| Neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD)and Huntington’s disease,are a group of diseases characterized by the progressive loss of neurons,leading to dysfunction of the central nervous system(CNS)and body behavior.Neurodegenerative diseases are caused by a series of complex functions,such as oxidative stress,excitatory amino acids and gene.Glutamate,an important excitatory transmitter in the central nervous system,which plays an important role in neuronal differentiation,migration,growth and survival.Glutamate is one of the vital factors that leading to neuronal damage,and the mechanism involved in neuronal death is very complex.When excessively released,glutamate induces both receptor-dependent excitotoxicity and non-receptor-mediated oxidative toxicity.Interestingly,emerging evidence indicates that oxidative glutamate toxicity in neurons and Ferroptosis share in a series of common changes.Ferroptosis is a recently recognized form of regulated cell death and is morphologically,biochemically,and genetically distinct from apoptosis,necrosis,and autophagy.The process of Ferroptosis is characterized by the accumulation of lethal reactive oxygen species(ROS).Thus,we think that the oxidative glutamate toxicity could induce the occurrence of neuron Ferroptosis,and influent oxidative inbalance of the body,ultimately lead to neurodegenerative diseases.All in all,the way and mechanism of the effect of glutamate on neuronal cell death is worth studying.In this study,we will investigate the role and possible mechanism of Ferroptosis in the model of HT-22 cells injured by glutamate.AIM:(1)To investigate the occurrence of oxidative damage on HT-22 cells injury induced by glutamate;(2)To study the relationship between glutamate induced neuronal cell death and Ferroptosis;(3)To investigate the protective effect and mechanism of Ferrostatin-1 on HT-22 cells injury induced by glutamate.Methods:HT-22 cells were treated with glutamate to establish injury model,MTT and Trypan blue staining assay was used to detect cell viability to establish the best suitable concentration of the model.We also investigated the effects of different inhibitors Z-VAD-FMK,3-Methyladenine,Necrostatin-1,Ferrostatin-1 and iron chelator DFO by MTT assay.LDH kit to detect LDH leakage rate;Inverted microscope and transmission electron microscopy was were used to observe cell morphology;Biochemical methods were used to analyze GSH,GPX,MDA,SOD activity;Release of cytosolic and lipid reactive oxygen species(ROS)were analyzed by the flow cytometry;the change of mitochondrial membrane potential and MDC level were detected by the flow cytometry;the morphological changes of cells were observed by fluorescence microscopy after DAPI staining;Western blotting to analyze expression of Ptgs2,Nrf2,GPX4,FP1 protein;RT-qPCR to analyze level of Ptgs2,GPX4 mRNA.Results:1.Results of MTT and Trypan blue staining assay indicated that growth inhibition rate of HT-22 cells nearly 50% after treated with 5 mM glutamate for 24 h.2.The study of cell morphology showed that 5 m M glutamate damage to HT-22 cells for 24 h,cells became smaller and round,the refractive index decreased,cells adherent cells showed poor damage state.3.Results of MTT assay showed that Ferrostatin-1 and DFO with different concentrations on injured HT-22 cells induced by glutamate showed a certain degree of protection,compared with the model group;The morphology observation results showed the morphology of 3-12μM Ferrostatin-1 treated cells was closer to the normal cells.4.Observing the cell morphology after staining DAPI,there was no significantly difference between 3-12μM Ferrostatin-1 group and model group in rate of apoptosis;5.Transmission electron microscopy test results found that after Glutamate treatment,the mitochondria structure was small significantly and its number was decreased,the density of its membrane were increased;3-12μM Ferrostatin-1 treatedcells was closer to the normal cells.6.MDC dyeing experiment results found that there were a large number of green fluorescent dot gathered marked by MDC in the cells with glutamate treatment while green fluorescent dot aggregation don’t change obviously in the cell with Ferrostatin-1.Ferrostatin-1 could inhibited glutamate-induced dissipation of the mitochondrial membrane potential using the JC-1 in HT-22 cells.7.Ferrostatin-1 ameliorated glutamate-induced oxidative stress,the intracellular and lipid ROS was analyzed by H2DCF-DA and BODIPY-C11 assay.When HT-22 cells pretreated with Ferrostatin-1,followed by exposure to glutamate,GSH level was significantly increased and GPX activity was significant increased as compared with the glutamate group(P<0.01).8.Western blot analysis indicated that Ferrostatin-1 decreases the expression of Ptgs2 protein as compared with model group.RT-qPCR indicated that glutamate could promote the expression of Ptgs2 gene as compared with control group.And Ferrostatin-1could decrease it.9.Western blot analysis indicated that Ferrostatin-1 concentration-dependently increases the expression of Nrf2/HO-1/GPX4 and FP1 protein.10.RT-qPCR results indicated that,comparing with control group,Ferrostatin-1treated group can significantly up-regulate expression of GPX4 mRNA induced by glutamate.Conclusion:Taken together,these findings suggest that glutamate is probably by increasing cytosolic and lipid reactive oxygen species(ROS),decreasing mitochondrial membrane potential and GSH-GPX level,inhibiting SOD and MDA activity,and inducing intracellular oxidative stress,upregulate the Ferroptosis-related proteins Ptgs2,causing cell Ferroptosis.Ferrostatin-1 shows an apparent protective effect on Ferroptosis of HT-22 cells injury induced by glutamate within a certain dosage range,its protective effect and mechanism may be related to reduce the releasing of ROS to prevent theoccurrence of oxidative stress,protect the normal structure of cell mitochondrial membrane,stabilize the level of mitochondrial membrane potential,decrease expression of Ptgs2 and up-regulated expression of GPX4. |
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