Astragali Radix–Chuanxiong Rhizoma mitigates ischemic stroke by inhibiting NET formation via the C5a/C5aR1 pathway

Background: Ischemic stroke (IS) is a leading cause of death and disability worldwide, posing a serious threat to human health. The herbal pair Astragali Radix–Chuanxiong Rhizoma (HQ–CX) is commonly used in the treatment of IS. However, the synergistic effects and underlying mechanisms of this herbal combination remain unclear. Objective: This study aimed to evaluate the therapeutic effects of the HQ–CX herbal pair on IS and to investigate its underlying mechanisms. Methods: A rat model of middle cerebral artery occlusion (MCAO) combined with Qi deficiency and blood stasis syndrome (QDBS) was established. The effects of HQ–CX on QDBS-MCAO rats, including improvements in QDBS symptoms, neurological function, and blood flow recovery in the ischemic area, were assessed by Qi deficiency syndrome score, blood stasis syndrome score, modified neurological severity score, cerebral blood flow measurement, and Nissl staining. Transcriptome analysis was conducted to predict the molecular mechanisms underlying the therapeutic effects of HQ–CX in IS. Additional experimental techniques, including enzyme-linked immunosorbent assay, immunofluorescence, Western blot analysis, and treatment with the pharmacological inhibitor deoxyribonuclease I, were employed to validate the efficacy of HQ–CX and explore its potential molecular targets. Furthermore, the intestinally absorbed chemical constituents of HQ–CX were identified by UPLC-Orbitrap-MS analysis, and its effects on neutrophil extracellular trap (NET) formation in vitro were evaluated through enzyme-linked immunosorbent assay, Western blot, and immunofluorescence assays. Results: In this study, HQ–CX not only alleviated symptoms associated with QDBS but also enhanced blood flow restoration in ischemic regions, thereby reducing infarct size in MCAO rats after ischemia. Transcriptomic analysis revealed that HQ–CX may exert neuroprotective effects by inhibiting NET formation, a potential therapeutic target for IS, through suppression of complement component 5a receptor 1, peptidyl arginine deiminase 4, and integrin subunit beta 3 expression. Furthermore, HQ–CX significantly reduced plasma levels of cell-free DNA and histone-DNA complexes in MCAO rats, and decreased the expression of myeloperoxidase and citrullinated histone H3 in the pia mater, cortex, and striatum of the ischemic hemisphere. Interestingly, HQ–CX failed to provide additional neuroprotection when NETs were degraded by deoxyribonuclease I, suggesting that its neuroprotective effects against ischemic injury are primarily mediated through inhibition of NET formation. Conclusion: HQ–CX exerts protective effects against IS by inhibiting NET formation through the complement component 5a/complement component 5a receptor 1 signaling pathway. These findings provide scientific evidence supporting the clinical efficacy of this herbal pair in the treatment of IS.