Neural Stem Cell Extracellular Vesicles Carrying YBX1 Inhibited Neuronal Pyroptosis Through Increasing m6A-modified GPR30 Stability and Expression in Ischemic Stroke.

IF 3.8 2区医学 Q1 CLINICAL NEUROLOGY

Translational Stroke Research Pub Date : 2025-04-01 Epub Date: 2023-11-15 DOI:10.1007/s12975-023-01210-z

Jun Peng, Jun He, Long Lin, You Li, Ying Xia

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Abstract

Neural stem cell-derived extracellular vesicles (NSC-derived EVs) alleviated ischemic stroke (IS) by suppressing the activation of nucleotide-binding domain leucine-rich repeats family protein 3 (NLRP3) inflammasome and neuronal pyroptosis. However, the specific mechanism needs further investigation. qRT-qPCR, Western blotting, and immunofluorescence detected related gene expression. Immunofluorescent analyzed the expression of Ki-67, βIII-Tubulin (Tuj1), and GFAP. Lactate dehydrogenase (LDH) release and IL-1β and IL-18 levels were analyzed by LDH and ELISA kits. TTC staining evaluated the infarction of brain tissues. Flow cytometric analysis measured caspase-1 activity. M6A methylated RNA immunoprecipitation PCR (MeRIP-PCR) measured methylation levels of G protein-coupled receptor 30 (GPR30). RIP and Co-IP analyzed the interactions of Y box binding protein (YBX1)/GPR30, YBX1/IGF2BP1 and NLRP3/speckle-type POZ protein (SPOP), as well as the ubiquitination levels of NLRP3. NSC-derived EVs inhibited the ischemia-reperfusion (I/R) injury of rats and the neuronal pyroptosis induced by oxygen-glucose deprivation/reoxygenation (OGD/R). Knockdown of EVs carrying YBX1 or GPR30 silencing abolished these inhibiting effects. GPR30 mRNA and IGF2BP1 protein were enriched by YBX1 antibody. YBX1 enhanced the stability of m6A-modified GPR30 by interacting with IGF2BP1 and thus promoting GPR30 expression. Knockdown of IGF2BP1 suppressed the binding between YBX1 and GPR30 mRNA. GPR30 promoted NLRP3 ubiquitination by interacting with SPOP. EVs carrying YBX1 could reduce the infarction of brain tissues and inhibit neuronal pyroptosis in rats with I/R injury. NSC-derived EVs carrying YBX1 increased the stability of m6A-modified GPR30 by interacting with IGF2BP1; the upregulation of GPR30 inhibited the activation of NLRP3 inflammasome through promoting NLRP3 ubiquitination by SPOP, ultimately suppressing the neuronal pyroptosis in IS.

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携带YBX1的神经干细胞胞外囊泡通过增加m6a修饰的GPR30在缺血性脑卒中中的稳定性和表达抑制神经元焦亡。

神经干细胞衍生的细胞外囊泡(NSC-derived EVs)通过抑制富含亮氨酸重复序列家族蛋白3 (NLRP3)炎症小体的激活和神经元焦凋亡来缓解缺血性卒中(IS)。但具体的机理还有待进一步研究。qRT-qPCR、Western blotting、免疫荧光检测相关基因表达。免疫荧光分析Ki-67、βIII-Tubulin (Tuj1)和GFAP的表达。采用乳酸脱氢酶(LDH)和酶联免疫吸附测定(ELISA)试剂盒检测乳酸脱氢酶(LDH)释放及IL-1β、IL-18水平。TTC染色评价脑组织梗死程度。流式细胞术检测caspase-1活性。M6A甲基化RNA免疫沉淀PCR (MeRIP-PCR)检测G蛋白偶联受体30 (GPR30)的甲基化水平。RIP和Co-IP分析Y盒结合蛋白(YBX1)/GPR30、YBX1/IGF2BP1和NLRP3/斑点型POZ蛋白(SPOP)的相互作用，以及NLRP3的泛素化水平。nsc源性ev可抑制大鼠缺血再灌注(I/R)损伤和氧-葡萄糖剥夺/再氧(OGD/R)所致的神经元焦亡。敲除携带YBX1或GPR30沉默的ev可消除这些抑制作用。YBX1抗体富集GPR30 mRNA和IGF2BP1蛋白。YBX1通过与IGF2BP1相互作用增强m6a修饰的GPR30的稳定性，从而促进GPR30的表达。IGF2BP1的敲低抑制了YBX1与GPR30 mRNA的结合。GPR30通过与SPOP相互作用促进NLRP3泛素化。携带YBX1的ev可减轻I/R损伤大鼠脑组织梗死，抑制神经元焦亡。携带YBX1的nsc衍生ev通过与IGF2BP1相互作用增加了m6a修饰的GPR30的稳定性;上调GPR30通过SPOP促进NLRP3泛素化，抑制NLRP3炎性体的活化，最终抑制IS的神经元焦亡。

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来源期刊

Translational Stroke Research CLINICAL NEUROLOGY-NEUROSCIENCES

CiteScore

13.80

自引率

4.30%

发文量

130

审稿时长

6-12 weeks

期刊介绍： Translational Stroke Research covers basic, translational, and clinical studies. The Journal emphasizes novel approaches to help both to understand clinical phenomenon through basic science tools, and to translate basic science discoveries into the development of new strategies for the prevention, assessment, treatment, and enhancement of central nervous system repair after stroke and other forms of neurotrauma. Translational Stroke Research focuses on translational research and is relevant to both basic scientists and physicians, including but not restricted to neuroscientists, vascular biologists, neurologists, neuroimagers, and neurosurgeons.