The Research And Mechanism Of Pilocarpine-induced Neurocyte Ferroptosis

Temporal lobe epilepsy(TLE)is the most common refractory epilepsy in China,which is a typical chronic nervous system disease.Glutamate Neurons in the target area of a patient appear repeated abnormal synchronous firing,resulting in irreversible damage and death after repeated abnormal firing.Due to the lack of effective drugs and means for the treatment of temporal lobe epilepsy,the condition of patients is generally gradually deteriorating,and a large number of nerve cells in the lesion area die in a very complex condition,including ferroptosis,autophagy,cell apoptosis,necrosis and other types.Pilocarpine is a hydrophilic and lipophilic agonist of non-selective muscarinic acetylcholine receptor 1(Mach R1).With small molecular weight,Pilocarpine can quickly penetrate the barrier formed by biofilm and induce significant cell depolarization.The animal temporal lobe epilepsy model constructed by Pilocarpine is one of the internationally recognized modeling methods,which can well simulate the pathological and behavioral characteristics of human temporal lobe epilepsy.The current research results have revealed in more detail the mechanism of various cell death modes,which cell death modes are the first to respond in the focal area of temporal lobe epilepsy patients and whether different cell death modes interact with each other.The Study of these questions is difficult because of the brain’s complex and sophisticated structure and interactions.In order to avoid the research difficulties caused by the complex structures and interactions in the brain,Neuro-2a,BV2 and U251,were treated in vitro with Pilocarpine.The degree of ferroptosis,autophagy,apoptosis and necrosis in different cells were investigated by controlling treatment time and concentration.To explore the mode of cell death induced by Pilocarpine in Neuro-2a cells and the interaction between different modes of death,and to compare the differences between BV2 and U251 in the existing mode of death.The experimental results are mainly divided into the following two parts:Part I: The mode of cell death in Neuro-2a cells induced by Pilocarpine and the interaction between different modes of death.1.Neuro-2a expression of MACHR1 was confirmed by Western blot.2.MTT was used to detect the cell viability curves after Pilocarpine treatment,and two treatments of 3.125 g/L and 6.25 g/L for 6 h and 3.125 g/L for 6 h,12 h and 24 h were determined.3.Detect the protein levels of various cell death modes after a short period of treatment.Compared with the control group,the expression levels of NCOA4,LC3 B and RIP1 were significantly up-regulated.Autophagy and ferritinophagy are activated.4.Detect intracellular LIP and total iron levels.Compared with the control group,the average free iron water in the two treatment concentrations was significantly decreased,and the total iron level in the inclusion was increased.5.Detect the protein levels of iron metabolism-related proteins in the two treatment groups.Compared with the control group,the protein levels of IRP2 and TFR1 were down-regulated,the protein levels of FTL were up-regulated,and the expression of DMT1+IRE was downregulated in the 6.25 g/L group.All are regulated by IRP2 except FTH.6.After extending the treatment time,the protein levels of various death forms after3.125 g/L Pilocarpine treatment were detected.Compared with 0 h,GPX4 was significantly down-regulated at 12 h and 24 h,COX-2 expression was down-regulated at 24 h,and ACSL4 expression had no significant change.LC3 b expression was up-regulated from 6 h and increased significantly with the extension of treatment time.The expression of NCOA4 was up-regulated at 6 h and down-regulated,but there was no significant difference at 24 h.This indicates that ferroptosis has occurred in cells,and autophagy and ferritinophagy are still taking place.7.Detection of intracellular LIP after treatment.Compared with 0 h,the free iron pool level decreased at 6 h,had no significant difference at 12 h,and increased significantly at 24 h.8.Detect the levels of iron metabolism-related proteins after treatment.Compared with the control group,the protein levels of IRP2,TFR1,DMT1+IRE were significantly decreased;The expression of FTL was up-regulated at 6 h,but had no significant change at 12 h and 24 h.FTH decreased significantly at 12 h and 24 h.9.The appropriate concentration of autophagy