Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy.

Xiuli Zhang,Zhenzhen Hu,Datong Zheng,Jinjin Xu,Ya Yuan,Na Dong,Xiuqin Jiang,Yifeng Lu,Víctor M. Mendoza-Núñez

doi:10.1155/2021/6219715

Abstract

Background Mesenchymal stem cell-derived exosomes (MSC-exos) have been recognized as a promising therapeutic strategy for neonatal hypoxic-ischemic brain damage (HIBD). Recently, microglial pyroptosis was shown to play a vital role in the progression of neonatal HIBD. However, whether MSC-exos improve HIBD by regulating microglial pyroptosis remains unknown. Methods Exosomes were isolated from human umbilical cord mesenchymal stem cells (huMSCs) and identified by transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). BV-2 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce microglial ischemia/reperfusion (I/R) in vitro. CCK-8, ELISA, western blot, and Hoechst 33342/PI double staining were performed to detect the pyroptosis of BV-2 cells. Conditioned medium (CM) from BV-2 cells exposed to different treatments was used to investigate its effect on neuronal injury. Moreover, 3-methyladenine (3-MA) and mitochondrial division inhibitor-1 (mdi-1) were used to verify the involvement of mitophagy in the protection of MSC-exos against OGD/R-induced pyroptosis in BV-2 cells. Finally, FOXO3a siRNA was used to investigate the involvement of FOXO3a in MSC-exo-induced mitophagy and pyroptosis inhibition. Results Exosomes from huMSCs were successfully extracted. In OGD/R-exposed BV-2 cells, MSC-exos increased cell viability and decreased the expression of NLRP3, cleaved caspase-1, and GSDMD-N as well as the release of IL-1β and IL-18. Compared with CM from OGD/R-exposed BV-2 cells treated with PBS, CM from OGD/R-exposed BV-2 cells treated with MSC-exos significantly increased the viability of SH-SY5Y cells and decreased LDH release. MSC-exos also increased the expression of TOM20 and COX IV in OGD/R-exposed BV-2 cells. Additionally, 3-MA and mdi-1 attenuated the inhibition of pyroptosis with MSC-exo treatment. Furthermore, FOXO3a siRNA partially abolished the neuroprotective effect of MSC-exos and attenuated mitophagy and pyroptosis inhibition induced by MSC-exo treatment. Conclusions Our findings demonstrated that MSC-exos increased FOXO3a expression to enhance mitophagy, therefore protecting microglia from I/R-induced pyroptosis and alleviating subsequent neuronal injury.

Highlights

Neonatal hypoxic-ischemic brain damage (HIBD) is a serious threat to the lives of newborns and the most important cause of long-term neurological dysfunction in infants [1, 2]
The transmission electron microscopy (TEM) results revealed that mesenchymal stem cell (MSC)-exos exhibited round-shaped morphology (Figure 1(a)), which is consistent with the typical exosomal morphology
MSC-exos significantly increased cell viability, inhibited the release of IL-1β and IL-18, and downregulated the expression levels of the key proteins associated with pyroptosis in oxygen-glucose deprivation (OGD)/Rexposed BV-2 cells. These findings suggested that the protective effect of MSC-exos against I/R-induced neuronal injury was associated with a reduced microglial pyroptosis

Summary

Introduction

Neonatal hypoxic-ischemic brain damage (HIBD) is a serious threat to the lives of newborns and the most important cause of long-term neurological dysfunction in infants [1, 2]. MSCderived exosomes (MSC-exos) have been identified as one of the key neuroprotective mechanisms of MSCs and can effectively ameliorate ischemia/reperfusion- (I/R-) induced brain injury by promoting angiogenesis, regulating immune responses, and inhibiting neuronal apoptosis [6,7,8,9,10]. Mesenchymal stem cell-derived exosomes (MSC-exos) have been recognized as a promising therapeutic strategy for neonatal hypoxic-ischemic brain damage (HIBD). 3-methyladenine (3-MA) and mitochondrial division inhibitor-1 (mdi-1) were used to verify the involvement of mitophagy in the protection of MSC-exos against OGD/R-induced pyroptosis in BV-2 cells. FOXO3a siRNA partially abolished the neuroprotective effect of MSC-exos and attenuated mitophagy and pyroptosis inhibition induced by MSC-exo treatment. Our findings demonstrated that MSC-exos increased FOXO3a expression to enhance mitophagy, protecting microglia from I/R-induced pyroptosis and alleviating subsequent neuronal injury

Methods

Results

Discussion

Conclusion