Supplementary Material for: RIP3 augments neuroinflammation by facilitating neutrophil infiltration during an ischemic stroke.

Introduction: Neutrophil infiltration is responsible for the neuroinflammation during an ischemic stroke. Here, we explored the role of receptor-interacting protein kinase 3 (RIP3) in neutrophil infiltration during an ischemic stroke. Methods: The rat middle cerebral artery occlusion (MCAO) model was utilized to identify pivotal proteins involved in neutrophil infiltration during an ischemic stroke. Neutrophils were isolated from the peripheral blood of mice, and a co-immunoprecipitation (co-IP) assay was performed to identify the proteins that interact with RIP3. Results: The rat MCAO model was successfully established. Myeloperoxidase (MPO) was significantly up-regulated in the MCAO group, indicating the presence of neutrophil infiltration. RIP3 protein level exhibited a similar trend to MPO protein level, suggesting that neuroinflammation might be partly activated by RIP3 through the promotion of neutrophil infiltration. Co-immunoprecipitation (co-IP) and mass spectrometry (MS) analyses suggested that RIP3 facilitated neutrophil infiltration partly by affecting protein kinases (Rock1 and Prkaca) downstream of RIP3, and the interaction between RIP3 and Rock1 or Prkaca was validated by IF and co-IP assays. Conclusion: In this study, it was observed that RIP3 affects neutrophil infiltration, a critical phenomenon associated with neuronal injury during ischemic stroke, partly by the modulation of downstream proteins such as Rock1 and Prkaca.引言：中性粒细胞浸润是缺血性脑卒中进程中神经炎症的核心诱因。本研究旨在探讨受体相互作用蛋白激酶3（receptor-interacting protein kinase 3, RIP3）在缺血性脑卒中中性粒细胞浸润过程中的作用。方法：本研究采用大鼠大脑中动脉闭塞（middle cerebral artery occlusion, MCAO）模型，筛选缺血性脑卒中进程中参与中性粒细胞浸润的关键蛋白。从小鼠外周血中分离中性粒细胞，通过免疫共沉淀（co-immunoprecipitation, co-IP）实验鉴定与RIP3相互作用的蛋白。结果：成功构建大鼠MCAO模型。大脑中动脉闭塞组的髓过氧化物酶（myeloperoxidase, MPO）表达显著上调，提示存在中性粒细胞浸润现象。RIP3蛋白的表达趋势与MPO蛋白高度相似，表明神经炎症可能部分通过RIP3促进中性粒细胞浸润而被激活。免疫共沉淀（co-IP）与质谱（mass spectrometry, MS）分析结果显示，RIP3可通过调控其下游蛋白激酶Rock1与Prkaca，部分介导中性粒细胞浸润；且RIP3与Rock1、Prkaca的相互作用已通过免疫荧光（immunofluorescence, IF）与co-IP实验得到验证。结论：本研究证实，RIP3可通过调控Rock1、Prkaca等下游蛋白，影响缺血性脑卒中进程中与神经元损伤密切相关的中性粒细胞浸润过程。