RNA 结合蛋白 RPS3 通过 SIRT1 激活 NLRP3 炎症小体介导缺血性脑卒中的小胶质细胞极化。
RNA binding protein RPS3 mediates microglial polarization by activating NLRP3 inflammasome via SIRT1 in ischemic stroke.
机构信息
Department of Neurology, The Second Affiliated Hospital of University of South China, 35 Jiefang Dadao, Zhengxiang District, Hengyang, Hunan, China.
Department of Neurology, The Second Affiliated Hospital of University of South China, 35 Jiefang Dadao, Zhengxiang District, Hengyang, Hunan, China.
出版信息
J Stroke Cerebrovasc Dis. 2023 Jun;32(6):107132. doi: 10.1016/j.jstrokecerebrovasdis.2023.107132. Epub 2023 Apr 21.
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.
背景
缺血性中风是一种高发病率的脑血流阻塞疾病。小胶质细胞极化是缺血性中风引起损伤的一个因素。在这里，我们专注于 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
对 SD 大鼠进行短暂性大脑中动脉闭塞（tMCAO）。TTC 染色检测梗死面积，神经评分评估。荧光染色检测神经元凋亡和小胶质细胞分化。氧葡萄糖剥夺/复氧（OGD/R）用于处理小胶质细胞。通过 RT-qPCR 测定 RPS3、SIRT1、M1 和 M2 极化标志物（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 RPS3 与 SIRT1 相互作用。CCK-8 测定细胞活力。流式细胞术和 ELISA 检测 M1 和 M2 极化标志物。比色法 CytoTox 96 细胞毒性试剂盒检测 LDH 释放。
结果
RPS3 耗竭改善 tMCAO 后大鼠的神经功能障碍并减少梗死面积。RPS3 敲低导致 SIRT1 表达增加，NLRP3 炎症小体激活减少，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 并降低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 通过 SIRT1 激活 NLRP3 炎症小体并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，提示缺血性中风治疗的新靶点。
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