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Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Published on: September 4, 2013
RNA binding protein RPS3 mediates microglial polarization by activating NLRP3 inflammasome via SIRT1 in ischemic
Dimi Zhou1, Lin Chen1, Yuzheng Wang1
1Department of Neurology, The Second Affiliated Hospital of University of South China, 35 Jiefang Dadao, Zhengxiang District, Hengyang, Hunan, China.
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.
Insights
RNA binding protein RPS3 exacerbates ischemic stroke injury by promoting M1 microglial polarization. Inhibiting RPS3 enhances M2 polarization, reduces brain infarction, and improves neurological function via the SIRT1/NLRP3 pathway.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Ischemic stroke causes significant morbidity due to obstructed cerebral blood flow.
- Microglial polarization plays a critical role in ischemic stroke-induced brain injury.
- The function of RNA binding protein RPS3 in microglial polarization following ischemic stroke requires further elucidation.
Purpose of the Study:
- To investigate the role and mechanism of RNA binding protein RPS3 in microglial polarization after ischemic stroke.
- To determine if RPS3 influences neuronal apoptosis and brain infarction in a rat model of ischemic stroke.
- To explore the potential of targeting RPS3 for therapeutic intervention in ischemic stroke.
Main Methods:
- Transient middle cerebral artery occlusion (tMCAO) model in Sprague-Dawley rats.
- Assessment of infarct area, neurological scores, and neuronal apoptosis.
- In vitro studies using oxygen and glucose deprivation/reoxygenation (OGD/R) on microglia.
- RT-qPCR and Western blot analysis for gene and protein expression (RPS3, SIRT1, NLRP3 inflammasome components, M1/M2 markers).
- RNA immunoprecipitation (RIP) assay to confirm RPS3-SIRT1 interaction.
Main Results:
- RPS3 depletion significantly improved neurological function and reduced infarct volume in tMCAO rats.
- Knockdown of RPS3 led to increased SIRT1 expression, decreased NLRP3 inflammasome activation, and promoted M2 microglial polarization.
- RPS3 directly targets and downregulates SIRT1 expression in microglia.
- RPS3 silencing protected neurons and microglia from OGD/R-induced cell death by modulating the SIRT1/NLRP3 pathway.
Conclusions:
- RPS3 plays a detrimental role in ischemic stroke by promoting M1 microglial polarization and neuronal injury.
- The SIRT1/NLRP3 pathway is a key mechanism through which RPS3 exerts its effects.
- RPS3 inhibition represents a promising therapeutic strategy for treating ischemic stroke.
