Bushen Huoxue Recipe enhances the immune microenvironment at the maternal-fetal interface via the Hippo signaling pathway in mice with recurrent spontaneous abortion.

Background and aim: Bushen Huoxue Recipe (BSHXR) has been proved clinical efficacy in treating recurrent spontaneous abortion (RSA), but the underlying mechanisms remain unknown. This study aimed to investigate the mechanism of BSHXR in the immune microenvironment of RSA. Experimental procedure: The chemical constituents of BSHXR were detected using high performance liquid chromatography (HPLC). The potential mechanisms were screened through network pharmacology. Female CBA/J mice were cohoused with male DBA/2 mice to establish an RSA model, or with BALB/c mice as the control group. The RSA mice were randomly divided into six groups and given distilled water, BSHXR-L (9.46 g/kg/d), BSHXR-M (18.92 g/kg/d), BSHXR-H (37.84 g/kg/d) or cyclosporin A (4.51 mg/kg/d) for 13 days. The embryo absorption rate was calculated. HE staining was used to evaluate decidual and labyrinthine tissues. Representative immunofluorescence staining, RT‒PCR and Western blotting were used to evaluate the expression of immune factors and proteins related to the Hippo signaling pathway. Results and conclusion: According to network pharmacology, BSHXR may act on the Toll-like receptor signaling pathway and Th17 cell differentiation. BSHXR significantly decreased the embryo absorption rate of RSA mice. The mRNA and protein expression levels of FoxP3 were increased in the BSHXR group, whereas the expression levels of TLR2, TLR4, TLR7 and RORγt were decreased. YAP/TAZ nuclear translocation was inhibited in the BSHXR group, and it might also regulate the Treg/Th17 immune balance by activating the Hippo pathway. Taken together, the findings of this study indicate that BSHXR may reduce pregnancy loss by regulating the immune microenvironment at the maternal-fetal interface, which provides a theoretical basis for its clinical application and future investigations.