GSK-3β inhibitor TWS119 alleviates hypoxic-ischemic brain damage via a crosstalk with Wnt and Notch signaling pathways in neonatal rats

Highlights

• TWS119 improves the tissue structure at 7 d after hypoxic-ischemic brain damage.

• TWS119 attenuates cell apoptosis at 7 d after hypoxic-ischemic brain damage.

• TWS119 up-regulates synaptic protein expression at 7 d after hypoxic-ischemic brain damage.

• TWS119 activates Wnt signaling pathway at 24 h after hypoxic-ischemic brain damage.

• TWS119 suppresses Notch signaling pathway at 7 d after hypoxic-ischemic brain damage.

Abstract

Preterm infant brain injury is a leading cause of morbidity and disability in survivors of preterm infants. Unfortunately, the effective treatment remains absent. Recent evidence suggests that GSK-3β inhibitor TWS119 has a neuroprotective role in adult brain injury by activation of Wnt/β-catenin signaling pathway. However, the role on neonatal brain injury is not yet explored. The study aims to evaluate the effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats. Three-day-old rats were randomly divided into 3 groups: sham group, HI group and TWS119 group. The neonatal rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O₂). A single dose of TWS119 (30 mg/kg) was intraperitoneally injected 20 min prior to hypoxia-ischemia (HI). At 7 d after HI, TWS119 improved the tissue structure, reduced cell apoptosis, up-regulated bcl-2 expression, up-regulated the expression of PSD-95 and Synapsin-1. At 24 h after HI, it activated Wnt/β-catenin signaling pathway by up-regulation of β-catenin protein expression and wnt3a/wnt5a/wnt7a mRNA expression. Simultaneously, it suppressed Notch signaling pathway by down-regulation of Notch1 and HES-1 proteins expression. Our study suggested that TWS119 performed a neuroprotective function at 7 d after hypoxic-ischemic brain damage via a crosstalk with Wnt/β-catenin and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.

Introduction

Preterm infant brain injury is a leading cause of disability in survivors of preterm infants. It has been reported that 5–10% of preterm survivors may suffer from severe neurological disability, such as cerebral palsy, and 25%-50% of survivors manifest with milder cognitive disabilities and behavioral problems (Back, 2015, Back, 2017). It places economic burden on the society and family. Previous evidence suggests that neuron death (Zhu et al., 2003, Zhu et al., 2005, Thornton et al., 2017) and synaptic injury (Wang et al., 2018, Liu et al., 2019) are involved in the pathogenesis of Preterm infant brain injury. However, the specific mechanism is still unclear and the effective therapy is very limited.

The Wnt and Notch pathways are two of highly conserved signaling pathways with a crosstalk in development and diseases (Collu et al., 2014). Evidence suggests that the activation of the Wnt signaling pathway may play an important role in the neuroprotective response after hypoxic-ischemic brain damage by diverse protective mechanisms, such as neurogenesis, neuroplasticity and angiogenesis (Lambert et al., 2016, Shruster et al., 2012, Sun et al., 2014). On the contrary, Notch signaling pathway is activated and plays a negative function in vivo and in vitro in hypoxic-ischemic brain damage (Xu et al., 2018). Evidence suggests that Notch pathway plays a role in neuronal apoptosis (Park et al., 2013). Moreover, Notch pathway interplays with other signaling pathways to induce cell death, such as Pin-1 (Baik et al., 2015) and p53 (Balaganapathy et al., 2018) pathways.

Evidence suggests that Wnt/β-catenin pathway is down-regulated by Glycogen synthase kinase-3β (GSK-3β) (Oliva et al., 2018), while Notch pathway is up-regulated by GSK-3β (Foltz et al., 2002). GSK-3β is a serine/threonine kinase and involved in the processes of neuronal plasticity and neurodegeneration (Jaworski et al., 2019). Inhibitors of GSK-3β perform a neuroprotective function on hypoxic-ischemic brain damage. Recently, TWS119, as one of GSK-3β inhibitors, plays an important neuroprotective role in adult stroke animal model by various mechanisms, such as anti-inflammatory activation (Song et al., 2019), attenuating hemorrhagic transformation (Wang et al., 2016), improving the blood-brain barrier (Wang et al., 2017). However, the role on neonatal brain injury is yet not explored. GSK-3β inhibitor SB216763 modulates Wnt and Notch pathways to equilibrate neurogenesis and gliogenesis in a rat model of Parkinson’s disease (Singh et al., 2018). Whether TWS119 plays a protective role in hypoxic-ischemic brain damage via modulation of Wnt and Notch pathways is unclear.

Some evidence suggests that the rat brain at postnatal days 10 is comparable to that of a term infant (Semple et al., 2013) which is the key period to assess brain maturation and injury of preterm infants (Duerden and Thompson, 2020). Therefore, in this study, postnatal day 10 (7 d after hypoxic-ischemic brain damage) is considered to assess the protective effect of TWS119 after hypoxic-ischemic brain damage. The aims of this study is to evaluate the neuroprotective effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.