Bone marrow mesenchymal stromal cell-derived exosomal NRF2 ameliorates cerebral ischemia-reperfusion injury by transcriptionally activating Lin28a.

Abstract

Cerebral ischemia-reperfusion (I/R) injury (CIRI) have severe consequences on brain function, and the exciting evidence has revealed protective role of acyl-CoA synthetase long chain family member 4 (Lin28a) against cerebral ischemia-reperfusion injury. The present work aims to reveal its molecular mechanism in regulating CIRI, with the hope of providing a therapeutic method for cerebral I/R injury. We hypothesized that the exosomal nuclear factor erythroid 2-related factor 2 (NRF2) derived from bone marrow mesenchymal stromal cells (BMSCs) could transcriptionally activate Lin28a, and thereby alleviate cerebral ischemia-reperfusion injury. This hypothesis was validated in the present work. Middle cerebral artery occlusion (MCAO) model was established using C57BL/6 J mice, and the neurological deficit, infarct volume, and brain water content were assessed to evaluate neuron injury. Human glioblastoma cells (A172) were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) treatment to mimic a cerebral I/R injury cell model. Exosome isolation reagent was used to isolate exosomes from cell supernatant of bone marrow mesenchymal stromal cells (BMSCs) through sequential centrifugation and filtration steps. mRNA expression level of Lin28a was detected by quantitative real-time polymerase chain reaction. Protein expression was analyzed by western blotting assay. TUNEL cell apoptosis detection kit was used to analyze cell apoptosis in brain tissues. Enzyme-linked immunosorbent assays and commercial kits were used to detect levels of inflammatory markers and oxidative stress markers. Ferrous Iron Colorimetric Assay Kit and Fe2+ colorimetric assay kit were used to analyze Fe2+ level. The association of Lin28a and NRF2 was identified by Chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter assay. The treatment of MCAO substantially augmented infarct volume in mice, impaired neurological function, and elevated brain water content. Lin28a was lowly expressed in brain tissues of mice with CIRI, and its overexpression protected against cerebral I/R injury of MCAO mice. Moreover, Lin28a overexpression protected A172 cells against OGD/R treatment-induced injury. Additionally, NRF2 transcriptionally activated Lin28a in A172 cells. BMSC-derived exosomes increased Lin28a expression in a NRF2-dependent manner. BMSC-derived exosomal NRF2 improved OGD/R-induced A172 cell injury by inducing Lin28a production. BMSC-derived exosomal NRF2 improved CIRI by transcriptionally activating Lin28a.