文章摘要
李钰,丁文倩,李琳,等.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[J].浙江中医药大学学报,2022,(5):473-482.
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
DOI:10.16466/j.issn1005-5509.2022.05.001
中文关键词: 黄芪甲苷  miR-199a-5p  神经干细胞  氧糖剥夺/再灌注  存活  凋亡
英文关键词: Astragaloside Ⅳ  miR-199a-5p  neural stem cells  oxygen-glucose deprivation/reperfusion  survival  apoptosis
基金项目: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)
作者单位
李钰 浙江中医药大学基础医学院 杭州 310053 
丁文倩 浙江中医药大学基础医学院 杭州 310053 
李琳 浙江中医药大学基础医学院 杭州 310053 
许家栋 浙江中医药大学基础医学院 杭州 310053 
方燕 浙江中医药大学基础医学院 杭州 310053 
杨琰 浙江中医药大学基础医学院 杭州 310053 
胡小伟 浙江中医药大学基础医学院 杭州 310053 
储利胜 浙江中医药大学基础医学院 杭州 310053 
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中文摘要:
      [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50 μmol·L-1黄芪甲苷效果最佳(P<0.01);与模型对照组比较,50 μmol·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。
英文摘要:
      [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-day-old 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. MiR-199a-5p antagonist and antagonist control were transfected into neural stem cells by lipofectamine 2000. 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 miR-199a-5p expression and inhibition of cleaved caspase-3 protein expression.
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