Protective effects of Jiao-Ai Decoction against progesterone withdrawal-induced pregnancy loss: Insights from rat model assays, bioactive component analysis, molecular simulation, and zebrafish angiogenesis evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiao-Ai Decoction (JAD), a classic Traditional Chinese Medicine (TCM) formula, has been applied traditionally for treating gynecological conditions such as menorrhagia, post-abortion bleeding, and threatened abortion (TA). Clinically, it yields an efficacy rate of over 90% for TA treatment, yet its associated molecular events have not been characterized. AIM OF THE STUDY: To explore the pharmacology and bioactive constituents of JAD against TA, focusing on angiogenesis, endometrial receptivity and hemodynamics, using three complementary platforms: rat in vivo models, zebrafish vascular functional assays, and in silico molecular binding prediction. MATERIALS AND METHODS: A mifepristone-induced progesterone withdrawal TA rat model was established, and rats were treated with JAD at either low, medium, or high doses (6, 12, 24 g/kg). Uterine morphology, embryo loss rate, blood viscosity, and histopathology (H&E staining, immunohistochemistry, immunofluorescence) were assessed. Angiogenic (VEGFA, p-VEGFR2, HIF-1α) and receptivity markers (Integrin β3, LIF, HOXA11) were evaluated by western blot and RT-qPCR. UPLC-Q-TOF-MS/MS was used to identify components distributed in blood and uterus. Molecular docking and molecular dynamics were performed to predict potential binding affinity between core bioactive components and vascular-related target proteins. Zebrafish models (vascular injury, erythrocyte injury and hemorrhage) were utilized to observe pro-angiogenic and hemostatic effects of JAD. RESULTS: JAD reduced embryo loss, restored uterine microvascularization (CD31, α-SMA) and improved hemorheology in rats. These phenotypic changes coincided with elevated VEGFA/p-VEGFR2, suppressed HIF-1α and greater placental angiogenesis. Meanwhile, higher pinopod density and upregulated Integrin β3, LIF, and HOXA11 collectively indicated improved endometrial receptivity after treatment. Molecular docking and molecular dynamics simulations predicted stable binding interactions between 19 major bioactive components of JAD and target proteins FLT1 and VWF. In zebrafish, JAD (12.5-200 μg/mL) and its representative constituents (e.g., adenosine, ferulic acid) stimulated angiogenesis and erythropoiesis while mitigating hemorrhage, correlating with elevated VEGFA/p-VEGFR2 protein and vascular-related mRNA levels (vwf, vegfa, flt1, kdr, kdrl). CONCLUSIONS: The anti-abortive effects of JAD are associated with the restoration of uterine vascular homeostasis, endometrial receptivity and hemodynamics. Similar expression trends of VEGF-related angiogenic markers were observed in both rat and zebrafish models after JAD treatment. This work provides experimental evidence linking the traditional use of JAD for pregnancy maintenance to the restoration of uterine vascular homeostasis, thereby bridging classical TCM theory with modern pharmacological insights for mechanistic interpretation.