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.

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. 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. 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. 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.

Liu M ，Li H ，Yang R ，Ji D ，Xia X ... -  《Journal of Neuroinflammation》

The neuroprotective effect of melatonin in glutamate excitotoxicity of R28 cells and mouse retinal ganglion cells.

Glaucoma is the leading cause of irreversible blindness. The progressive degeneration of retinal ganglion cells (RGCs) is the major characteristic of glaucoma. Even though the control of intraocular pressure could delay the loss of RGCs, current clinical treatments cannot protect them directly. The overactivation of N-methyl-D-aspartic acid (NMDA) receptors by excess glutamate (Glu) is among the important mechanisms of RGC death in glaucoma progression. Melatonin (MT) is an indole neuroendocrine hormone mainly secreted by the pineal gland. This study aimed to investigate the therapeutic effect of MT on glutamate excitotoxicity of mouse RGCs and R28 cells. The Glu-induced R28 cell excitotoxicity model and NMDA-induced retinal injury model were established. MT was applied to R28 cells and the vitreous cavity of mice by intravitreal injection. Cell counting kit-8 assay and propidium iodide/Hoechst were performed to evaluate cell viability. Reactive oxygen species and glutathione synthesis assays were used to detect the oxidative stress state of R28 cells. Retina immunofluorescence and hematoxylin and eosin staining were applied to assess RGC counts and retinal structure. Flash visual-evoked potential was performed to evaluate visual function in mice. RNA sequencing of the retina was performed to explore the underlying mechanisms of MT protection. Our results found that MT treatment could successfully protect R28 cells from Glu excitotoxicity and decrease reactive oxygen species. Also, MT rescued RGCs from NMDA-induced injury and protected visual function in mice. This study enriches the indications of MT in the treatment of glaucoma, providing practical research ideas for its comprehensive prevention and treatment.

Wang C ，An Y ，Xia Z ，Zhou X ，Li H ，Song S ，Ding L ，Xia X ... -  《-》

Ripa-56 protects retinal ganglion cells in glutamate-induced retinal excitotoxic model of glaucoma.

Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.

Feng L ，Dai S ，Zhang C ，Zhang W ，Zhu W ，Wang C ，He Y ，Song W ... -  《-》

Pathogenesis of lupus nephritis: RIP3 dependent necroptosis and NLRP3 inflammasome activation.

RIP3 activation leads to activation of necroptosis and the NLRP3 inflammasome pathways. The activation of RIP3 in lupus nephritis (LN) has not been investigated. In this study, RIP3 and necroptosis pathway activations were demonstrated in podocytes in renal biopsies from patients with class IV LN and in the diseased kidneys from lupus-prone NZM2328 and MRL/lpr mice. RIP3 activation was accompanied with the activation of MLKL, the effector molecule of the necroptosis pathway, and activation of caspase-1, the effector of the NLRP3 inflammasome pathway. Podocyte activation of RIP3 was detected readily with the development of LN in NZM2328 mice, suggesting this activation may play a significant role in the pathogenesis of LN. GSK872, a RIP3 specific inhibitor, inhibited the development of LN in MRL/lpr mice with down-regulation of RIP3 activation in podocytes, decreased the splenic sizes and weights and anti-dsDNA antibody titers. IgG from pooled sera of diseased NZM2328 mice succumbing to LN induced both the necroptosis pathway and NLRP3 inflammasome activation in a podocyte cell line and this activation was specifically blocked by GSK872. These results indicate that the necroptosis pathway and the RIP3 dependent NLRP3 inflammasome pathway are activated in podocytes during LN. Inhibition of RIP3 kinase may be a novel therapeutic approach to treat LN and systemic lupus erythematosus (SLE).

Guo C ，Fu R ，Zhou M ，Wang S ，Huang Y ，Hu H ，Zhao J ，Gaskin F ，Yang N ，Fu SM ... -  《-》

Necrostatin-1 Protects Against Paraquat-Induced Cardiac Contractile Dysfunction via RIP1-RIP3-MLKL-Dependent Necroptosis Pathway.

Zhang L ，Feng Q ，Wang T 《-》