目的: 研究黄芪甲苷(AS-Ⅳ)通过影响DNA甲基转移酶表达变化来上调血管内皮生长因子A(VEGFA)表达水平减轻急性低氧造成的脑损伤。方法: 以小鼠海马神经元细胞(HT22细胞)为研究对象,分为对照组(Control)、低氧组(Hypoxia)和黄芪甲苷处理组(H+AS-Ⅳ)。将HT22细胞低氧13 h复氧6 h构建低氧模型,H+AS-Ⅳ组于复氧的同时给与100 μmol/L的黄芪甲苷药物处理。荧光倒置显微镜观察细胞形态变化;采用Real-time PCR 检测DNA甲基化转移酶1(DNMT1)、DNA甲基化转移酶3A(DNMT3A)、DNA甲基化转移酶3B(DNMT3B)、VEGFA在mRNA水平上的表达变化;采用蛋白免疫印迹法(Western blot)检测DNMT1、DNMT3A、DNMT3B在蛋白水平上的表达变化。结果: 与对照组相比,低氧后,HT22细胞胞体变小,触角变短。DNMT1、DNMT3A的mRNA水平和蛋白水平均有所下降(P＜0.05),DNMT3B则表达上升(P＜0.05),VEGFA的mRNA水平下降(P＜0.05)。与低氧组相比,给予AS-Ⅳ治疗后细胞形态有所改善。DNMT1、DNMT3A的mRNA水平和蛋白水平均无变化,DNMT3B则有所降低(P＜0.05),VEGFA的mRNA水平上升(P＜0.05)。结论: 黄芪甲苷可以下调低氧后DNMT3B的表达水平,发挥神经保护作用,这一保护机制可能是通过上调VEGFA的表达来发挥的。
Objective: To investigate the effect of Astragaloside IV (AS-Ⅳ) on the expression of DNA methyltransferase to up-regulate the expression level of vascular endothelial growth factor A (VEGFA) to reduce brain injury caused by acute hypoxia. Methods: Mice hippocampal neurons (HT22 cells) were divided into Control group, Hypoxia group and Astragaloside IV group (H+AS-Ⅳ). HT22 cells were hypoxic for 13 h and reoxygenated for 6 h to establish a hypoxic model. Group H+AS-Ⅳ was treated with 100 μmol/L astragaloside IV at the same time of reoxygenation. The morphological changes of cells were observed by fluorescence inverted microscope. Real-time PCR was used to detect the expression changes of DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3A (DNMT3A), DNA methyltransferase 3B (DNMT3B) and VEGFA at mRNA level. The expression of DNMT1, DNMT3A and DNMT3B at the protein level was detected by Western blot. Results: Compared with the control group, the cell body of HT22 cells became smaller and the antennae became shorter after hypoxia. The mRNA and protein levels of DNMT1 and DNMT3A were decreased (P＜0.05), while the expression of DNMT3B was increased (P＜0.05), and the mRNA level of VEGFA was decreased (P＜0.05). Compared with the hypoxic group, the cell morphology was improved after AS-Ⅳ treatment. The mRNA level and protein level of DNMT1 and DNMT3A were unchanged, while the mRNA level of DNMT3B was decreased (P＜0.05), and the mrna level of VEGFA was increased (P＜0.05). Conclusion: Astragaloside Ⅳ can downregulate the expression of DNMT3B after hypoxia, exerting a neuroprotective effect, and this protective mechanism may be exerted by upregulating the expression of VEGFA.
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