Ethyl acetate extract of Poecilobdella manillensis Lesson ameliorates ischemia stroke through inhibiting cell apoptosis and suppressing TLR4/NF-κB-mediates neuroinflammation.

Poecilobdella manillensis Lesson is a well-recognized medicinal leech in traditional Chinese medicine and Guangxi Zhuang ethnic medicine. It has long been used to activate blood circulation and remove blood stasis for the treatment of ischemic stroke. Modern pharmacological research has verified its potent anticoagulant and anti-inflammatory activities. Current studies mainly focus on its polypeptide components that exert antithrombotic effects to improve cerebral ischemia, while the neuroprotective potential and related mechanisms of its small-molecule constituents remain largely unclear. This study aimed to investigate the therapeutic effects of the ethyl acetate extract (EA) of P. manillensis on cerebral ischemia-reperfusion injury and to clarify its underlying molecular mechanism. The chemical constituents of EA were identified by UPLC-Q-TOF-MS/MS. Network pharmacology and molecular docking were used to predict and verify core targets and pathways. Neuroprotective and anti-inflammatory effects of EA were evaluated in a rat MCAO/R model, OGD/R-injured SH-SY5Y cells, and LPS-stimulated BV2 cells, using histological staining, Western blot, immunohistochemistry, and RT-qPCR. Seven small-molecule components were identified in EA, and 314 overlapping targets related to ischemic stroke were screened. Network analysis showed that TLR4 was the core target, and the main enriched pathways included NF-κB, Toll-like receptor, apoptosis and TNF signaling pathways. Consistent with the predicted results, EA significantly reduced cerebral infarct volume and improved neurological deficits in MCAO/R rats, and inhibited neuronal apoptosis and microglial inflammation in vivo. In vitro, EA notably improved the survival of OGD/R-injured neurons and suppressed LPS-induced inflammatory responses in BV2 cells. Meanwhile, EA markedly downregulated the expression of TLR4/NF-κB and NLRP3 inflammasome-related molecules. The present study demonstrated that EA protects against cerebral ischemia-reperfusion injury by inhibiting neuronal apoptosis and TLR4/NF-κB-mediated neuroinflammation. These findings provide a scientific basis for the traditional clinical application of P. manillensis and suggest that EA could serve as a potential therapeutic candidate for ischemic stroke.