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GSK872 and necrostatin-1 protect retinal ganglion cells against necroptosis through inhibition of RIP1/RIP3/MLKL pathway in glutamate-induced retinal excitotoxic model of glaucoma

Authors :
Mengyuan Liu
Haibo Li
Rongliang Yang
Dan Ji
Xiaobo Xia
Source :
Journal of Neuroinflammation, Vol 19, Iss 1, Pp 1-22 (2022)
Publication Year :
2022
Publisher :
BMC, 2022.

Abstract

Abstract Background Glaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-d-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear. Methods The glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting. Results We found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1β (IL-1β), was markedly inhibited. Conclusions Our findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.

Details

Language :
English
ISSN :
17422094
Volume :
19
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Journal of Neuroinflammation
Publication Type :
Academic Journal
Accession number :
edsdoj.bb35fe7875624ef38999a467456f95ff
Document Type :
article
Full Text :
https://doi.org/10.1186/s12974-022-02626-4