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

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

JOURNAL=Frontiers in Pharmacology

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2020.00812

DOI=10.3389/fphar.2020.00812

ISSN=1663-9812

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 OGD-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 related to the reduced expression of inflammatory-related cytokines and to the interference with interactions between GSK3β- and RIP1K-mediated pathways.