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补阳还五汤通过调节自噬促进OGD/R损伤大鼠NSCs增殖、分化能力
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作者单位:

广州中医药大学 基础医学院,广州 510006

作者简介:

秦彬喻,硕士,从事中医药防治心脑血管疾病的研究,E-mail:20181104430@stu.gzucm.edu.cn

通讯作者:

关莉,博士,教授,硕士生导师,从事中医药防治心脑血管疾病研究,E-mail:guanli@gzucm.edu.cn

中图分类号:

R2-0;R22;R285.5;R289;R33

基金项目:

国家自然科学基金项目(8167140990)


Buyang Huanwutang Promoted Proliferation and Differentiation of Neural Stem Cells via Regulating Autophagy Following Oxygen-glucose Deprivation/Reoxygenation Injury
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College of Basic Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,China

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    摘要:

    目的 探讨补阳还五汤(BDT)对神经干细胞(NSCs)糖氧剥夺/复氧(OGD/R)损伤后增殖、分化的影响。方法 从SD大鼠海马区域分离培养NSCs,将细胞随机分为5组,分别为常氧组,模型组,BDT组,雷帕霉素(Rapa)组和自噬抑制剂3-甲基腺嘌呤(3-MA)联合BDT组,常氧组和模型组使用20%空白血清,BDT组使用20%含药血清,Rapa剂量1 μmol·L-1,3-MA剂量5 mmol·L-1。除常氧组外,其余各组进行糖氧剥夺/复氧。光镜观察细胞形态的变化;免疫荧光检测巢蛋白(nestin)表达,鉴定NSCs;细胞增殖与活性检测法(CCK-8)检测NSCs的存活率;5-乙炔基-2-脱氧尿嘧啶苷(EdU)标记法检测NSCs增殖;Ad-mCherry-GFP-LC3B检测自噬活性;蛋白免疫印记法检测BDT对自噬相关蛋白的影响;免疫荧光法检测脑源性神经营养因子(BDNF),β-微管蛋白Ⅲ(β-tubulin Ⅲ)和胶质纤维酸性蛋白(GFAP)。结果 与常氧组比较,糖氧剥夺/复氧会显著降低大鼠NSCs的细胞活力,与模型组比较,20%BDT含药血清明显改善了大鼠NSCs存活(P<0.01)。BDT可以诱导OGD后NSCs自噬小体的产生,与常氧组比较,微管相关蛋白轻链3Ⅱ(LC3Ⅱ),Beclin-1表达升高(P<0.05,P<0.01),p62变化不明显;与模型组比较,BDT组LC3Ⅱ,Beclin-1明显上调(P<0.05,P<0.01),p62表达下调(P<0.01);Rapa组起到类似效果(P<0.05,P<0.01),3-MA组抑制了自噬的活性(P<0.01)。Ad-mCherry-GFP-LC3B结果显示,与常氧组比较,模型组荧光强度显著增加(P<0.01);与模型组比较,20%BDT含药血清和Rapa组显著增加自噬荧光强度(P<0.01),3-MA抑制了自噬活性(P<0.01)。免疫荧光结果显示,与常氧组比较,EdU,β-tubulin Ⅲ,GFAP和BDNF阳性细胞数显著减少(P<0.01);与模型组比较,20%BDT含药血清和Rapa起到了类似的保护和促进作用(P<0.05,P<0.01);3-MA组可以部分阻断BDT的神经保护和分化能力(P<0.05)。结论 BDT预保护可以通过上调自噬减少糖氧剥夺造成的大鼠NSCs损伤,促进增殖、分化。

    Abstract:

    Objective To investigate the effect of Buyang Huanwutang (BHT) on proliferation and differentiation in neural stem cells (NSCs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury.Method NSCs isolated from the hippocampus of SD rats were cultured and randomly divided into a normoxia group, a model group, a BHT group, a rapamycin (Rapa) group, and a combination group [autophagy inhibitor 3-methyladenine (3-MA) combined with BHT]. The 20% blank serum was used in the normoxia group, and 20% BHT-medicated serum in the BHT group. The doses of Rapa and 3-MA were 1 μmol·L-1 and 5 mmol·L-1, respectively. The cells were subjected to OGD/R except those in the normoxia group. The cell morphology was observed under a light microscope. NSCs were confirmed by immunofluorescence detection of nestin expression. The viability and proliferation of NSCs were assessed by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2-deoxyuridine (EdU) labeling, respectively. Furthermore, Ad-mCherry-GFP-LC3B fluorescence assay was performed to investigate autophagy. The effect of BHT on autophagy-related protein expression was detected by western blot assay. Brain derived neurotrophic factor (BDNF), β-tubulin Ⅲ, and glial fibrillary acidic protein (GFAP) were evaluated by immunofluorescence assay.Result OGD/R significantly reduced the cell viability of rat NSCs as compared with the normoxia group. Compared with the model group, the BHT group exhibited significantly improved viability of rat NSCs (P<0.01). BHT induced the production of autophagosomes in NSCs after OGD. The BHT group showed increased expression of microtuble-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) and Beclin-1 (P<0.05,P<0.01) and slightly changed p62 compared with the normoxia group, and significantly up-regulated LC3Ⅱ and Beclin-1 (P<0.05,P<0.01) and down-regulated expression of p62 (P<0.01) compared with the model group. The Rapa group had similar effect as the BHT group (P<0.05,P<0.01), while the combination group inhibited the activity of autophagy (P<0.01). As indicated by the results of ad-mCherry-GFP-LC3B, compared with the normoxia group, the model group showed increased fluorescence intensity (P<0.01), and the BHT and Rapa groups could further increased the fluorescence intensity of autophagy (P<0.01), while the combination group inhibited autophagy activity (P<0.01). Immunofluorescence results revealed that compared with the normoxia group, the model group displayed significantly reduced positive cells of EdU, β-tubulin Ⅲ, GFAP, and BDNF (P<0.01), and the BHT and Rapa groups exerted similar protective and promoting effects (P<0.05,P<0.01), while the combination group partially blocked the neuroprotection and differentiation ability of BHT (P<0.05).Conclusion BHT pretreatment can effectively protect rat NSCs against OGD-induced injury and promoted proliferation and differentiation by up-regulating autophagy.

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秦彬喻,彭冬,王逸雪,章时杰,王奇,关莉.补阳还五汤通过调节自噬促进OGD/R损伤大鼠NSCs增殖、分化能力[J].中国实验方剂学杂志,2021,27(11):9~18

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  • 收稿日期:2021-01-05
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  • 在线发布日期: 2021-05-06
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