inhibitor 3-MA was screened by MTT.When the concentration of 3-MA was no more than 40 μM,the cell activity was not affected after treatment for 24 h.10.Ferritinophagy were detected by immunofluorescence double-label LAMP1 and FTH at the presence of autophagy inhibitor 3-MA.3-MA was found to be effective in reducing degradation of Ft H caused by Pilocarpine.11.MTT assay showed that Neuro-2a cell viability was significantly up-regulated after3-MA and Pilo combined treatment,compared with the Pilo treatment group.12.Transcription levels of TNF-α,IL-6 and IL-1β were detected after treatment.Compared with 0 h group,TNF-α and IL-6 were extremely significantly down-regulated at 6h,and then up-regulated with the prolongation of time,but not as much as 0 h group.IL-1βwas significantly up-regulated at 6 h and down-regulated at 12 h and 24 h,but it was significantly more than that at 0 h.Part II: Differentiation of modes of death between Pilocarpine-induced BV2 and U251.1.The expression of MACHR1 in both BV2 and U251 cells was confirmed by Western blot.2.MTT was used to detect the cell viability curves of BV2 and U251 after Pilocarpine treatment at different concentrations and times.BV2 was found to be more sensitive to Pilocarpine treatment.3.The levels of BV2 and U251 necrosis marker proteins in the two treatment groups were detected.Compared with the control group,the RIP1 expression of BV2 was down-regulated in 6.25 g/L group.Both RIP1 and RIP3 were upregulated in U251.This indicated that necrosis was activated in U251,but not in BV2.4.Apoptosis marker protein levels of BV2 and U251 cells in the two treatment groups were detected.Compared with control group,Bax/Bcl2 expression of BV2 was up-regulated in 6.25 g/L group,and activated Caspase-3 was up-regulated significantly.Bax/Bcl2 expression of U251 was upregulated in 3.125 g/L group,while activated Caspase-3 had no significant change.This indicated that apoptosis was activated in BV2,but not in U251.5.The protein levels of ferroptosis marker proteins in BV2 and U251 cells were detected.Compared with control group,ACSL4 expression of BV2 was up-regulated in 3.125 g/L group,and COX-2 expression was down-regulated in 6.25 g/L group.GPX4 expression of U251 was up-regulated,and COX-2 expression was up-regulated in 6.25 g/L group.Suggesting that ferroptosis did not occur and some pathways began to respond in different way.6.The levels of LIP and total iron in BV2 and U251 cells of both treatment groups were detected.Compared with the control group,the LIP of BV2 was significantly up-regulated,and the intracellular total iron level was down-regulated.The LIP of U251 was upregulated in6.25 g/L group,and there was no significant change in intracellular total iron level.7.The levels of iron metabolism-related proteins in BV2 and U251 cells of the two treatment groups were detected.Compared with the control group,IRP1,IRP2 and DMT1+IRE of the two kinds of cells were all down-regulated in 6.25 g/L group.TFR1 was down-regulated in both treatment groups.BV2 was significantly decreased at FTH.FPN1,FTH and FTL of U251 were decreased in the 6.25 g/L group.It indicated the ferritin changes were not regulated by IRP-IRE regulation.8.The levels of ferritinophagy marker proteins of BV2 and U251 in the two treatment groups were detected.Compared with the control group,BV2 and U251 were down-regulated in both treatment groups.It indicates that NCOA4 is consumed.In conclusion,Pilocarpine induced rapid and intense autophagy in Neuro-2a cells,and autophagy-dependent ferritin degradation was rapidly activated.This increases the level of intracellular free iron pool,and at the same time leads to the disorder of iron flow in and out of cells,and the increase of intracellular total iron level.Both induce iron death induced by iron divalent ions.Pilocarpine induced rapid apoptosis of BV2 cells and was more sensitive to the stimulation of Pilocarpine than that of U251.Pilocarpine induced rapid activation of the necrosis pathway of U251,which may be related to its low iron content and low iron intake.In this study,we described the process of ferroptosis in Neuro-2a cells induced by pilocarpine,which was used to construct a classical temporal lobe epilepsy model.We also described the differences form of rapid activation of cell death between BV2 and U251 after pilocarpine treatment,which may provide some new ideas for exploring the treatment of temporal lobe epilepsy in the future.