补肾安胎冲剂对人胎盘微血管内皮细胞VEGF/PI3K/Akt信号通路表达的影响  

The effects of Bushen Antai Granules on the expression of VEGF/PI3K/Akt signaling pathway in human placental microvascular endothelial cells

作　　者：李元琪 李伟莉[1] 储继军[1] 余欣慧[1] 吴俊 陆莎莎 LI Yuanqi;LI Weili;CHU Jijun;YU Xinhui;WU Jun;LU Shasha(The First Affiliated Hospital of Anhui Medical University,Hefei 230000,China)

机构地区：[1]安徽中医药大学第一附属医院妇产科,合肥230000 [2]蒙城县中医院妇科

出　　处：《环球中医药》2024年第12期2441-2448,共8页Global Traditional Chinese Medicine

基　　金：国家自然科学基金(81574017);国家卫生健康委配子及生殖道异常研究重点实验室生殖医联体专项基金(NHC-RMC-2021-6);国家中医药管理局全国名老中医药专家李伟莉传承工作室项目;安徽中医药大学临床科研项目(2021yfylc19);安徽省高等学校科学研究项目(2024AH051049);中华中医药学会妇科分会青年培英计划项目。

摘　　要：目的基于VEGFR-2沉默探讨补肾安胎冲剂(后文简称BSAT)对人胎盘微血管内皮细胞(human placental microvascular endothelial cells,HPMVECs)的影响,通过血管内皮生长因子(vascular endothelial growth factor,VEGF)/磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)/丝苏氨酸蛋白激酶(protein kinase B,Akt)通路探索其治疗复发性流产(recurrent spontaneous abortion,RSA)的作用机制。方法在体外构建人胎盘微血管内皮细胞,随后采用转染小干扰RNA(siRNA)技术沉默VEGFR-2基因表达,并给予补肾安胎冲剂含药血清或空白血清干预,将人胎盘微血管内皮细胞分为空白血清组(PBS+空白血清)、siRNA空白血清组(PBS+VEGFR-2 siRNA+空白血清)、siRNA NC空白血清组(PBS+NC siRNA+空白血清)、siRNA药物血清组(PBS+VEGFR-2 siRNA+BSAT含药血清)、药物血清组(PBS+BSAT含药血清)予对应处理,实时荧光定量PCR、免疫荧光法及Western blot分别检测VEGF、VEGFR-2、PI3K、Akt mRNA及蛋白表达,观察在VEGFR-2基因沉默时,补肾安胎冲剂是否能通过PI3K/Akt通路介导血管新生。结果空白血清组和siRNA NC空白血清组相比,VEGF、VEGFR-2、PI3K、Akt mRNA及蛋白无明显差异(P>0.05);药物血清组细胞VEGF、VEGFR-2、PI3K、Akt mRNA及蛋白的表达明显高于空白血清组(P<0.05)。与空白血清组相比,siRNA空白血清组VEGF、VEGFR-2、PI3K、Akt mRNA及蛋白的表达明显下降(P<0.01)。siRNA药物血清组与siRNA空白血清组相比,VEGF、VEGFR-2、PI3K、Akt有明显上升(P<0.01),补肾安胎冲剂能显著提高VEGF/PI3K/Akt的低表达。结论补肾安胎冲剂通过激活HPMVECs中VEGFR-2,靶向上调PI3K/Akt表达,介导血管新生,促进母胎界面血管重构,从而对RSA发挥治疗作用。Objective To investigate the effect of Bushen Antai(BSAT)Granules on human placental microvascular endothelial cells(HPMVECs)based on VEGFR-2 silencing,and to explore its mechanism of action in treating recurrent spontaneous abortion(RSA)through the vascular endothelial growth factor(VEGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Methods Human placental microvascular endothelial cells were constructed in vitro,and then the expression of VEGFR-2 gene was silenced by transfection of small interfering RNA(siRNA)technology.The cells were intervened with Bushen Antai Granules drug containing serum or blank serum.The human placental microvascular endothelial cells were divided into blank serum group(PBS+blank serum),siRNA blank serum group(PBS+VEGFR-2 siRNA+blank serum),siRNA NC blank serum group(PBS+NC siRNA+blank serum),siRNA drug serum(PBS+VEGFR-2 siRNA+BSAT drug containing serum),and drug serum group(PBS+BSAT drug containing serum)for corresponding treatment.VEGF and VEGFR-2 were detected by real-time fluorescence quantitative PCR,immunofluorescence,and Western blotting.Observe the expression of PI3K,Akt mRNA and protein,and observe whether Bushen Antai Granules can mediate angiogenesis through the PI3K/Akt pathway when VEGFR-2 gene is silenced.Results There were no significant differences in VEGF,VEGFR-2,PI3K,Akt mRNA and protein between the blank serum group and the siRNA NC blank serum group(P>0.05);The expression of VEGF,VEGFR-2,PI3K,Akt mRNA and protein in the drug serum group was significantly higher than that in the blank serum group(P<0.05).Compared with the blank serum group,the siRNA blank serum group showed a significant decrease in the expression of VEGF,VEGFR-2,PI3K,Akt mRNA and protein(P<0.01).Compared with the siRNA blank serum group,the siRNA drug serum group showed a significant increase in VEGF,VEGFR-2,PI3K,and Akt(P<0.01),and the Bushen Antai Granules could significantly increase the low expression of VEGF/PI3K/Akt.Conclusion Bushen Antai Granules activate VEGFR-2 in HPMVE

关 键 词：补肾安胎冲剂 复发性流产 胎盘微血管内皮细胞 磷脂酰肌醇3激酶/丝苏氨酸蛋白激酶信号通路 血管内皮生长因子 

分 类 号：R285.5[医药卫生—中药学]