18α-GA激活Nrf2对成体神经干细胞的影响  

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells

作　　者：杨娜[1] 赵云鹤[1] 黄菲菲 刘琦玮 陆利[1] 

机构地区：[1]山西医科大学人体解剖学教研室,山西太原030001 [2]山西医科大学生物制药系,山西太原030001

出　　处：《中国病理生理杂志》2017年第10期1738-1745,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81571381);山西省自然科学基金资助项目(No.2015011132);山西医科大学校级博士启动基金(No.03201604)

摘　　要：目的:明确18α-甘草次酸(18α-GA)对核因子E2相关因子2(Nrf2)的激活作用,观察上调Nrf2对成体神经干细胞(a NSCs)增殖和分化能力的影响,探讨维持a NSCs活力的有效途径。方法:体外培养新生、成年和老年小鼠的侧脑室室膜下区神经干细胞(NSCs),观察随年龄增长NSCs中Nrf2的表达变化。以18α-GA作用a NSCs,用real-time PCR和Western blot实验比较18α-GA组与DMSO对照组a NSCs的Nrf2表达变化。构建携带绿色荧光蛋白(GFP)基因的Nrf2-shRNA慢病毒载体(LV)感染a NSCs,用real-time PCR和Western blot实验检测shRNA对Nrf2的沉默效率。将a NSCs按照干预条件不同分为DMSO组、18α-GA组、LV-GFP组和LV-Nrf2-shRNA组,运用Brd U掺入实验、Tuj1免疫荧光染色、CCK-8实验、Hoechst 33342/PI双染色和活性氧簇(ROS)水平测定等方法评价18α-GA是否通过激活Nrf2对a NSCs的增殖、分化、细胞活力、细胞凋亡以及氧化应激水平产生影响。结果:随年龄增长,成年和老年小鼠a NSCs的Nrf2 mRNA表达水平较新生小鼠NSCs显著降低(P<0.01),但a NSCs的ROS水平显著高于新生小鼠NSCs(P<0.05)。应用18α-GA后,a NSCs的Nrf2 mRNA与蛋白表达水平较DMSO组明显升高(P<0.01),并且a NSCs的Brd U阳性率也显著增加(P<0.01)。2 mg/L 18α-GA作用后,a NSCs的Tuj1阳性率和细胞活力较DMSO组显著升高(P<0.05),而凋亡率和ROS水平显著下降(P<0.05和P<0.01)。但是,敲减a NSCs的Nrf2并应用18α-GA干预后,LV-Nrf2-shRNA组Brd U阳性率、Tuj1阳性率以及细胞活力均较LV-GFP组显著下降(P<0.05),ROS水平却显著上升(P<0.05)。结论:18α-GA可能通过激活Nrf2提高a NSCs的抗氧化能力,促进a NSCs增殖和分化,维持a NSCs潜能。AIM：To investigate the effect of nuclear factor E2-related factor 2 （Nrf2） activation by 18α-gly-cyrrhetinic acid （18α-GA） on the proliferation and self-renewal of adult neural stem cells （aNSCs）, and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS：NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector （LV） car-rying green fluorescent protein （GFP） gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species （ ROS） were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS：The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs （P〈0.01）, while the ROS level of aNSCs was significantly higher （P〈0.05）.After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group （ P〈0.01）.Increased number of BrdU ＋and Tuj1 ＋cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability （P〈0.05）.Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group （P〈0.05）.After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU ＋and Tuj1 ＋cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group （P〈0.05）.Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group （P�

关 键 词：18α-甘草次酸 成体神经干细胞 核因子E2相关因子2 细胞增殖 细胞分化 小鼠 

分 类 号：R741[医药卫生—神经病学与精神病学] R363[医药卫生—临床医学]