Abstract
Excitatory toxicity due to excessive glutamate release is considered the core pathophysiological mechanism of cerebral ischemia. It is primarily mediated by N-methyl-D-aspartate receptors (NMDARs) on neuronal membranes. Our previous studies have found that icaritin (ICT) exhibits neuroprotective effects against cerebral ischemia in rats, but the underlying mechanism is unclear. This study aims to investigate the protective effect of ICT on glutamate-induced neuronal injury and uncover its possible molecular mechanism. An excitatory toxicity injury model was created using rat primary cortical neurons treated with glutamate and glycine. The results showed that ICT has neuroprotective effects on glutamate-treated primary cortical neurons by increasing cell viability while reducing the rate of lactate dehydrogenase (LDH) release and reducing apoptosis. Remarkably, ICT rescued the changes in the ERK/DAPK1 signaling pathway after glutamate treatment by increasing the expression levels of p-ERK, p-DAPK1 and t-DAPK1. In addition, ICT also regulates NMDAR function during glutamate-induced injury by decreasing the expression level of the GluN2B subunit and enhancing the expression level of the GluN2A subunit. As cotreatment with the ERK-specific inhibitor U0126 and ICT abolishes the beneficial effects of ITC on the ERK/DAPK1 pathway, NMDAR subtypes and neuronal cell survival, ERK is recognized as a crucial mediator in the protective mechanism of ICT. In conclusion, our findings demonstrate that ICT has a neuroprotective effect on neuronal damage induced by glutamate, and its mechanism may be related to inactivating GluN2B-containing NMDAR through the ERK/DAPK1 pathway. This study provides a new clue for the prevention and treatment of clinical ischemic cerebrovascular diseases.
Highlights
Stroke is currently a prevalent disease that severely threatens human health and life expectancy
The protein expression levels of p-ERK, p-Death-associated protein kinase 1 (DAPK1) and t-DAPK1, as well as DAPK1 mRNA, decreased significantly 14 h after excitatory toxicity injury, and ICT treatment was able to significantly recover these changes (Figures 4C,D,F–H), which was consistent with previous results
Our study found that ICT increases the phosphorylation levels of Extracellular signal-regulated kinase 1/2 (ERK1/2), DAPK1 and GluN2A and reduces the phosphorylation of GluN2B (Figures 2, 3); collectively, these may be the mediators of the protective effect of ICT against glutamate-induced neuronal damage
Summary
Stroke is currently a prevalent disease that severely threatens human health and life expectancy. Drugs for the treatment of ischemic stroke and improving the prognosis that have remarkable curative effects and few side effects remain scarce. Cerebral ischemia/reperfusion injury is a complex pathophysiological process that involves multiple events, such as brain energy disorder, cell acidosis, excitatory amino acid toxicity, intracellular calcium overload, free radical damage, inflammatory cytokine damage, and activation of apoptosis genes (Khoshnam et al, 2017; Meng et al, 2019; Wei et al, 2019). Excitatory toxicity plays an important role in cerebral ischemia/reperfusion injury, especially in the early stage (Amantea and Bagetta, 2017; Huang et al, 2017b). Effective inhibition of excitatory toxicity is a key strategy for reducing neuronal damage in ischemic brain injury
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