Inhibition of GSK3β and RIP1K Attenuates Glial Scar Formation Induced by Ischemic Stroke via Reduction of Inflammatory Cytokine Production.

Jin Liu,Hui-Ling Zhang,Feng Gu,Yong-Ming Zhu,Yi-Fan Wang,Yi Guo,Xin-Yi Dong,Liang Lin,Ming Zhou,Zhan-Xiang Wang

doi:10.3389/fphar.2020.00812

Abstract

In the chronic phase following ischemic stroke, glial scars can prevent axonal regeneration and the intensification of inflammation. The protective effect of inhibition of glycogen synthase kinase-3β (GSK3β) or receptor-interacting protein 1 kinase (RIP1K) on ischemic stroke has been previously reported. The current study examined the effects of RIP1K and GSK3β on ischemic stroke-induced glial scar formation. To investigate this, we used an in vivo model of ischemic stroke based on middle cerebral artery occlusion for 90 min followed by reperfusion for 7 d, and an in vitro model in primary cultured astrocytes involving oxygen and glucose deprivation for 6 h followed by reoxygenation for 24 h. Both in vivo and in vitro, we found that SB216763, a GSK3β inhibitor, and necrostatin-1 (Nec-1), a RIP1K inhibitor, decreased levels of glial scar markers, including glial fibrillary acidic protein (GFAP), neurocan, and phosphacan. SB216763 and Nec-1 also decreased levels of inflammatory related cytokines, including interleukin-6 (IL-6), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α). However, only Nec-1 increased the level of interleukin-1 receptor antagonist. Concurrent neutralization of TNF-α, IL-1β, and IL-6 with their antibodies provided better reduction in oxygen and glucose deprivation-induced increases in scar markers than obtained with separate use of each antibody. Further investigations showed that SB216763 reduced the levels of necroptosis-related proteins, including RIP1K, p-RIP1K, RIP3K, p-RIP3K, mixed lineage kinase domain-like protein (MLKL), and p-MLKL, while Nec-1 decreased the expression of p-GSK3β. Compared with Nec-1 (10 μM) and SB216763 (1 μM) alone, Nec-1 and SB216763 in combination reduced levels of GFAP, neurocan, and inflammatory-related cytokines. In conclusion, inhibition of GSK3β or RIP1K reduced glial scar formation induced by ischemic stroke. The underlying mechanisms might be at least, partially related to reducing levels of inflammatory-related cytokines and to blocking an interaction between GSK3β- and RIP1K-mediated pathways.

Highlights

Ischemic stroke is the third most common cause of death in most Western countries (Durukan and Tatlisumak, 2007; Sommer, 2017) and has become the most common cause of death in China (Wang et al, 2017), usually resulting from an atherosclerotic thrombus formation in a major cerebral artery
lactate dehydrogenase (LDH) results showed that application of SB216763 at 1, 5, or 10 mM protected astrocytes following oxygen and glucose deprivation (OGD)/reinjury, as shown by the reduction of LDH leakage (Figure 1)
The results showed that both SB216763 and Nec-1 suppressed levels of inflammatory cytokines in astrocytes, including interleukin-1 b (IL-1b) (Figures 9B, F), IL-6 (Figures 9C, G), and tumor necrosis factor-a (TNF-a) (Figures 9D, H), but only Nec-1 increased the level of Interleukin-1 receptor antagonist (IL-1Ra) (Figures 9A, E)

Summary

Introduction

Ischemic stroke is the third most common cause of death in most Western countries (Durukan and Tatlisumak, 2007; Sommer, 2017) and has become the most common cause of death in China (Wang et al, 2017), usually resulting from an atherosclerotic thrombus formation in a major cerebral artery. Only 20% of patients with ischemic stroke receive tPA treatment because of its limited therapeutic window of 4.5 h and its severe side effects, including an increased risk of symptomatic intracerebral hemorrhage (Deng et al, 2019). Astrocytes are the most abundant glial cell type in the brain and play a key role in reactive astrogliosis and glial scar formation after ischemic stroke (Barreto et al, 2011; Sims and Yew, 2017). Reactive astrogliosis helps to protect brain cells against severe injury (Mohn and Koob, 2015) and to avoid oxidative stress in the acute stage. Reactive astrocytes express inhibitory molecules, including chondroitin sulfate proteoglycans (CSPGs) such as neurocan, phosphacan, and aggrecan, which inhibit axonal regeneration (Liu and Chopp, 2016). Exploring the molecular mechanisms underlying glial scar formation is critical for improving our understanding of ischemic stroke

Methods

Results

Conclusion