BACKGROUND: Ischemic stroke is a life-threatening cerebrovascular disease with limited therapeutic options. During the progression of acute ischemic stroke (AIS), neutrophil-involved inflammation mediated by high mobility group box 1 (HMGB1) considerably contributes to intensification of neuronal injury. Siegesbeckia orientalis L. (SO), one of the primary sources of Sigesbeckiae Herba, is promising in anti-neuroinflammation and neutrophil function modulation. Consequently, it is supposed that SO could fight against neuronal inflammatory injury following AIS. PURPOSE: The current study struggles to explore the ameliorative effects of ethanol extract of SO (EESO) on neuronal inflammatory injury following AIS, and dissect the related mechanisms focusing on HMGB1-driven neutrophil recruitment and neutrophil extracellular traps (NETs) generation. METHODS: Murine photothrombotic stroke model was established to evaluate the ameliorative effects of EESO administration against AIS. Histopathological examination and immunofluorescence staining were conducted for the observation of cerebral neuronal injury, neutrophil infiltration and NETs generation. Additionally, inflammatory indexes and serum HMGB1 levels were also detected through qPCR and ELISA, respectively. In vitro, the effects of EESO-containing serum administration on neutrophil migration and NETs generation were also assessed. HMGB1-overexpressed mimic transfection, cellular thermal shift assay and coimmunoprecipitation were employed to investigate whether the compounds from EESO-containing serum targeted HMGB1 to block the receptor for advanced glycation end products (RAGE)-CD11b interaction. Furthermore, potential active compounds of EESO targeting HMGB1 were screened and verified. RESULTS: EESO administration alleviated photochemically induced murine AIS as revealed by remarkably reducing infract volume as well as improving cerebral blood flow and neurological functions. Moreover, EESO administration prominently mitigated secondary neuronal injury, restrained neutrophil infiltration and NETs generation, as well as lowered the levels of serum pro-inflammatory mediators and HMGB1. In vitro, the compounds in EESO-containing serum directly interacted with neuron-derived HMGB1. HMGB1-driven neutrophil migration and NETs generation through the RAGE-CD11b interaction were also reversed by EESO-containing serum administration. Additionally, isoimperatorin, 4,7-dimethyltetral-1-one, perillartine and darutigenol, as the active components, contributed to the suppressive effects of EESO on neutrophil migration and NETs generation driven by HMGB1. CONCLUSION: In the present study, it was demonstrated that HMGB1 promoted interaction between CD11b and RAGE to drive NETs generation for the first time. Furthermore, EESO was proved to target neuron-derived HMGB1 to inhibit neutrophil infiltration and NETs generation against neuronal inflammatory injury poststroke, which was attributed to the components absorbed in the blood including isoimperatorin, 4,7-dimethyltetral-1-one, perillartine and darutigenol.