Neural Stem Cell Extracellular Vesicles Carrying YBX1 Inhibited Neuronal Pyroptosis Through Increasing m6A-modified
1Department of Neurosurgery, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Hainan Province, Haikou, 570208, People's Republic of China.
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.
