The Study Of The Mechanism Of Necrostatin-1Protects Glutamate-mediated Toxicity

Background and significance: Glutamate is a very important amino acid which isinvolved in many processes of life activities, like energy metabolism and nerveconduction. The disorder of glutamate metabolism results in many illness. But themechanism of toxicity of glutamate is not very clear.Objective: To determin the role of necrostatin-1and RIP1/RIP3in glutamateinduced MEF and HT22cells death.Methods: The survival rates of wild type and RIP3-/-mouse embryonic fibroblast(MEF) and HT22cells after treating with glutamate and TNF-were tested to check thesensitivities of these cells to glutamate and TNF-.To figure out whether the target ofnecrostatin-1in protecting glutamate induced cells death was the same as TNF-induced necroptosis, RIP1was knocked down with siRNA while necrostatin-1waspreteated before treating with glutamate and TNF-And the efficiency of RIP1RNAiwas examined by western blot. To figure out whether RIP3was involved in glutamateand TNF-induced cells death, RIP3was deleted in MEF cells and knocked down withsiRNA in HT22cells and and then tested their sensitivities to glutamate and TNF-. Tofigure out whether AIF was involved in glutamate and TNF-induced cells death, AIFwas knocked down in MEF and HT22cells and then tested their sensitivities toglutamate and TNF-. To determine the role of autophagy or lysosome and ROS inglutamate and TNF-induced cells death, the lysosome or autophagy inhibitors andROS inhibitors were used to test whether they can block the glutamate andTNF-induced cells death.Results:1) Glutamate induced cell death in MEF and HT22cells, and caspaseinhibitor z-VAD enhanced the cell death.2) Necrostatin-1can block glutamate inducedcell death in MEF and HT22cells, while the cell death was unable to be blocked by theknockdown of RIP1.3)The deletion of RIP3, which regulates necroptosis throughinteracting with RIP1, had no effect on glutamate induced cell death, but suppressed TNF-induced necroptosis in MEF cells. Moreover, RIP3RNAi did not blockglutamate induced death of HT22cells.4）The knockdown of AIF exerted no effect ongultamate induced death in MEF and HT22cells.5) Glutamate induced death of MEFand HT22cells was greatly blocked by autophage/lysosome inhibitors such asbalfilomycin-A1(BFA),3-Methyladenine(3-MA).6) ROS inhibitors such as butylatedhydroxyanisole(BHA), N-acetylcysteine(NAC)significantly suppressed both glutamateinduced death and TNF-induced necroptosis in MEF and HT22cells.Conclution: Taken together, these data not only provided the first evidence that thetarget of necrostatin-1protecting glutamate-mediated toxicity of MEF and HT22cellswas not RIP1, but also demonstrated that glutamate-mediated toxicity of MEF or HT22cells was independently of RIP1/RIP3signaling pathway. We further pointed thatglutamate induced cell death depended on autophagy or lysosome, and ROS may beinvolved in the downstream of the pathway.