Background

Ischemic stroke is the obstruction of cerebral blood flow with a high morbidity. Microglial polarization is a contributing factor for ischemic stroke-induced injury. Here, we focused on function and mechanism of RNA binding protein RPS3 in microglial polarization after ischemic stroke.

Methods

Transient middle cerebral artery occlusion (tMCAO) was conducted in SD rats. Infarct area was detected by TTC staining and neurological score was assessed. Fluorescence staining tested neuronal apoptosis and microglial differentiation. Oxygen and glucose deprivation/reoxygenation (OGD/R) was applied for treating microglia. Levels of RPS3, SIRT1, M1 and M2 polarization markers (CD86, iNOS, CD206, Arg-1) were determined by RT-qPCR. Western blot detected RPS3, SIRT1, NLRP3, ASC and Cleaved-caspase-1 expression. RIP assay validated that RPS3 interacted with SIRT1. CCK-8 measured cell viability. Flow cytometry and ELISA assessed M1 and M2 polarization markers. LDH release was detected using colorimetric CytoTox 96 Cytotoxicity kit.

Results

RPS3 depletion improved neurological dysfunction and reduced infarction area in rats after tMCAO. Knockdown of RPS3 resulted in increased SIRT1 expression and decreased NLRP3 inflammasome activation, and induced microglia M2 polarization after ischemia-reperfusion (I/R). Besides, RPS3 directly targeted SIRT1 and reduced its expression in microglia. RPS3 silencing suppressed OGD/R-triggered neuronal and microglial cell death through SIRT1. Moreover, RPS3 activated NLRP3 inflammasome and regulated microglial polarization via SIRT1.

Conclusion

RPS3 regulates microglial polarization and neuronal injury through SIRT1/NLRP3 pathway, suggesting a novel target for ischemic stroke treatment.

中文翻译：

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RNA结合蛋白RPS3通过SIRT1激活NLRP3炎症小体介导缺血性中风中的小胶质细胞极化

背景

缺血性中风是脑血流受阻，发病率很高。小胶质细胞极化是缺血性中风引起的损伤的一个促成因素。在此，我们重点研究RNA结合蛋白RPS3在缺血性中风后小胶质细胞极化中的功能和机制。

方法

在 SD 大鼠中进行短暂性大脑中动脉闭塞（tMCAO）。通过TTC染色检测梗塞面积并评估神经学评分。荧光染色测试神经元凋亡和小胶质细胞分化。氧糖剥夺/复氧（OGD/R）用于治疗小胶质细胞。通过 RT-qPCR 测定 RPS3、SIRT1、M1 和 M2 偏振标记物（CD86、iNOS、CD206、Arg-1）的水平。Western blot检测RPS3、SIRT1、NLRP3、ASC和Cleaved-caspase-1的表达。RIP 测定验证了 RPS3 与 SIRT1 相互作用。CCK-8 测量细胞活力。流式细胞术和 ELISA 评估了 M1 和 M2 极化标记。使用比色 CytoTox 96 细胞毒性试剂盒检测 LDH 释放。

结果

RPS3 耗竭可改善 tMCAO 后大鼠的神经功能障碍并减少梗塞面积。RPS3 的敲低导致 SIRT1 表达增加，NLRP3 炎性体激活减少，并在缺血再灌注 (I/R) 后诱导小胶质细胞 M2 极化。此外，RPS3直接靶向SIRT1并减少其在小胶质细胞中的表达。RPS3 沉默通过 SIRT1 抑制 OGD/R 触发的神经元和小胶质细胞死亡。此外，RPS3 激活 NLRP3 炎性体并通过 SIRT1 调节小胶质细胞极化。

结论

RPS3 通过 SIRT1/NLRP3 通路调节小胶质细胞极化和神经元损伤，为缺血性中风治疗提供了一个新靶点。