18α-甘草次酸通过抗氧化作用促进成年小鼠室管膜下区神经干细胞增殖

18α-glycyrrhetinic acid promoting neural stem cells proliferation through antioxidant in the subventricular zone of adult mice

目的探讨18α-甘草次酸(18α-GA)对成年小鼠室管膜下区(SVZ)神经干细胞(NSCs)增殖的影响及其机制。方法100只6月龄BALB/c小鼠随机平均分为18α-GA组(腹腔注射18α-GA 40 mg/kg两个月,以DMSO为溶解介质)和DMSO对照组(腹腔注射含等体积DMSO溶解介质的PBS溶液),每组50只,其中15只用于在体实验,其余35只小鼠断头取脑,分离培养SVZ区NSCs。采用Ki-67染色、成球实验、BrdU掺入实验及CCK-8实验检测SVZ区NSCs增殖活力,二氢乙啶(DHE)染色等方法检测活性氧簇(ROS)及超氧化物歧化酶1(SOD1)水平,Real-time PCR及Western blotting检测SVZ区NSCs的核因子E2相关因子2(Nrf2)表达。结果在体实验结果显示,腹腔注射18α-GA后,小鼠SVZ区Ki-67阳性细胞数较DMSO对照组明显增多,ROS水平较DMSO对照组明显下降,而SOD1在mRNA和蛋白水平均明显升高,分别是对照组的(3.17±0.073)倍和(2.12±0.02)倍(P<0.05和P<0.001)。细胞实验结果表明,18α-GA组小鼠SVZ区的NSCs成球速度快,数量多,其中直径在40~60μm和≥60μm的神经球数量分别是DMSO对照组的2.1倍和4倍(P<0.001);18α-GA组小鼠SVZ区的NSCs活力明显提高(P<0.001),BrdU阳性率是DMSO对照组的(1.75±0.17)倍(P<0.01);18α-GA组小鼠SVZ区NSCs的ROS水平较DMSO对照组下降18.91%±4.33%(P<0.05),而SOD1在mRNA和蛋白水平均明显升高(P<0.05和P<0.01)。此外,腹腔注射18α-GA后小鼠SVZ区及其NSCs中Nrf2的mRNA和蛋白水平均高于对照组(P<0.01和P<0.05)。结论在体给予18α-GA可提高成年NSCs的SOD1活性,减少细胞内ROS堆积,维持和改善成年小鼠SVZ区NSCs的增殖潜能。

Objective To explore the effect of 18α-glycyrrhetinic acid(18α-GA)on the proliferation of adult mice neural stem cells(NSCs)and its underlying mechanism.Methods One hundred 6-month BALB/c mice were randomly divided into DMSO control group and 18α-GA group(mice were intraperitoneally injected with 40 mg/kg 18α-GA every day for 2 months).The proliferation capability,oxidative status and nuclear factor E2-related factor 2(Nrf2)level of NSCs in the adult mice subventricular zone(SVZ)were measured through both in vivo and in vitro experiments,including Ki-67 staining,neurosphere formation assay,BrdU incorporation,CCK-8 assay,reactive oxygen species(ROS)detection,superoxide dismutase 1(SOD1)determination,Real-time PCR and Western blotting.Results Elevated Ki-67 positive cells were observed in SVZ of mice with 18α-GA application.Meanwhile,ROS level attenuated but SOD1 mRNA and protein level increased significantly in the SVZ of 18α-GA group mice,the latter of which were(3.17±0.073)and(2.12±0.02)times respectively than that of the control group(P<0.05 and P<0.001).Likewise,the similar changes were exhibited in vitro data.NSCs of 18α-GA group mice displayed higher proliferation potency confirmed by accelerated neurosphere formation and increased neurosphere number(P<0.001),as well as higher BrdU positive ratio(P<0.01)and NSCs vitality(P<0.001).NSCs of mice with 18α-GA injection exhibited decreased ROS level by 18.91%±4.33%(P<0.05)and enhanced SOD1 level,compared with those in NSCs of DMSO group mice.Furtherly,the Nrf2 expression in SVZ and NSCs of 18α-GA group mice was higher than that of the control group.Conclution 18α-GA administration plays a vital role in the maintainence and amelioration of adult mice NSCs proliferation through activating SOD1 and diminishing ROS aggregations.