Danshen-Chuanxiong alleviates cerebral ischemia by regulating carnitine metabolism and angiogenesis via activating CPT1/HIF-1α signaling axis.

Cerebral ischemia remains a leading cause of mortality and long-term disability. Current therapeutic interventions are still limited by a narrow therapeutic time window, hemorrhagic risk, and incomplete functional recovery against the complex pathological cascade of ischemic injury. Salvia miltiorrhiza-Ligusticum chuanxiong (Danshen-Chuanxiong, DC) herb pair, widely recognized in clinical practice, is extensively employed as a therapeutic intervention for cerebral ischemia. Nevertheless, the bioactive ingredients of DC and its underlying mechanism in cerebral ischemia remain largely unclear. This study aimed to discover the active ingredients and mechanisms of DC against ischemic stroke through in vivo and in vitro chemical characterization, network pharmacology, multi-omics profiling, and experimental validation. First, the UHPLC-QTOF-MS technique was employed to comprehensively profile the chemical constituents of DC and to characterize its absorbed prototypes within mouse brain and serum tissues. Subsequently, the middle cerebral artery occlusion/reperfusion (MCAO/R) murine model was utilized to evaluate the therapeutic effects of DC through TTC staining, neurological deficit scoring, rotarod and balance-beam tests, Evans blue extravasation, and histological assessment. To clarify the active substances and molecular mechanisms, the integrated analysis of network pharmacology, transcriptomics, and metabolomics was conducted. Candidate pathways and targets were validated by qRT-PCR, Western blotting, and immunofluorescence. The role of carnitine metabolism was investigated through L-carnitine supplementation and treatment with the Cpt1 inhibitor in vivo, and further validated in an oxygen-glucose deprivation/reoxygenation (OGD/R) model using bEnd.3 cells using both pharmacological inhibition and Cpt1-specific siRNA-mediated knockdown. CETSA was performed to evaluate the interaction between DC and CPT1. Molecular docking and molecular dynamics simulations were employed to evaluate the binding affinity and stability of the absorbed prototype compounds toward CPT1 and HIF-1α. Seventy-nine chemical ingredients were characterized from the DC extract. Among them, sixteen prototypes were discovered and identified in mice biological samples. DC administration mitigated cerebral ischemia in a dose-dependent manner, evidenced by reduced infarct size, improved neurological and motor performance, as well as blood-brain barrier integrity. Integrated multi-omics analysis revealed that the effects of DC treatment were significantly enriched in pathways related to carnitine metabolism and angiogenesis. Pathway enrichment profiling identified CPT1 as a pivotal regulator within the carnitine metabolism pathway. L-carnitine supplementation promotes angiogenesis and alleviates cerebral ischemia. In contrast, pharmacological inhibition of carnitine metabolism effectively abrogates the protective and pro-angiogenic effects of DC on MCAO/R mice and OGD/R-induced vascular endothelial cells. CETSA profiles confirmed that DC promoted the thermal stability of CPT1. Moreover, immunofluorescence and Western blotting demonstrated that DC alleviated cerebral ischemia through activating the CPT1/HIF-1α signaling pathway. Molecular docking simulations revealed that the 16 prototype compounds displayed robust binding affinities toward CPT1 and HIF-1α. Among the 16 compounds, salvianolic acid B (SalB), tanshinone I (Tan I), and ligustilide (Lig) were identified as key bioactive constituents contributing to the therapeutic efficacy of DC. This study provides the first evidence that DC exerts therapeutic effects by activating the CPT1/HIF-1α signaling axis, which subsequently modulates carnitine metabolism and angiogenesis-related pathways. Taken together, these findings establish a solid theoretical foundation supporting the clinical translation and further development of DC for treating cerebral ischemia.