The Neuroprotective Effect Of GSK872 And Necrostatin-1 In Glutamate-induced Mice Model Of Glaucoma

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.Necroptosis is a novel form of programmed cell death,which contributes to neuronal death in many neurodegenerative diseases,while its role in RGCs death in glaucoma remains elusive.The present study aimed to investigate the potential role of necroptosis in glutamate-induced RGCs death and the underlying mechanisms,as well as the neuroprotective effects of necroptosis inhibitors GSK872 and Nec-1 on glutamate excitotoxicity in RGCs by establishing the glutamate-induced excitotoxicity glaucoma model.Methods:The glutamate-induced R28 cell excitotoxicity model and NMDA-induced mice glaucoma model were established in this study.Cell counting kit-8,lactate dehydrogenase(LDH)release assay and Hoechst 33342/PI dual staining 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 detected by using DCFH-DA probe and GSH assay kit respectively,indicating the extent of oxidative stress in R28 cells.Levels of pro-inflammatory cytokines were measured by qRT-PCR.Transmission electron microscopy(TEM)was used to detect necroptotic morphological changes in R28 cells and RGCs.Retinal RGCs numbers were detected by immunofluorescence.Hematoxylin and eosin staining(HE)was used to detect retinal morphological changes.The expression levels of RIP1,RIP3,MLKL and NLRP3 inflammasome-related proteins were measured by Western blotting and immunofluorescence.Further treatment was administered using the RIP3 inhibitor GSK872 and the RIP1 inhibitor Nec-1,and changes in the above-mentioned indicators were subsequently detected.Results:CCK-8 and LDH release assay showed that glutamate significantly inhibited the viability of R28 cells,promoted the release of LDH,and exhibited dose dependence.Hoechst 33342/PI double staining and Annexin V-FITC/PI double staining further indicated that the death rate of R28 cells increased continuously with the prolonged treatment time of glutamate.Transmission electron microscopy revealed that R28 cells exhibited typical necroptosis morphology after glutamate treatment.In addition,glutamate induced an increase in ROS and a decrease in GSH in R28 cells.qRT-PCR showed that the expression of inflammatory cytokines TNF-α,IL-6,and IL-1β in R28 cells increased significantly after glutamate treatment.HE staining showed that the total retinal thickness and ganglion cell complex(GCC)thickness were significantly decreased at different time points after intravitreal injection of NMD A compared with the saline-treated group.Whole-mount staining of the retina showed that the number of RGCs in NMDA-injected mice was significantly reduced compared with the saline group.Transmission electron microscopy showed that RGCs in NMD A-injected mice exhibited typical necroptotic changes.Western blotting and immunofluorescence staining of retinal sections showed that glutamate excitotoxicity significantly increased the expression of RIP 1,RIP3,p-MLKL,and key proteins of NLRP3 inflammasome activation(including NLRP3,pro-caspase-1,cleaved-caspase-1,and IL-1β)in R28 cells and mice retina.GSK872 and Nec-1 could effectively inhibit glutamate-induced R28 cells death,reduce ROS and inflammatory cytokines production,and increase GSH content.Intravitreal injection of GSK872 and Nec-1 could significantly increase the total thickness of retina and thickness of GCC in NMDA-injured mice and reduce the loss of RGCs.GSK872 and Nec-1 could significantly downregulate the expression levels of RIP1,RIP3,p-MLKL,and key proteins of NLRP3 inflammasome activation induced by glutamate excitotoxicity in R28 cells and mice retina.Conclusions:Glutamate excitotoxicity induces intracellular oxidative stress and production of pro-inflammatory cytokines,leading to retinal damage and RGCs necroptosis in mice.The RIP1/RIP3/MLKL pathway mediates activation of NLRP3 inflammasome and RGCs necroptosis induced by glutamate excitotoxicity.GSK872 and Nec-1 can suppress the activation of NLRP3 inflammasome by inhibiting RIP 1/RIP3/MLKL pathway,reduce retinal oxidative stress and inflammatory response,and protect RGCs from necroptosis induced by glutamate excitotoxicity,showing significant neuroprotective effects.