BMSCs-derived exosomal Egr2 inhibited OGD/R-induced neuronal cell injury through the RNF8/DAPK1 axis in ischemic stroke

Abstract

Bone marrow-derived mesenchymal stem cells (BMSCs) can facilitate functional rehabilitation after ischemic stroke (IS) by secreting exosomes. This study aimed to elucidate the specific mechanism of action of BMSC-derived exosomal Egr2 in OGD/R-induced neuronal cell damage. Exosomes were isolated from BMSCs. OGD/R-treated N2a cells were used for in vitro experiments. CCK8 and flow cytometry analysis were applied to measure cell viability, apoptosis, and BMSCs surface markers. Protein levels were analyzed using western blotting. The binding sites of Egr2 on the RNF8 promoter were predicted using the JASPAR database and verified using ChIP and dual-luciferase reporter assays. Co-IP was used to validate the relationship between RNF8 and DAPK1. Cellular localization of RNF8 and DRPK1 was confirmed by immunofluorescence staining. Egr2 was enriched in BMSC-derived exosomes. Exosomal Egr2 isolated from BMSCs increased the viability and reduced the apoptosis of OGD/R-treated N2a cells. However, these effects were abrogated by Egr2 knockdown. Egr2 activated RNF8 by binding to its promoter. In addition, RNF8 negatively regulated DAPK1 by promoting DAPK1 ubiquitination to alleviate OGD/R-stimulated neuronal cell damage. RNF8 overexpression or DAPK1 knockdown reversed OGD/R and Egr2 knockdown in BMSC co-treated with neuronal cell injury. BMSCs-derived exosomal Egr2 relieved OGD/R-treated neuronal cell injury by regulating the RNF8/DAPK1 axis.

Keywords: BMSCs; DAPK1; Egr2; Exosome; Ischemic stroke; RNF8.