补肾活血方改善复发性自然流产的血栓前状态：网络药理学与实验验证

The Bushenhuoxue formula improves prothrombotic state in recurrent spontaneous abortion: network pharmacology and experimental validation.

作者信息

Yang Xuan, Su Shulan, Liu Lijing, Liu Pan, Lu Maoqing, Wei Yinuo, Gao Yaqiong

机构信息

Department of Gynecology, Shanghai University of Traditional Chinese Medicine affiliated Shuguang Hospital Anhui Hospital, Anhui, Hefei - China.

Jiangsu Provincial Key Laboratory of Prescription High Technology Research, Nanjing University of Traditional Chinese Medicine, National and Local Joint Engineering Research Center for the Industrialization of Traditional Chinese Medicine Resources and Innovative Prescriptions, Jiangsu Province Traditional Chinese Medicine Resource Industrialization Process Collaborative Innovation Center, Nanjing, Jiangsu - China.

出版信息

Drug Target Insights. 2025 Aug 19;19:59-70. doi: 10.33393/dti.2025.3562. eCollection 2025 Jan-Dec.

INTRODUCTION

Bushenhuoxue formula is a traditional Chinese medicine formula with relatively safe clinical effects, but its mechanism in recurrent spontaneous abortion (RSA) is still unclear. Our present study used Network pharmacology an experimental validation to discuss how Bushenhuoxue formula improves prethrombotic state in RSA.

MATERIALS AND METHODS

The active ingredients of Bushenhuoxue formula (Drug) were acquired from our previous study. The putative targets of ZYP relevant to AS were obtained from TCMSP, Swiss Target Prediction, STITCH, DisGeNET, and Gene Cards databases. Protein-protein interactions (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted using the Cytoscape software. Furthermore, experiments were carried out for target validation in BALB/c mice, collecting placental tissue from different groups, the cell apoptosis by TUNEL assay; the pathology by HE staining; relative mRNA expression by qRT-PCR assay; relative protein expression by IHC and WB assay.

RESULTS

Animal experiments, compared with the NC group, the AT-III, PROG, HCG, APC and t-PA concentrations were significantly depressed (P˂0.05, respectively), Apoptosis cell numbers were significantly up-regulated with PI3K/AKT/HIF-1 VEGF significantly depressing (P˂0.001, respectively). With Bushenhuoxue formula supplement, AT-III, PROG, HCG, APC and t-PA concentrations were significantly improved in RSA model mice; and improved pathological changes and apoptosis cell number in placenta tissues (P˂0.05, respectively). However, with LY294002 supplement, the drug treatment effects were disappeared.

CONCLUSION

Bushenhuoxue formula improves prethrombotic state in RSA stimulating PI3K/AKT/HIF-1α/VEGF pathway

引言

补肾活血方是一种临床疗效相对安全的中药方剂，但其在复发性自然流产（RSA）中的作用机制仍不清楚。我们目前的研究使用网络药理学和实验验证来探讨补肾活血方如何改善RSA中的血栓前状态。

材料与方法

补肾活血方（药物）的活性成分来自我们之前的研究。从中药系统药理学数据库与分析平台（TCMSP）、瑞士靶点预测、STITCH、疾病基因数据库（DisGeNET）和基因卡片数据库中获取与抗磷脂综合征（AS）相关的滋肾育胎丸（ZYP）的假定靶点。使用Cytoscape软件进行蛋白质-蛋白质相互作用（PPI）网络、基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析。此外，在BALB/c小鼠中进行了靶点验证实验，收集不同组的胎盘组织，通过TUNEL法检测细胞凋亡；通过苏木精-伊红（HE）染色检测病理变化；通过实时定量聚合酶链反应（qRT-PCR）检测相对mRNA表达；通过免疫组织化学（IHC）和蛋白质免疫印迹（WB）检测相对蛋白质表达。

结果

动物实验中，与正常对照组（NC组）相比，抗凝血酶III（AT-III）、孕酮（PROG）、人绒毛膜促性腺激素（HCG）、活化蛋白C（APC）和组织型纤溶酶原激活剂（t-PA）浓度显著降低（P均<0.05），凋亡细胞数显著上调，磷脂酰肌醇-3激酶/蛋白激酶B/低氧诱导因子-1α/血管内皮生长因子（PI3K/AKT/HIF-1α/VEGF）显著降低（P均<0.001）。给予补肾活血方后，RSA模型小鼠的AT-III、PROG、HCG、APC和t-PA浓度显著改善；胎盘组织的病理变化和凋亡细胞数得到改善（P均<0.05）。然而，给予LY294002后，药物治疗效果消失。