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BackgroundMicroglia polarization plays a crucial role in the progression of cerebral ischemia-reperfusion injury (CIRI), but the mechanisms remain largely undefined. The preset study aimed to investigate the mechanism of microglia polarization following CIRI.MethodsCIRI was modeled in C57BL/6J mice through middle cerebral artery occlusion-reperfusion and in BV2 cells via oxygen and glucose deprivation/reoxygenation. Reverse transcription-quantitative PCR, western blotting, flow cytometry and fluorescence staining were used to detect the expression levels of key proteins associated with microglia polarization, as well as the expression of TNFAIP3 and RACK1. The interaction between TNFAIP3 and RACK1 was verified by co-immunoprecipitation. TNFAIP3 or RACK1 gene interference (overexpression and/or silencing) was employed to examine the role of the TNFAIP3/RACK1 axis in microglia polarization following CIRI.ResultsThe results revealed that Arg-1 expression decreased, inducible nitric oxide synthase expression increased and TNFAIP3 was upregulated 24 h after CIRI. Furthermore, TNFAIP3 interacted with RACK1 to deubiquitinate and increase the expression of RACK1. These results indicate that knocking down either TNFAIP3 or RACK1 promotes microglia M1 polarization, and overexpression of RACK1 can promote microglia M2 polarization. RACK1 exerts its neuroprotective effects through NF-κB, as demonstrated by the use of NF-κB inhibitors.ConclusionThe present findings indicate that TNFAIP3 inhibits M1 microglial polarization via deubiquitination of RACK1 after CIRI, RACK1 exerts its effects through NF-κB.研究背景：小胶质细胞极化在脑缺血再灌注损伤（cerebral ischemia-reperfusion injury, CIRI）的进展过程中发挥关键调控作用，但其具体分子机制尚未完全阐明。本研究旨在探讨脑缺血再灌注损伤后小胶质细胞极化的潜在调控机制。 研究方法：本研究通过大脑中动脉闭塞再灌注法构建C57BL/6J小鼠脑缺血再灌注损伤模型，同时采用氧糖剥夺/复氧法构建BV2细胞损伤模型。采用逆转录定量PCR（reverse transcription-quantitative PCR, RT-qPCR）、蛋白质印迹法、流式细胞术及荧光染色技术，检测小胶质细胞极化相关关键蛋白的表达水平，以及TNFAIP3与RACK1的表达情况。通过免疫共沉淀实验验证TNFAIP3与RACK1之间的相互作用。通过对TNFAIP3或RACK1进行基因干预（过表达及/或沉默），探讨TNFAIP3/RACK1信号轴在脑缺血再灌注损伤后小胶质细胞极化中的调控作用。 研究结果：结果显示，脑缺血再灌注损伤后24小时，精氨酸酶1（Arg-1）的表达水平显著下调，诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）的表达水平显著上调，同时TNFAIP3的表达亦明显升高。进一步研究发现，TNFAIP3可与RACK1直接结合，通过去泛素化修饰上调RACK1的表达水平。上述结果表明，沉默TNFAIP3或RACK1均可促进小胶质细胞向M1型极化，而过表达RACK1则可诱导小胶质细胞向M2型极化。通过NF-κB抑制剂实验证实，RACK1可通过NF-κB信号通路发挥神经保护作用。 研究结论：本研究结果证实，脑缺血再灌注损伤后，TNFAIP3通过对RACK1进行去泛素化修饰，抑制小胶质细胞向M1型极化；而RACK1则通过NF-κB信号通路发挥其调控功能。