International immunopharmacology · 2025
Taohong Siwu decoction alleviates cerebral ischemia-reperfusion injury by inhibiting the formation of NETs through STAT1/NLRP3/GSDMD pathway.
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Ji Zhaojie, Chen Mengyuan, Zhou Xian, Chen Xinru, Wang Huifang, Han Lan, Chen Weidong
Аннотация
BACKGROUND: Ischemic stroke (IS) is a significant global health burden, contributing to disability and imposing financial strain on societies. Taohong Siwu decoction (THSWD), a classical TCM formula, has shown neuroprotective effects in IS therapy, particularly in mitigating inflammation, but its precise mechanisms remain unclear. Neutrophil extracellular traps (NETs) have been implicated in post-stroke neuroinflammation and brain damage, making NETs a potential therapeutic target for IS.
OBJECTIVE: This study focuses on the protective effect and molecular mechanism of THSWD on IS injury, focuses on its inhibitory effect on the formation of NETs, and explores the potential molecular mechanism of THSWD regulating the formation of NETs.
METHODS: Network pharmacology was employed to predict the interactions between THSWD and disease-associated target genes. In vivo experiments were conducted using a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) to evaluate the effects of THSWD on infarct volume, neurological deficits, cerebral microvascular injury, inflammatory cytokine secretion, and NETs formation. In vitro experiments were performed to investigate the effects of THSWD serum on NETs formation in rat neutrophils stimulated by phorbol myristate acetate (PMA). The underlying mechanism of THSWD in regulating NETs was further investigated via transcriptome sequencing (RNA-seq).
RESULTS: Utilizing network pharmacology, 264 overlapping targets of THSWD and IS were identified, with key pathways including neutrophil-mediated immunity and NETs formation. Experimental validation in a rat MCAO/R model demonstrated that THSWD significantly reduced cerebral infarct volume, improved neurological deficits, and attenuated blood-brain barrier (BBB) disruption by restoring vascular integrity (ZO-1, Occludin) and enhancing hemodynamic recovery. Furthermore, THSWD downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-2, IL-5, IL-18, and TNF-β) and neutrophil infiltration while downregulating NETs markers Myeloperoxidase (MPO) and Citrullinated histone H3(CitH3) in serum and ischemic brain tissue. In vitro, THSWD-containing serum inhibited PMA-induced NETosis in neutrophils by reducing extracellular DNA release and ROS production. RNA-seq of peripheral blood neutrophils identified 326 differentially expressed genes (DEGs) commonly altered by MCAO/R injury and rescued by THSWD treatment. KEGG enrichment revealed significant involvement of the NOD-like receptor signaling pathway, with bioinformatic analysis identifying STAT1 as a core hub gene. Subsequent mechanistic validation confirmed that THSWD suppressed the activation of the STAT1/NLRP3/GSDMD signaling axis in neutrophils.
CONCLUSION: THSWD ameliorates IS by inhibiting NETs formation via the STAT1/NLRP3/GSDMD signaling pathway, reducing inflammatory cytokine release and protecting against cerebral microvascular injury.
OBJECTIVE: This study focuses on the protective effect and molecular mechanism of THSWD on IS injury, focuses on its inhibitory effect on the formation of NETs, and explores the potential molecular mechanism of THSWD regulating the formation of NETs.
METHODS: Network pharmacology was employed to predict the interactions between THSWD and disease-associated target genes. In vivo experiments were conducted using a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) to evaluate the effects of THSWD on infarct volume, neurological deficits, cerebral microvascular injury, inflammatory cytokine secretion, and NETs formation. In vitro experiments were performed to investigate the effects of THSWD serum on NETs formation in rat neutrophils stimulated by phorbol myristate acetate (PMA). The underlying mechanism of THSWD in regulating NETs was further investigated via transcriptome sequencing (RNA-seq).
RESULTS: Utilizing network pharmacology, 264 overlapping targets of THSWD and IS were identified, with key pathways including neutrophil-mediated immunity and NETs formation. Experimental validation in a rat MCAO/R model demonstrated that THSWD significantly reduced cerebral infarct volume, improved neurological deficits, and attenuated blood-brain barrier (BBB) disruption by restoring vascular integrity (ZO-1, Occludin) and enhancing hemodynamic recovery. Furthermore, THSWD downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-2, IL-5, IL-18, and TNF-β) and neutrophil infiltration while downregulating NETs markers Myeloperoxidase (MPO) and Citrullinated histone H3(CitH3) in serum and ischemic brain tissue. In vitro, THSWD-containing serum inhibited PMA-induced NETosis in neutrophils by reducing extracellular DNA release and ROS production. RNA-seq of peripheral blood neutrophils identified 326 differentially expressed genes (DEGs) commonly altered by MCAO/R injury and rescued by THSWD treatment. KEGG enrichment revealed significant involvement of the NOD-like receptor signaling pathway, with bioinformatic analysis identifying STAT1 as a core hub gene. Subsequent mechanistic validation confirmed that THSWD suppressed the activation of the STAT1/NLRP3/GSDMD signaling axis in neutrophils.
CONCLUSION: THSWD ameliorates IS by inhibiting NETs formation via the STAT1/NLRP3/GSDMD signaling pathway, reducing inflammatory cytokine release and protecting against cerebral microvascular injury.