黄芪注射液对缺氧缺糖/复氧复糖大鼠海马神经元钙调蛋白表达的影响

摘要

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钙调蛋白(CaM)表达的影响,探讨黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元凋亡的机制.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 h、0.5 h、2 h、6 h、24 h、48 h、72 h和120 h采用免疫组织化学染色法检测caspase-3的表达,采用Western blotting和RT-PCR方法检测CaM的表达.结果:与正常对照组相比,除0 h和0.5 h之外,缺氧缺糖/复氧复糖组各时点海马caspase-3蛋白阳性神经元数目、占神经元总数的百分率及平均吸光度值均明显增多(P<0.05),于24 h达到高峰.黄芪注射液组除0 h和0.5 h之外,各时点以上指标比缺氧缺糖/复氧复糖组显著减少(P<0.05).黄芪注射液溶剂对照组以上指标与缺氧缺糖/复氧复糖组无差异(P>0.05).各时点缺氧缺糖/复氧复糖组海马神经元CaM 蛋白表达均较正常对照组明显升高(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点CaM蛋白表达无明显变化(P>0.05),而黄芪注射液组在各个时点CaM蛋白表达明显降低(P<0.05).各时点缺氧缺糖/复氧复糖组海马神经元CaM mRNA表达均较正常对照组明显降低(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点CaM mRNA表达无明显变化(P>0.05),而黄芪注射液组在各个时点CaM mRNA表达明显升高(P<0.05).结论:黄芪注射液抑制CaM表达,可能是其减轻缺氧缺糖/复氧复糖大鼠海马神经元凋亡的机制之一.%AIM: To investigate the effect of Astragalus injection on the expression of calmodulin( CaM ) after hypoxia/ hypoglycemia and reoxygenation in rat hippocampal neurons.METHODS: The hippocampal neurons were cultured for 8 days and divided into 4 groups: normal control group ( normal control ), hypoxia/hypoglycemia and reoxygenation group ( model ), Astragalus injection solution group ( solution control ) and Astragalus injection group ( Astragalus ).The cells in all groups were treated with reoxygenation and normal medium after deprived of oxygen and glucose for 30 min except normal control group.The method of immunohistochemistry was used to measure the number of caspase - 3 positive neurons.The expression of CaM at mRNA and protein levels was measured at time points of 0 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h and 120 h after hypoxia/hypoglycemia and reoxygenation by RT - PCR and Western blotting, respectively.RESULTS: No difference of the parameters at all time points between model group and solution control group was found.Compared with normal control group, the numbers and the percentages of caspase -3 positive cells at all time points obviously increased in model group except at 0 h and 0.5 h ( P ＜0.05 ).Compared with model group, the numbers and the percentages of caspase - 3 positive cells were decreased in Astragalus injection group except at 0 h and 0.5 h ( P ＜ 0.05 ).Compared with normal control group, the protein expression of CaM in rat hippocampal neurons at all time points obviously increased in model group ( P ＜ 0.05 ).However, the protein expression of CaM in rat hippocampal neurons at all time points obviously decreased in Astragalus injection group as compared with model group ( P ＜0.05 ).Compared with normal control group, the mRNA expression of CaM in rat hippocampal neurons at all time points obviously decreased in model group ( P ＜0.05 ).The mRNA expression of CaM in rat hippocampal neurons at all time points obviously increased in Astragalus injection group as compared with model group ( P ＜0.05 ).CONCLUSION: Astragalus injection inhibits the protein expression of CaM, the calcium overload and the expression of caspase - 3 after hypoxia/hypoglycemia and reoxygenation, thus inhibiting hippocampal neuronal apoptosis.