arrived at 24 h; the change of neuronal configuration.the numbers.the percentage of caspase-3 protein positive neuronal cells in solution group accorded with those in model group( P＞ 0.05 ); compared with model group, the numbers, the percentage of caspase-3 protein were less obvious in the astragalus injection group except 0 h and 0.5 h( P＜ 0.05 ).Western blot results showed that:compared with normal group , the expression of caspase3 protein in hippocampal neurons of rats at every time point increased obviously in model group except Oh and 0.5 h( P ＜ 0.05 ), and peaked at 24 h; compared with model group, the expression of caspase-3 protein at every time point in astragalus injection solution group had no obvious change( P＞ 0.05 ), however that in hippocampal neurons of rats at every time point decreased obviously in the astragalus injection group except 0 h and 0.5 h( P＜0.05 ).Conclusion Astragalus injection can inhibit the expression of caspase-3 after hypoxia/hypoglycemia and reoxygenation , and accordingly inhibits hippocampal neuronal apoptosis.%目的 观察黄芪注射液对缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因caspase-3表达的影响.方法 取原代培养8 d的大鼠海马神经元,随机分为4组:正常对照组、缺氧缺糖/复氧复糖组、黄芪注射液溶剂对照组和黄芪注射液组.除正常对照组外均进行缺氧缺糖0.5 h,再复氧复糖.各组于复氧复糖后0、0.5、2、6、24、48、72 h和120 h采用免疫组织化学染色法和Western blot检测caspase-3蛋白的表达,采用原位杂交检测海马神经元caspase-3 mRNA的表达.结果 免疫组化结果显示:与正常对照组相比,除0 h和0.5 h之外,缺氧缺糖/复氧复糖组各时间点海马caspase-3阳性神经元数目占神经元总数的百分率均明显增多(P<0.05),于24 h达到高峰.黄芪注射液溶剂对照组以上指标的变化趋势与缺氧缺糖/复氧复糖组相一致.黄芪注射液组除0 h和0.5 h之外,各时间点以上指标均比缺氧缺糖/复氧复糖组减少(P<0.05).Western blot检测结果显示:除0 h和0.5 h外,各时间点缺氧缺糖/复氧复糖组海马神经元caspase-3 蛋白的平均灰度值均较正常对照组明显增加(P<0.05),24 h最高;与缺氧缺糖/复氧复糖组相比,黄芪注射液溶剂对照组各时间点caspase-3蛋白的平均灰度值无变化(P>0.05),而黄芪注射液组除0 h和0.5 h外,在各个时间点caspase-3蛋白的平均灰度值明显降低(P<0.05).原位杂交检测结果显示:caspase-3 mRNA阳性神经元形态、数目占神经元总数的百分率的变化趋势与caspase-3蛋白的变化趋势完全一致.结论 黄芪注射液可抑制缺氧缺糖/复氧复糖大鼠海马神经元凋亡相关基因caspase-3的表达,从而抑制缺氧缺糖/复氧复糖大鼠海马神经元的凋亡.
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