Overexpression of EphB4 promotes neurogenesis, but inhibits neuroinflammation in mice with acute ischemic stroke

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Overexpression of EphB4 promotes neurogenesis, but inhibits neuroinflammation in mice with acute ischemic stroke

JIN WANG1
, ZUN ZHANG2
, SHAOJING FU1
, XIAOJIE LI1
, XINHUI LI3
, SHAOBIN WANG1
 and LIHE YUAN1
Departments of 1
Neurology and 2
Orthopedics, Inner Mongolia Baogang Hospital, Baotou, Inner Mongolia 014010;
3
Department of Neurology, First Affiliated Hospital of Baotou Medical College, Baotou, Inner Mongolia 014016, P.R. China
Received January 22, 2021; Accepted June 10, 2021
DOI: 10.3892/mmr.2021.12396

Abstract. 

Ischemic stroke is one of the most common diseases that has a high rate of mortality, and has become a burden to the healthcare system. Previous research has shown that EPH receptor B4 (EphB4) promotes neural stem cell proliferation and differentiation in vitro. However, little is known regarding
its role in the neurogenesis of ischemic stroke in vivo. Thus, the present study aimed to verify whether EphB4 was a key regulator of neurogenesis in ischemic stroke in vivo. Cerebral ischemia was induced in C57BL/6J mice via middle cerebral artery occlusion (MCAO), followed by reperfusion.
Immunofluorescence staining was performed to evaluate the effect of EphB4 on the neurogenesis in cerebral cortex. The levels of inflammatory cytokines were determined using an ELISA kit. The expression levels of ABL proto‑oncogene 1, non‑receptor tyrosine kinase (ABL1)/Cyclin D1 signaling
pathway‑related proteins were detected via western blotting. The current findings indicated that EphB4 expression was significantly increased in the cerebral cortex of MCAO model mice in comparison with sham‑operated mice. Moreover, EphB4 appeared to be expressed in neural stem cells (Nestin+),
and persisted as these cells became neuronal progenitors (Sox2+), neuroblasts [doublecortin (DCX)+], and eventually mature neurons [neuronal nuclei (NeuN)+]. Overexpression of EphB4 elevated the number of proliferating (bromodeoxyuridine+, Ki67+) and differentiated cells (Nestin+, Sox2+,
DCX+  and NeuN+), indicating the promoting effect of EphB4 on the neurogenesis of ischemic stroke. Furthermore, EphB4 overexpression alleviated the inflammation injury in MCAO model mice. The expression levels of proteins‑related to the ABL1/Cyclin D1 signaling pathway were significantly
increased by the overexpression of EphB4, which suggested that restoration of EphB4 promoted the activation of the ABL1/Cyclin D1 signaling pathway. In conclusion, this study contributes to the current understanding of the mechanisms of EphB4 in exerting neurorestorative effects and may recommend a potential new strategy for ischemic stroke treatment.

Introduction

Stroke remains the second major cause of mortality worldwide and is the leading cause of death in China (1). According to a Global Burden of Disease study, there were 5.5 million deaths
and 116.4 million disability adjusted life years due to stroke in 2017 (2). Although rapid progress has been achieved in the understanding of the pathogenesis of ischemic stroke, there is currently no effective therapeutic approach for nerve repair (3). Accumulating evidence has shown that cerebral ischemia can stimulate the activation of quiescent neural stem cells (NSCs) in the subventricular zone (SVZ) into transient amplifying progenitors, which become neuroblasts after several divisions (4,5). The neuroblasts migrate into the damaged cortex and differentiate into mature neurons to promote the recovery of the neurological function (6). The growth peak of NSCs in the adult brain after ischemia is 7‑10 days (7). However, this internal response cannot functionally compensate for the ischemic damage, and the migrating cells do not differentiate into mature neurons in the cortex (8,9). Thus, it is critical to search for therapeutic approaches and specific targets to augment the differentiation of endogenous NSCs into neurons. Neuroinflammation participates in the pathophysiological progress of secondary brain injury after ischemic stroke by increasing pro‑inflammatory cytokines and neuronal apoptosis (10). A previous study has shown that the levels of inflammatory cytokines gradually increase within 0‑24 h of cerebral ischemia reperfusion in rats, and reach a peak at 24 h (11). The interaction between neuroinflammation and subsequent neurogenesis remains unknown, and thus, has gained significant interest in recent years. Moreover, further understanding of neuroinflammation and its association with neurogenesis could provide a novel approach for brain repair. Previous studies have reported that ephrin (Eph) receptors can regulate the proliferation of stem cells and progenitor cells in the central nervous system (CNS) (12). The Eph receptor family is the largest known receptor tyrosine kinase family. The members of this family bind their ligand ephrins to initiate bidirectional signaling and regulate different physiological activities, which have become popular research subjects internationally (13). Previous studies have shown that ephrinB/EphB signaling served an early role in the regulation of stem cell behavior. For instance, ephrinB3/EPH receptor B3 (EphB3) signaling exhibited an inhibitory effect on NSC proliferation in the developing SVZ (14). In the adult CNS, overexpression of ephrinB2 or EphB2 promotes NSC proliferation and represses neuroblast migration (15). However, research regarding EphB4 focuses on tumor cells. Restoration of EphB4 promotes tumor cell proliferation, migration and angiogenesis (16,17). Moreover, a previous study revealed that EphB4 regulated the self‑renewal, proliferation and neuronal differentiation of human embryonic NSCs in vitro (18).The present study aimed to investigate the role of EphB4 in the neurogenesis and neuroinflammation of ischemic stroke in vivo.