中药药理与临床2025，Vol.41Issue(10)：17-25,9.
通过3D体外共培养模型探讨补肾调经方含药血清经子宫内膜基质HIF-2α调控滋养层细胞侵袭的作用机制
Mechanism of Bushen Tiaojing Fang Containing Serum in Regulating Trophoblast Cell Invasion by Endometrial Stromal HIF-2α via 3D In Vitro Co-Culture Model
摘要
Abstract
Objective:To explore the role and mechanism of Bushen Tiaojing Fang(补肾调经方)containing serum in regulating human chorionic trophoblast cells(HTR-8)invasion via the hypoxia-inducible factor-2α(HIF-2α)-mediated pathway in human endometrial stromal cells(HESC)via a 3D in vitro co-culture model.Methods:The cell counting kit-8(CCK-8)method was used to screen the optimal concentration of drug-containing serum in Bushen Tiaojing Fang rats.Afterwards,differentiation mixtures were used in vitro to induce HESC decidualiza-tion,which was co-cultured with human trophoblast blastoid spheroids(BLS)formed by HTR-8 to establish a 3D in vitro co-culture model.Under hypoxic conditions,a specific siRNA was applied for targeted knockdown of HIF-2α in decidualized HESC that was later intervened by Bushen Tiaojing Fang containing serum.Then,the conditioned medium of decidualized HESC was collected to stimulate HTR-8.The experi-ment included a normal control serum group,a drug-containing serum group of 10%Bushen Tiaojing Fang,a negative control group,a siRNA-HIF-2α group,and a group of siRNA-HIF-2α+10%Bushen Tiaojing Fang containing serum.The 3D in vitro co-culture model was used to observe the effects of Bushen Tiaojing Fang containing serum on the migration and invasion capabilities of BLS.Enzyme-linked immunosor-bent assay(ELISA)was performed to measure vascular endothelial growth factor A(VEGFA)content in HESC conditioned medium.West-ern blot and RT-qPCR were used to detect the protein and mRNA expressions of HIF-2α,and downstream molecules lipoxygenase(LOX)and VEGFA in decidualized HESC,respectively,as well as the protein and mRNA expressions of survival signals PI3K and p-AKT in HTR-8,and invasion signals EGFL7,mitogen-activated protein kinase(MAPK)and NOTCH1.Results:Compared with the normal control serum group,the drug-containing serum group of 10%Bushen Tiaojing Fang decoction showed an expanded range of placental villi-like cell extension in BLS at 24 h and 48 h,with increased migration diameter(P<0.05).The protein and mRNA expressions of HIF-2α,LOX,and VEGFA were significantly upregulated in decidualized HESC(P<0.05 or P<0.01),and the VEGFA content was significantly increased in the conditioned medium(P<0.01).The protein and mRNA expressions of PI3K,p-AKT,EGFL7,MAPK,and NOTCH1 were significantly upregulated in HTR-8(P<0.05 or P<0.01).Compared with the negative control group,the siRNA-HIF-2α group exhibited a smaller range of placental vil-li-like cell extension in BLS at 24 h and 48 h,with reduced migration diameter(P<0.05)and adhesion rate(P<0.01).The protein and mRNA expressions of HIF-2α,LOX,and VEGFA were downregulated in decidualized HESC(P<0.05,P<0.01),the VEGFA content was re-duced in the conditioned medium(P<0.01).Similarly,the protein and mRNA expressions of PI3K and p-AKT were downregulated in HTR-8(P<0.05 or P<0.01).Compared with the siRNA-HIF-2α group,the group of siRNA-HIF-2α+10%Bushen Tiaojing Fang decoction showed a significantly expanded range of placental villi-like extension in BLS at 24 h and 48 h(P<0.01),with increased migration diameter(P<0.01)and adhesion rate(P<0.01).The protein and mRNA expressions of HIF-2α,LOX,and VEGFA were significantly elevated in de-cidualized HESC(P<0.05 or P<0.01),the VEGFA content was significantly increased in the conditioned medium(P<0.01),and the pro-teins and mRNA expressions of PI3K and p-AKT were significantly elevated in HTR-8(P<0.05 or P<0.01).Conclusion:Bushen Tiaojing Fang Fang containing serum may promote HTR-8 migration and invasion by activating PI3K/AKT,a survival signal of HTR-8,and enhancing the expression of EGFL7/MAPK/NOTCH1,an invasion signal of HTR-8,via the HIF-2α/LOX/VEGFA pathway in decidualized HESC.关键词
补肾调经方含药血清/3D体外共培养模型/缺氧诱导因子2α/人子宫内膜基质细胞/人绒毛膜滋养层细胞/基质-滋养层细胞对话/侵袭Key words
Bushen Tiaojing Fang containing serum/3D in vitro co-culture model/Hypoxia-inducible factor 2 alpha/Human endometrial stro-mal cell/Human chorionic trophoblast cell/Stroma-trophoblast cell dialog/Invasion引用本文复制引用
张鑫铭,刘章铭,杨海军,潘威,徐华洲,马惠荣,杜惠兰,李文雅,宋翠淼..通过3D体外共培养模型探讨补肾调经方含药血清经子宫内膜基质HIF-2α调控滋养层细胞侵袭的作用机制[J].中药药理与临床,2025,41(10):17-25,9.基金项目
国家自然科学基金面上项目(编号:82174419) （编号:82174419）
河北省自然科学基金项目(编号:H2020423077) （编号:H2020423077）
河北省中医药管理局重点项目(编号:Z2022001). （编号:Z2022001）
