中国中药杂志2025，Vol.50Issue(14)：3847-3856,10.DOI:10.19540/j.cnki.cjcmm.20250213.705
补阳还五汤通过小窝蛋白1调控YAP1/HIF-1α信号通路促进bEnd.3细胞糖氧剥夺/复氧损伤后血管新生的机制研究
Buyang Huanwu Decoction promotes angiogenesis after oxygen-glucose deprivation/reoxygenation injury of bEnd.3 cells by regulating YAP1/HIF-1α signaling pathway via caveolin-1
摘要
Abstract
This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD)in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R)of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3,bEnd.3)based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP 1)/hypoxia-inducible factor-1α(HIF-1α)signaling pathway.Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS)was used to analyze the blood components of BHD.The cell counting kit-8(CCK-8)method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3.The lentiviral transfection method was used to construct a Cav1 silent stable strain,and Western blot and polymerase chain reaction(PCR)methods were used to verify the silencing efficiency.The control bEnd.3 cells were divided into a normal group(sh-NC control group),an OGD/R model+blank serum group(sh-NC OGD/R group),and an OGD/R model+drug-containing serum group(sh-NC BHD group).Cav1 silent cells were divided into an OGD/R model+blank serum group(sh-Cav1 OGD/R group)and an OGD/R model+drug-containing serum group(sh-Cav1 BHD group).The cell survival rate was detected by the CCK-8 method.The cell migration ability was detected by a cell migration assay.The lumen formation ability was detected by an angiogenesis assay.The apoptosis rate was detected by flow cytometry,and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot.Finally,co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α.The results showed astragaloside Ⅳ,formononetin,ferulic acid,and albiflorin in BHD can all enter the blood.The drug-containing serum of BHD at a mass fraction of 10％may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells.Compared with the sh-NC control group,the sh-NC OGD/R group showed significantly decreased cell survival rate,cell migration rate,mesh number,node number,and lumen length,significantly increased cell apoptotic rate,significantly lowered phosphorylation level of YAP1 at S127 site,and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α,vascular endothelial growth factor(VEGF),and vascular endothelial growth factor receptor 2(VEGFR2).Compared with the same type of OGD/R group,the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate,cell migration rate,mesh number,node number,and lumen length,a significantly decreased cell apoptotic rate,a further decreased phosphorylation level of YAP1 at S127 site,and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α,VEGF,and VEGFR2.Compared with the sh-NC OGD/R group,the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate,cell migration rate,mesh number,node number,and lumen length,a significantly increased cell apoptotic rate,a significantly increased phosphorylation level of YAP1 at S127 site,and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α,VEGF,and VEGFR2.Compared with the sh-NC BHD group,the sh-Cav1 BHD group showed significantly decreased cell survival rate,cell migration rate,mesh number,node number,and lumen length,a significantly increased cell apoptotic rate,a significantly increased phosphorylation level of YAP1 at the S127 site,and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α,VEGF,and VEGFR2.YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody,and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody.The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.关键词
补阳还五汤/小窝蛋白1/YAP1/HIF-1α信号通路/bEnd.3细胞/糖氧剥夺/复氧损伤/血管新生Key words
Buyang Huanwu Decoction/caveolin-1/YAP1/HIF-1α signaling pathway/bEnd.3 cells/oxygen-glucose deprivation/reoxygenation injury/angiogenesis引用本文复制引用
陈博威,欧阳银,曾繁佐,刘英飞,田丰铭,徐雅倩,易健,刘柏炎..补阳还五汤通过小窝蛋白1调控YAP1/HIF-1α信号通路促进bEnd.3细胞糖氧剥夺/复氧损伤后血管新生的机制研究[J].中国中药杂志,2025,50(14):3847-3856,10.基金项目
国家自然科学基金项目(82074251) （82074251）
湖南省自然科学基金项目(2022JJ30357,2024JJ6357,2024JJ9413) （2022JJ30357,2024JJ6357,2024JJ9413）
长沙市自然科学基金项目(kq2403104) （kq2403104）
湖南省教育厅优秀青年项目(24B0353) （24B0353）
