黄芪甲苷上调miR-199a-5p表达抑制氧糖剥夺/再灌注诱导的神经干细胞凋亡  

Astragaloside Ⅳ Inhibits the Apoptosis of Neural Stem Cells Induced by Oxygen-glucose Deprivation/Reperfusion through Upregulating miR-199a-5p

作　　者：李钰 丁文倩 李琳[1] 许家栋[1] 方燕[1] 杨琰[1] 胡小伟 储利胜[1] LI Yu;DING Wenqian;LI Lin(College of Basic Medical Sciences,Zhejiang Chinese Medical University,Hangzhou(310053),China)

机构地区：[1]浙江中医药大学基础医学院,杭州310053

出　　处：《浙江中医药大学学报》2022年第5期473-482,共10页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金项目(81073075、82104426);浙江省自然科学基金项目(LY22H280009);浙江中医药大学科研项目(2021JKZDZC01)。

摘　　要：[目的]探讨黄芪甲苷是否通过上调miR-199a-5p抑制氧糖剥夺/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)诱导的神经干细胞凋亡。[方法]分离14 d胎龄SD大鼠的大脑皮层神经干细胞并鉴定,随机分为正常对照组、模型对照组、黄芪甲苷组、黄芪甲苷+miR-199a-5p拮抗剂组(拮抗剂组)和黄芪甲苷+miR-199a-5p拮抗剂对照组(拮抗剂对照组)。除正常对照组外,其余组神经干细胞先行氧糖剥夺8 h,再灌注72 h,拮抗剂组和拮抗剂对照组分别采用脂质体2000转染miR-199a-5p拮抗剂和拮抗剂对照。采用2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt,CCK-8]法检测神经干细胞活性,异硫氰酸荧光素标记磷脂结合蛋白/碘化丙啶(Annexin-fluorescein isothiocyanate isomer/propidium iodide,AnnexinⅤ-FITC/PI)双染法检测神经干细胞凋亡,实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测miR-199a-5p表达,免疫印迹法检测切割型半胱氨酸蛋白酶-3(cleaved caspase-3)和半胱氨酸蛋白酶-3(caspase-3)蛋白表达。[结果]分离培养的神经干细胞纯度为98.41%,黄芪甲苷能够提高OGD/R损伤的神经干细胞存活率,其中50μmol·L^(-1)黄芪甲苷效果最佳(P<0.01);与模型对照组比较,50μmol·L^(-1)黄芪甲苷能抑制OGD/R损伤的神经干细胞凋亡(P<0.01),上调miR-199a-5p表达(P<0.01),下调cleaved caspase-3蛋白表达(P<0.01),而miR-199a-5p拮抗剂能显著逆转上述效应(P<0.01)。[结论]黄芪甲苷能够促进OGD/R损伤的神经干细胞存活,抑制其凋亡,作用机制可能与其上调miR-199a-5p表达,抑制cleaved caspase-3蛋白表达有关。[Objective] To investigate whether Astragaloside Ⅳ inhibits the apoptosis of neural stem cell injury induced by oxygen-glucose deprivation/reperfusion(OGD/R) through upregulation of miR-199a-5p. [Methods] The neural stem cells from the brain cortex of 14-dayold fetal rats were isolated and identified. Neural stem cells were randomly divided into normal control group, model control group,Astragaloside Ⅳ group, Astragaloside Ⅳ+miR-199a-5p antagonist group(antagonist group) and Astragaloside Ⅳ+miR-199a-5p antagonist control group(antagonist control group). Neural stem cells were deprived of oxygen and glucose for 8 h and reperfusion for 72 h in all groups except for normal control group. Mi R-199a-5p antagonist and antagonist control were transfected into neural stem cells by lipofectamine2000. The viability of neural stem cells was detected by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt(CCK-8) assay. The apoptosis of neural stem cells was determined by Annexin-fluorescein isothiocyanate isomer/propidium iodide(Annexin Ⅴ-FITC/PI) double staining. The expression of miR-199a-5p was examined by Real-time quantitative polymerase chain reaction(Real-time qPCR). The protein expression levels of caspase-3 and cleaved caspase-3 were detected by Western blot. [Results] The purity of the cultured neural stem cells was 98.41%. Astragaloside Ⅳ improved the survival rate of OGD/R injured neural stem cells, and 50 μmol·L^(-1)Astragaloside Ⅳ had the best effect(P<0.01). Compared with model control group, 50 μmol·L^(-1)Astragaloside Ⅳ inhibited apoptosis of neural stem cells injured by OGD/R(P<0.01), up-regulated miR-199a-5p expression(P<0.01), down-regulated cleaved caspase-3 protein expression(P<0.01). However, miR-199a-5p antagonist could significantly reverse the above effects(P<0.01). [Conclusion] Astragaloside Ⅳ can promote the survival and inhibit the apoptosis of neural stem cells induced by OGD/R, which may be related to up-regulation of m

关 键 词：黄芪甲苷 miR-199a-5p 神经干细胞 氧糖剥夺/再灌注 存活 凋亡 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]