S1P-mediated cerebral microarteriogenesis induced by Tong-Qiao-Huo-Xue decoction after ischemia-reperfusion

Background Tong-Qiao-Huo-Xue Decoction (TQHXD), a classical Chinese medicine formula, is widely used to enhance blood circulation, remove stasis, and improve outcomes in ischemic stroke. Its mechanisms in cerebral microarteriogenesis remain unclear. Purpose This study investigated the role of TQHXD in promoting cerebral microarteriogenesis after ischemic stroke and explored S1P-mediated communication between brain microvascular endothelial cells (BMECs) and brain microvascular smooth muscle cells (BMSMCs). Study design An integrated approach combining metabolomics, molecular docking, MCAO/R rat models, and in vitro BMSMC assays was employed to elucidate TQHXD neurovascular protective mechanisms. Methods Stroke-related targets were predicted via metabolomics, and UHPLC-MS/MS identified brain-penetrating active components. Molecular docking evaluated their binding affinity to S1P1. MCAO/R rats were assessed for neurological function, neuronal apoptosis, and cerebral microvascular morphometry. In vitro, BMSMC viability, proliferation, migration, phenotypic switching, and angiogenic factor expression were analyzed. Western blotting, co-immunoprecipitation, and pull-down assays validated key signaling pathways and protein–protein interactions. Results TQHXD improved neurological deficits, reduced cortical neuronal apoptosis, and increased microvascular density, length, and perfusion. CSF-detectable components (muscone, amygdalin, ligustilide, paeoniflorin, and hydroxysafflor yellow A) exhibited high S1P1 affinity. In vitro, TQHXD enhanced BMSMC viability, proliferation, migration, and phenotypic switching, activated S1P1/RAS/RAF/MEK/ERK signaling, and upregulated angiogenic and neurotrophic factors (PDGF, VEGF, bFGF, BDNF). Co-immunoprecipitation and pull-down assays confirmed specific protein–protein interactions within the S1P-mediated cascade. Conclusion TQHXD confers neurovascular protection by activating S1P-mediated S1P1/RAS/RAF/MEK/ERK signaling, promoting cerebral microarteriogenesis and collateral circulation restoration, providing mechanistic evidence supporting its clinical application in ischemic stroke.