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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