Objective To investigate the effect of astragalus injection on the activity of SOD and the content of MDA after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats. Methods The hippocampal neurons cultured for eight days were divided into normal control (normal), hypoxia/hypoglycemia and reoxygenation (model), astraglus injection solution (solution), astragalus injection (astragalus) groups. All groups were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 min except normal group. The activity of SOD and the MDA content of the rats hippocampal neurons were detected after hypoxia/hypoglycemia and reoxygenation 0, 0. 5, 2, 6, 24, 48, 72, 120 h. Western blotting was used to measure the expression of caspase-3 protein, in situ hybridization was used to measure the expression of caspase-3 mRNA after hypoxia/hypoglycemia and reoxygenation 0, 0. 5, 2, 6, 24, 48, 72, 120 h Results Compared with those of normal control group, the SOD activity was decreased obviously and the MDA content was increased obviously in hippocampal neurons of rats at every time points in hypoxia/hypoglycemia and reoxygenation group( P ＜0. 05 ); Compared with those of hypoxia/hypoglycemia and reoxygenation group, SOD activity and MDA content at every time points in original astragalus injection group had no obvious change( P ＞0. 05 ), however SOD activity was increased obviously and MDA content was decreased obviously in hippocampal neurons of rats at every time points in astragalus injection group (P ＜0. 05 ). In situ hybridization results showed that: compared with those of normal control group, the numbers, the percentage and the MOD of caspase-3 protein positive neuronal cells at every time points were increased obviously in hypoxia/hypoglycemia and reoxygenation group except 0 h and 0. 5 h( P ＜0. 05 ), and peak at 24 h. The change of neuronal configuration, the numbers, the percentage and the MOD of caspase-3 protein positive neuronal cells in astragalus injection solution group were accorded with hypoxia/hypoglycemia and reoxygenation group ( P ＞ 0. 05 ); Compared with those of hypoxia/hypoglycemia and reoxygenation group, the numbers, the percentage and the MOD of caspase-3 protein were less obviously in astragalus injection group except 0 h and 0. 5 h ( P ＜0. 05 ). Western blotting results showed that: compared with that of normal group, the expression of caspase-3 protein in hippocampal neurons of rats at every time points was increased obviously in hypoxia/hypoglycemia and reoxygcnation group except 0 h and 0. 5 h ( P ＜ 0. 05 ), and peaks at 24 h. Compared with hypoxia/hypoglycemia and reoxygenation group, the expression of caspase-3 protein at every time points in astragalus injection solution group had no obvious change ( P ＞ 0. 05 ), however the expression of caspase-3 protein in hippocampal neurons of rats at every time points was decreased obviously in astragalus injection group except 0 h and 0. 5 h ( P ＜ 0. 05 ).Conclusions Astragalus injection could inhibit the generation and release free radicals after hypoxia/hypoglycemia and reoxygenation, inhibit the expression of caspase-3, accordingly inhibited 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超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量的影响及其抑制神经元损伤的机制.方法 原代培养8 d的大鼠海马神经元随机分为正常对照组、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测定SOD活力、MDA含量,Western印迹检测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 各时间点缺氧缺糖/复氧复糖组海马神经元SOD活性均较正常对照组明显降低(P<0.05),MDA含量升高(P<0.05);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SOD的活性、MDA含量无明显变化(P>0.05),而黄芪注射液组在各个时间点SOD的活性明显升高(P<0.05),MDA含量降低(P<0.05).原位杂交结果显示: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及平均光密度值均明显增多(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/复氧复糖组显著减少(P<0.05).Western印迹检测结果显示: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明显变化(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).结论 黄芪注射液可通过减少自由基生成,抑制caspase-3的表达,进而抑制缺氧缺糖/复氧复糖大鼠海马神经元的凋亡.
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