Zishen Yutai Pills Promote Angiogenesis at the Maternal-Fetal Interface in Recurrent Spontaneous Abortion Mice by Regulating miR-187/VEGF Axis

Abstract

Ethnopharmacological Relevance

Zishen Yutai pills (ZYP), a traditional Chinese patent medicine, was listed in China in 1981. It is composed of 15 traditional Chinese medicines and has the effects of regulating menstruation, helping pregnancy, and preventing abortion. In clinical practice, it is effective in preventing habitual and threatened miscarriages, and continuing to explore its mechanism of action is very meaningful research.

Aim of the Study

To explore the possible mechanism of ZYP promoting angiogenesis at the maternal-fetal interface in recurrent spontaneous abortion (RSA).

Materials and Methods

In vitro experiments, placental trophoblast cells (PTCs) were isolated from the placental tissue of RSA mice and divided into six groups: Control group, Model group, ZYP group, miR-187 inhibitor NC group, miR-18 7 inhibitor group, and miR-187 inhibitor+ZYP group. Cell viability and cell cycle were measured using CCK8 and flow cytometry, respectively. The expression levels of miR-187, VEGF, VEGF-R1, and VEGF-R2 were measured using RT-qPCR, WB, and IF staining. Animal experiments first establish an RSA mice model (CBA/J × DBA/2) and then randomly divide the mice into four groups (n=10): normal pregnancy group, RSA model group, ZYP group, and progesterone capsule group. Observed the changes in embryo absorption rate, pathological morphology of decidual tissue, and ultrastructure of vascular endothelial cells in each group of mice. RT-qPCR, WB, and IF staining methods were used to determine the expression of miR-187, VEGF, VEGF-R1, and VEGF-R2.

Results

In vitro, ZYP promoted the viability of PTCs and regulated their cell cycle, and ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2 levels. miR-187 inhibitor showed the same effects, and further ZYP intervention enhanced the effects. In vivo, ZYP remarkably reduced embryo resorption rates, and improved the pathological morphology of decidual tissues and ultrastructure of vascular endothelial cells. Moreover, ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2.

Conclusion

In summary, ZYP can regulate the expression of VEGF via miR-187, then promote the angiogenesis at the maternal-fetal interface, and playing a therapeutic role in RSA.

Graphical Abstract

Keywords:

• recurrent spontaneous abortion
• Zishen Yutai pills
• miR-187
• vascular endothelial growth factor
• VEGF
• angiogenesis

Abbreviations

ANOVA, analysis of variance; CCK, cholecystokinin; CHM, Chinese herbal medicines; DBA, Data Base Administrator; DMEM, dulbecco modified eagle medium; HE, hematoxylin-eosin; HPLC, high performance liquid chromatography; IF, immunofluorescence; mean ± SD, mean ± standard deviation; miRNAs, microRNAs; NC, National Certificate; OD, optical density; PTCs, placental trophoblast cells; PVDF, polyvinylidene fluoride; RIPA, Radio immunoprecipitation assay; RSA, recurrent spontaneous abortion; RT-qPCR, real time qPCR; SD, Sprague-Dawley; TCM, traditional Chinese medicine; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor; WT, wild-type; WB, Western blot. ZYP, Zishen Yutai pills.

Consent for Publication

This manuscript has been approved by all authors, and there are no conflicts of interest in the submitted content.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

The present work was financially supported by grants from Luo Yuankai-Zishen Yutaiwan-Research Fund for Young Scholars (20190805); Key Project of Natural Science Research of Anhui Provincial Higher Education Institutions (KJ2020A0408) and Clinical Research Fund of Anhui University of Chinese Medicine (2021yfylc09).