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1μmol·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-tubulinⅢ)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-tubulinⅢ,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。
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) and5 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-LC3 B 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 3 Ⅱ(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 upregulating autophagy.
作者
秦彬喻
彭冬
王逸雪
章时杰
王奇
关莉
QIN Bin-yu;PENG Dong;WANG Yi-xue;ZHANG Shi-jie;WANG Qi;GUAN Li(College of Basic Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第11期9-18,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(8167140990)。
