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目的研究葛根素注射液对H_2O_2诱导海马神经元损伤的保护作用及其潜在机制。方法将细胞随机分成4组:空白组(细胞未处理组),阴性对照组(100 mg·L~(-1)葛根素),阳性对照组(40μmol·L~(-1)H_2O_2)和实验组(100mg·L~(-1)葛根素+40μmol·L~(-1)H_2O_2)。通过MTT法确立H_2O_2诱导海马神经元损伤模型的最佳条件。显微镜观察细胞形态变化,用MTT法测定神经元活性。以生化法测定超氧化物歧化酶(SOD)、过氧化氢酶(HP)、丙二醛(MDA)、谷胱甘肽(GSH)和活性氧(ROS)的水平,以实时荧光定量PCR检测凋亡相关基因Bcl-2和Bax的表达。结果阳性对照组的H_2O_2最佳处理条件为40μmol·L~(-1)处理1 h,细胞存活率为(69.67±4.93)%。实验组中葛根素最佳浓度为100mg·L~(-1)。与空白组(100%)相比,阳性对照组的HP、SOD和GSH分别为(56±4.58)%,(60.33±6.11)%,(51±7)%,均显著降低;而MDA、ROS分别为(152.33±6.03)%,(149.67±10.79)%,显著升高,差异均有统计学意义(均P<0.01)。与阳性对照组比较,实验组的HP、SOD和GSH分别为(88.67±4.73)%,(89.33±4.51)%,(90.33±3.21)%,显著升高;而MDA、ROS分别为(108.00±4.58)%,(111.00±8.32)%,显著降低,差异均有统计学意义(均P<0.01)。与空白组相比,阳性对照组的Bcl-2 mRNA表达为0.37±0.06,明显下降;而Bax mRNA表达为2.64±0.46,明显升高。与阳性对照组相比,实验组的Bcl-2mRNA、Bax mRNA的表达水平分别为0.75±0.08,1.44±0.08,差异均有统计学意义(均P<0.01)。结论葛根素可能通过调节Bax和Bcl-2基因的表达水平对H_2O_2所致海马神经元的损伤起到保护作用。
Objective To study the protective effect of puerarin on hippocampus neuron injury induced by H 2 O 2 and its potential mechanism. Methods The cells were randomly divided into 4 groups: blank group (untreated cells), negative control group (100 mg · L -1) puerarin and positive control group (40 μmol·L -1 H 2 O 2) Group (100 mg · L -1 Puerarin + 40 μmol·L -1 H 2 O 2). The optimal conditions of H 2 O 2 -induced neuronal damage model were established by MTT assay. The morphological changes of cells were observed with a microscope and the activity of neurons was measured by MTT method. The levels of superoxide dismutase (SOD), catalase (HP), malondialdehyde (MDA), glutathione (GSH) and reactive oxygen species (ROS) were measured by biochemical methods. Expression of apoptosis related genes Bcl-2 and Bax. Results The optimum conditions of H 2 O 2 in positive control group were 40 μmol·L -1 treatment for 1 h, the cell survival rate was (69.67 ± 4.93)%. The best concentration of puerarin in the experimental group was 100 mg · L -1. Compared with the blank group (100%), the HP, SOD and GSH of the positive control group were (56 ± 4.58)%, (60.33 ± 6.11)%, (51 ± 7)%, (152.33 ± 6.03)% and (149.67 ± 10.79)%, respectively, which were significantly increased (all P <0.01). Compared with the positive control group, the levels of HP, SOD and GSH in the experimental group were (88.67 ± 4.73)%, (89.33 ± 4.51)% and (90.33 ± 3.21)%, respectively, 4.58)%, (111.00 ± 8.32)%, respectively, which were significantly lower (all P <0.01). Compared with the blank group, the positive control group Bcl-2 mRNA expression was 0.37 ± 0.06, decreased significantly; and Bax mRNA expression was 2.64 ± 0.46, significantly higher. Compared with the positive control group, the expression levels of Bcl-2mRNA and Bax mRNA in the experimental group were 0.75 ± 0.08 and 1.44 ± 0.08, respectively (all P <0.01). Conclusion Puerarin may protect hippocampal neurons induced by H_2O_2 by regulating the expression of Bax and Bcl-2 genes.