Phytomedicine : international journal of phytotherapy and phytopharmacology · 2026
Elucidating the NETs-BNIP3-pyroptosis axis in cerebral ischemia-reperfusion injury and the intervention of Bu Yang Huan Wu decoction.
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Zhang Tianci, Guo Shu Han, Zheng Tiangang, Chen Xiaohan, Xu Zhuorui, Zhou Xiaohong, Gao Weijuan
Аннотация
BACKGROUND: Cerebral ischemia‑reperfusion injury (CIRI) leads to poor stroke outcomes, partly due to sustained endothelial damage triggered by neutrophil extracellular traps (NETs) during the subacute phase. However, the exact downstream pathways through which NETs drive endothelial cell death, and whether Buyang Huanwu Decoction (BHD) intervenes in this process, remain unclear.
OBJECTIVE: To investigate how NETs induce endothelial injury in CIRI and to evaluate the protective effects of BHD.
METHODS: Chemical profiling of BHD aqueous extract and BHD‑containing serum was performed by UHPLC‑HRMS. A rat transient middle cerebral artery occlusion (tMCAO) model and an in vitro model of primary brain microvascular endothelial cells (BMECs) subjected to oxygen‑glucose deprivation/reperfusion (OGD/R) together with isolated NETs were used. Network pharmacology and transcriptomics were applied to predict key targets. NET formation was dynamically monitored. Functional experiments included a pyroptosis inhibitor, mitophagy modulators, BHD, and lentivirus‑mediated BNIP3 knockdown/overexpression.
RESULTS: Absorbable components of BHD included calycosin‑7‑O‑β‑D‑glucoside, hydroxysafflor yellow A, and paeoniflorin. BHD improved neurological deficits, reduced infarct volume, and preserved blood‑brain barrier integrity in tMCAO rats. NETs peaked on day 3 after reperfusion and were suppressed by BHD. Mechanistically, NETs synergized with OGD/R to overactivate BNIP3, leading to dysregulated mitophagic flux and subsequent NLRP3/caspase‑1/GSDMD‑mediated endothelial pyroptosis. BNIP3 knockdown attenuated this cascade, whereas BNIP3 overexpression mimicked the damaging effects. BHD inhibited this NETs‑BNIP3‑pyroptosis cascade. In parallel, BHD partially restored mitochondrial dynamics (p‑DRP1, FIS1, MFN1) and promoted mitochondrial biogenesis (PGC‑1α, SIRT1, TFAM), consistent with its multi‑target regulation of mitochondrial quality control. Furthermore, under BNIP3 overexpression, BHD still reduced mitophagy and pyroptosis markers, indicating BNIP3‑independent protective effects.
CONCLUSION: These findings suggest that in CIRI, delayed NET formation overactivates BNIP3‑driven mitophagy and triggers endothelial pyroptosis. BHD exerts neurovascular protection by interfering with this NETs‑BNIP3‑pyroptosis axis while also improving mitochondrial dynamics and biogenesis as parallel protective mechanisms. This study provides experimental evidence for the pathogenic role of NETs and the multi‑target action of BHD in CIRI.
OBJECTIVE: To investigate how NETs induce endothelial injury in CIRI and to evaluate the protective effects of BHD.
METHODS: Chemical profiling of BHD aqueous extract and BHD‑containing serum was performed by UHPLC‑HRMS. A rat transient middle cerebral artery occlusion (tMCAO) model and an in vitro model of primary brain microvascular endothelial cells (BMECs) subjected to oxygen‑glucose deprivation/reperfusion (OGD/R) together with isolated NETs were used. Network pharmacology and transcriptomics were applied to predict key targets. NET formation was dynamically monitored. Functional experiments included a pyroptosis inhibitor, mitophagy modulators, BHD, and lentivirus‑mediated BNIP3 knockdown/overexpression.
RESULTS: Absorbable components of BHD included calycosin‑7‑O‑β‑D‑glucoside, hydroxysafflor yellow A, and paeoniflorin. BHD improved neurological deficits, reduced infarct volume, and preserved blood‑brain barrier integrity in tMCAO rats. NETs peaked on day 3 after reperfusion and were suppressed by BHD. Mechanistically, NETs synergized with OGD/R to overactivate BNIP3, leading to dysregulated mitophagic flux and subsequent NLRP3/caspase‑1/GSDMD‑mediated endothelial pyroptosis. BNIP3 knockdown attenuated this cascade, whereas BNIP3 overexpression mimicked the damaging effects. BHD inhibited this NETs‑BNIP3‑pyroptosis cascade. In parallel, BHD partially restored mitochondrial dynamics (p‑DRP1, FIS1, MFN1) and promoted mitochondrial biogenesis (PGC‑1α, SIRT1, TFAM), consistent with its multi‑target regulation of mitochondrial quality control. Furthermore, under BNIP3 overexpression, BHD still reduced mitophagy and pyroptosis markers, indicating BNIP3‑independent protective effects.
CONCLUSION: These findings suggest that in CIRI, delayed NET formation overactivates BNIP3‑driven mitophagy and triggers endothelial pyroptosis. BHD exerts neurovascular protection by interfering with this NETs‑BNIP3‑pyroptosis axis while also improving mitochondrial dynamics and biogenesis as parallel protective mechanisms. This study provides experimental evidence for the pathogenic role of NETs and the multi‑target action of BHD in CIRI.