Study On The Role Of Ferroptosis In Neurotoxicity Induced By Subchronic Exposure To Aluminum In Rats

Objective:Aluminum（Al）is a metal element widely present in daily life including drinking water,food additives,and aluminum-containing drugs.It is closely related to neurodegenerative diseases for its strong pro-oxidant activity.Excessive aluminum would cause neurotoxic effects,interfere with nerve conduction,and induce oxidative damage to cells.The brain is sensitive to reactive oxygen species（ROS）and the oxidative damage mainly manifest as lipid peroxidation for its high metabolic rate and low antioxidant capabilities.Ferroptosis is a newly definited cell death.It is characterized by the accumulation of iron,lipid hydroperoxide,and ROS together with its metabolites in the cytoplasm,and it is simultaneously affected by the peroxidation of polyunsaturated fatty acids on the membrane,resulting in the loss of selective permeability of the cells plasma membrane and lesion.Aluminum can strengthen the damage of transition metal such as iron to the body and produces hydroxyl radicals,which injures the lipid molecular membrane,and finally manifests as the body’s oxidative damage.Studies have shown that amino acid metabolism pathways such as the regulation of the Xc-/GSH/GPX4 axis,lipid metabolism pathways such as the regulation of ACSL4,Keap1/NRF2pathway,and ferritin autophagy pathway can effectively regulate intracellular iron and ROS formation,and further regulate ferroptosis.Studies have suggested that aluminum may cause ferroptosis in nerve cells,but the specific mechanism is still unclear.Based on the previous work of our researching group,we established sub-chronic aluminum exposure rat models.The rats’learning and memory ability,and ferroptosis-related features such as the ultrastructure of neurons and synapses,iron deposition,and lipid peroxidation levels,and the related m RNA and protein levels of the Sirt1/NRF2,amino acid and lipid metabolism,ferritin autonomy pathways induced by aluminum exposure were evaluated by using neurobehavioral,morphological,biochemical and molecular biology techniques.Resveratrol and deferiprone were used to interfere with the above indicators.The possible mechanisms of ferroptosis in neurotoxicity induced by subchronic aluminum exposure were explored to clarify the possible mechanism and intervention measures of ferroptosis in cognitive impairment caused by aluminum.Methods:In this study,we established sub-chronic（12 weeks）aluminum exposure rat models through drinking water.The rats’learning and memory were detected by Morris water maze system;ROS content in hippocampus were measured with ELISA kit;Iron deposition in hippocampus were detected by using Prussian blue staining method and iron,MDA and GSH/GSSG content in hippocampus were detected by kit method;Real-time PCR method was used to detect Sirt1,Nrf2,Gpx4,Slc7a11,Fth1 mRNA levels of rat hippocampal nerve cells;SIRT1,NRF2,HO-1,GPX4,FTH1,NCOA4,SLC7A11,P53,ACSL4 protein levels were measured by Western Blot.Ultrastructure of neurons was detected by transmission electron microscope.Simultaneously,the Sirt1 agonist resveratrol and iron chelator deferiprone were used to intervene to detect the role of different regulatory pathways of iron death in the impairment of learning and memory induced by aluminum.Results:1.Hippocampal coefficient:Change of hippocampal coefficient in each group was exhibited;2.Learning and memory test:With the increase of AlCl₃exposure,in Morris water maze test,the escape latency for rats increased,and learning ability declined during training period;In the positioning navigation test,it showed a trend of increasing escape latency of the aluminum-stained rats,the swimming distance increases,and the swimming path becomes more complicated;In the space exploration test,the tendency of the aluminum-stained rats to find the platform decreases,and the search strategy changes;The number of entering the target quadrant is reduced,as well as the staying time in the target quadrant is reduced;3.The ultrastructure of hippocampal neurons in the AlCl₃exposure group changes:With the increase of the AlCl₃exposure dose,the mitochondria in hippocampal neurons appear obvious feature of ferroptosis such as outer membrane breakage and membrane density increase and disappearance of cristae,and ultrastructure damage of hippocampal neurons and nerve synapses;4.With the increase of AlCl₃ exposure dose,the tissue iron deposition in rat hippocampus increases.5.The levels of ROS,GSH/GSSG,as well as the MDA in the AlCl₃ exposure groups increased.6.SIRT1/NRF2 pathway changes:SIRT1,NRF2 transcription and protein expression levels decreased,HO-1 protein levels decreased;7.Xc-/GPX4 pathway changes:P53protein levels increased,SLC7A11,GPX4 transcription and protein levels decreased;9.Lipid metabolism pathway changes:ACSL4 protein levels increased,9.Ferritin autophagy pathway changes:FTH1 transcription and protein levels decreased,and NCOA4 protein levels decreased;10.After pretreatment with resveratrol or deferiprone,the above-mentioned damage was reduced.Conclusion:1.Sub-chronic aluminum exposure could impair the rats learning and memory ability;2.Aluminum exposure can damage the neurons and neural synaptic ultrastructure,leading to neuronal ultrastructures with ferroptosis characteristics;3.Aluminum exposure could cause iron deposition in the hippocampus of rats.4.The oxidative injury in rat hippocampus and lipid peroxidation are aggravated after aluminum exposure;5.After treating with deferiprone or resveratrol,the above-mentioned damage was improved;6.The SIRT1/NRF2 pathway,the Xc-/GPX4 pathway,the lipid metabolism pathway,and the ferritin autophagy pathway all play certain roles in ferroptosis caused by aluminum exposure.