Chinese Journal of Experimental Traditional Medical Formulae
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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    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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History
  • Received:January 05,2021
  • Revised:
  • Adopted:
  • Online: May 06,2021
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