As a sensitive cold-shock protein, RNA binding protein motif 3 (RBM3) exhibits a neuroprotective function in the condition of brain injury. However, how RBM3 is involved in acute ischemic stroke by affecting stress granules (SGs) remains unclear. Here, we established an oxygen-glucose deprivation/reperfusion (OGD/R) model in rat primary cortical neurons and PC12 cells to explore the potential mechanism between RBM3 and SG formation in acute ischemic/reperfusion (I/R) condition. The immunofluorescence results showed that the SG formation significantly decreased in rat primary cortical neurons and PC12 cells during the reperfusion period after 6 h of OGD stimulation. The western blot results, flow cytometry analysis, and cell viability assessment showed that the RBM3 expression and ratio of cell viability significantly decreased, while the rate of apoptosis increased in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Co-immunoprecipitation (Co-IP) and immunofluorescence indicated that RBM3 and GTPase-activating protein-binding protein 1 (G3BP1) colocalized cytoplasm of PC12 cells after 6 h of OGD stimulation when the SGs formation reached the highest level. Besides, overexpression and knockdown of the RBM3 were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. The results of overexpression and knockdown of RBM3 gene illustrated the pivotal role of RBM3 in affecting SG formation and apoptosis level in OGD-treated PC12 cells. In conclusion, RBM3 could combine with G3BP1 resulted in increasing stress granules generation in rat primary cortical neurons and PC12 cells after 6 h of oxygen-glucose deprivation (OGD) injury, which ultimately reduced the apoptosis in OGD-induced cells. Our study may enable a new promising target for alleviating ischemia-reperfusion injury in cells.

作为一种敏感的冷休克蛋白，RNA结合蛋白基序3（RBM3）在脑损伤的情况下表现出神经保护功能。但是，RBM3如何通过影响应激颗粒（SGs​​）参与急性缺血性卒中仍不清楚。在这里，我们在大鼠原代皮层神经元和PC12细胞中建立了氧葡萄糖剥夺/再灌注（OGD / R）模型，以探讨在急性缺血/再灌注（I / R）条件下RBM3和SG形成之间的潜在机制。免疫荧光结果显示，在OGD刺激6小时后，大鼠原代皮层神经元和PC12细胞的SG形成明显减少。Western印迹结果，流式细胞仪分析和细胞生存力评估表明，RBM3表达和细胞生存率显着降低，OGD刺激6 h后，再灌注期间PC12细胞凋亡率增加。免疫共沉淀（Co-IP）和免疫荧光表明，当SGs形成达到最高水平时，OGD刺激6 h后，RBM3和GTPase激活蛋白结合蛋白1（G3BP1）将PC12细胞的细胞质共定位。此外，RBM3的过表达和敲除分别通过质粒转染和CRISPR-Cas9技术实现。RBM3基因的过表达和敲低的结果说明了RBM3在OGD处理的PC12细胞中影响SG形成和凋亡水平的关键作用。综上所述，RBM3可以与G3BP1结合，导致缺氧-葡萄糖剥夺（OGD）损伤6小时后，大鼠原代皮层神经元和PC12细胞的应激颗粒产生增加，最终减少了OGD诱导的细胞的凋亡。我们的研究可能为减轻细胞的缺血再灌注损伤提供一个新的有希望的靶标。