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.