Pharmacological basis of Suhexiang Wan in ischemic stroke: identification and functional characterization of piperlongumine as a brain-distributed bioactive constituent associated with STAT3-mediated blood-brain barrier protection.

ETHNOPHARMACOLOGICAL RELEVANCE: Suhexiang Wan (SHXW) is a classical traditional Chinese medicine (TCM) formula that has been traditionally used for stroke-related disorders, including sudden loss of consciousness, neurological dysfunction, and hemiplegia. Although modern pharmacological studies have suggested its neuroprotective and anti-inflammatory properties, the brain-distributed constituents potentially associated with its therapeutic effects in ischemic stroke (IS) and the underlying molecular mechanisms remain unclear.

AIM OF THE STUDY: This study aimed to elucidate the pharmacological basis of SHXW in IS by identifying brain-distributed constituents and investigating a candidate constituent potentially associated with its protective effects against cerebral ischemia/reperfusion injury (CIRI), with particular emphasis on blood-brain barrier (BBB) protection.

MATERIALS AND METHODS: SHXW-derived constituents detected in brain tissue under middle cerebral artery occlusion/reperfusion (MCAO/R) conditions were characterized using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Integrated network pharmacology analysis was performed to predict potential therapeutic targets and signaling pathways. Piperlongumine (PIP) was further confirmed and quantified in SHXW and brain tissue using an authentic standard-based LC-MS/MS approach. Molecular docking, molecular dynamics simulations, biophysical binding assays, and cellular target-engagement experiments were used to investigate the interaction between PIP and signal transducer and activator of transcription 3 (STAT3). The protective effects and associated mechanisms were evaluated in a mouse MCAO/R model and an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cell injury model.

RESULTS: Among 1,991 putatively annotated constituents of SHXW, 55 SHXW-derived constituents were detected in brain tissue under MCAO/R conditions. In the MCAO/R mouse model, SHXW significantly improved neurological deficits, reduced infarct volume and brain edema, decreased BBB permeability, and restored tight junction protein expression. Among the brain-detected constituents, PIP, a natural alkaloid derived from Piper longum L., was selected for further investigation based on its detection in brain tissue, network topology characteristics, predicted association with STAT3, and reported pharmacological relevance to ischemic injury. Authentic standard-based LC-MS/MS confirmed PIP in SHXW and brain tissue, with a PIP content of 48.21 ± 1.10 μg/g in SHXW and a brain concentration of 159.67 ± 8.37 ng/g at 2 h after SHXW administration. Biophysical and cellular target-engagement assays supported the interaction between PIP and STAT3. PIP partially recapitulated the protective effects of SHXW by alleviating neurological deficits, preserving BBB integrity, restoring endothelial barrier function, and suppressing STAT3-associated inflammatory responses. STAT3 knockdown partially phenocopied the endothelial protective effects of PIP, whereas restoration of STAT3 expression in STAT3-knockdown cells reversed the protective phenotype. PIP treatment attenuated the injury-associated changes following STAT3 restoration, supporting the functional involvement of STAT3-associated signaling in PIP-mediated BBB protection.

CONCLUSIONS: This study provides new insights into the pharmacological basis of SHXW in IS by integrating chemical profiling, brain distribution analysis, and pharmacological validation. SHXW exerted protective effects against CIRI and preserved BBB integrity. Among the SHXW-derived constituents detected in brain tissue, PIP was identified and functionally characterized as a bioactive candidate associated with BBB protection. The findings support the involvement of PIP-STAT3 interaction and STAT3-associated inflammatory signaling in PIP-mediated BBB protection. These results provide pharmacological evidence linking the traditional application of SHXW with its modern molecular mechanisms while highlighting brain-distribution-guided approaches for investigating the therapeutic basis of complex TCM formulas.