GSK872 和 necrostatin-1 通过抑制谷氨酸诱导的青光眼视网膜兴奋性毒性模型中的 RIP1/RIP3/MLKL 通路保护视网膜神经节细胞免受坏死性凋亡,Journal of Neuroinflammation

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GSK872 和 necrostatin-1 通过抑制谷氨酸诱导的青光眼视网膜兴奋性毒性模型中的 RIP1/RIP3/MLKL 通路保护视网膜神经节细胞免受坏死性凋亡
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2022-10-26 , DOI: 10.1186/s12974-022-02626-4
Mengyuan Liu _{1,

2,

3} , Haibo Li _{1,

2,

3} , Rongliang Yang _{1,

2,

3} , Dan Ji _{1,

2,

3} , Xiaobo Xia _{1,

2,

3}

Affiliation

青光眼是全球不可逆失明的主要原因，其特征是视网膜神经节细胞 (RGC) 进行性退化。目前对青光眼的治疗只能减缓或部分预防疾病进展，未能完全预防 RGC 死亡和视野缺损。通过 N-甲基-d-天冬氨酸 (NMDA) 受体的谷氨酸兴奋性毒性在青光眼中 RGCs 死亡中起着至关重要的作用，这通常伴随着氧化应激和 NLRP3 炎性体激活。然而，确切的机制仍不清楚。本研究建立了谷氨酸诱导的R28细胞兴奋性毒性模型和NMDA诱导的小鼠青光眼模型。进行细胞计数试剂盒 8、Hoechst 33342/PI 双染色和乳酸脱氢酶释放测定以评估细胞活力。Annexin V-FITC/PI双染色检测细胞凋亡和坏死率。活性氧 (ROS) 和谷胱甘肽 (GSH) 用于检测 R28 细胞中的氧化应激。通过qRT-PCR测量促炎细胞因子的水平。透射电子显微镜 (TEM) 用于检测 RGC 中的坏死形态变化。通过免疫荧光检测视网膜 RGC 数量。苏木精和伊红染色用于检测视网膜形态变化。通过免疫荧光和蛋白质印迹测量RIP1、RIP3、MLKL和NLRP3炎症小体相关蛋白的表达水平。我们发现谷氨酸兴奋性毒性通过在体内和体外激活 RIP1/RIP3/MLKL 通路诱导 RGCs 坏死性凋亡。给予 RIP3 抑制剂 GSK872 和 RIP1 抑制剂 necrostatin-1 (Nec-1) 可防止谷氨酸诱导的 RGC 损失、视网膜损伤、神经炎症、ROS 的过度产生和 GSH 的减少。此外，在 GSK872 和 Nec-1 抑制 RIP1/RIP3/MLKL 通路后，谷氨酸诱导的参与 NLRP3 炎性体激活的关键蛋白上调，包括 NLRP3、pro-caspase-1、cleaved-caspase-1 和白细胞介素- 1β（IL-1β）被显着抑制。我们的研究结果表明 RIP1/RIP3/MLKL 通路介导 RGC 的坏死性凋亡并调节由谷氨酸兴奋性毒性诱导的 NLRP3 炎性体活化。此外，GSK872 和 Nec-1 可以通过抑制 RIP1/RIP3/MLKL 通路保护 RGC 免受坏死性凋亡并抑制 NLRP3 炎性体激活，从而为青光眼提供一种新的神经保护治疗。

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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

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.

更新日期：2022-10-27

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