[Mechanism study on regulation of maternal-fetal immune balance in treatment of recurrent spontaneous abortion by Wenyang Jianpi Formula targeting CREB/TLRs/Th17/Treg axis].

This study aims to investigate the molecular targets of the Wenyang Jianpi Formula in treating recurrent spontaneous abortion(RSA) and its regulatory mechanisms on maternal-fetal immune balance. An RSA model was established using CBA/J×DBA/2 mice, which were randomly assigned to the model group, low-dose(10.14 g·kg~(-1)), medium-dose(20.28 g·kg~(-1)), high-dose(40.56 g·kg~(-1)) Wenyang Jianpi Formula groups, and a normal control group(n=10 per group). In vivo results demonstrated that, compared with the model group, the high-dose Wenyang Jianpi Formula group significantly reduced embryo resorption rate(P<0.01) and effectively suppressed the abnormal overexpression of the transcription factor cAMP-response element-binding protein(CREB) in peripheral blood and decidual tissue(P<0.01), suggesting CREB may be a key target for the formula's action. In vitro experiments utilizing CREB overexpression/knockdown cell models revealed that serum containing the formula significantly inhibited excessive activation of key molecules of the Toll-like receptors(TLRs) signaling pathway [TLR2/4/7/9, myeloid differentiation factor 88(MyD88), nuclear factor-κB(NF-κB), and mitogen-activated protein kinases(MAPKs)](P<0.05). Concurrently, it effectively corrected the imbalance between T helper 17 cells(Th17) and regulatory T cells(Treg), thereby significantly reducing the secretion of pro-inflammatory cytokines interleukin-17A(IL-17A), IL-23, and IL-6, while significantly increasing the secretion of anti-inflammatory cytokines transforming growth factor-β(TGF-β) and IL-10(all P<0.01). Genetic intervention experiments further demonstrated that CREB overexpression enhanced TLR pathway activity(P<0.05) and exacerbated Th17/Treg imbalance. Conversely, interfering with CREB expression reversed these abnormal effects(all P<0.05). In summary, this study provides multi-level in vivo and in vitro evidence that the Wenyang Jianpi Formula blocks abnormal activation of the TLRs/MyD88/NF-κB/MAPKs signaling pathway by targeting CREB downregulation, thereby regulating Th17/Treg immune balance. This improves the maternal-fetal interface immune microenvironment, offering novel experimental support and potential therapeutic targets for TCM-based immunomodulatory treatment of RSA.