The Role Of Endoplasmic Reticulum Stress And ATF3 Pathway In Ferroptosis Of Hippocampal Nerve Cells Induced By Lanthanum Exposure

Objective:Rare earth elements（REEs）are 17 metal elements in Ggroup IIIB in the periodic table,with atomic numbers of 21,39 and 57～71.Studies have shown that REEs enter the body through the food chain,causing damage to the body’s nervous system,and the IQ level and memory of children in rare earth area are significantly lower than normal children.Lanthanum（La）is relatively active in nature and is often used to study the neurotoxic mechanism of REEs.Ferroptosis is an iron-dependent regulatory cell death driven by lipid peroxidation as a result of an imbalance between cell metabolism and REDOX homeostasis.Activating transcription factor 3（ATF3）is a stress-induced transcription factor,whose expression can be rapidly induced by cellular stress,and which further regulates the expression and transcription of downstream genes.Er stress can be activated by La,and when ER stress occurs,ATF3 is upregulated.This study investigated whether La promoted the upregulation of ATF3 through influencing endoplasmic reticulum stress level,and then up-regulated nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4）to promote reactive oxygen species（ROS）.Meanwhile,down-regulation of cystine/glutamate antiporter system（System Xc^-）leads to depletion of cysteine and glutathione（GSH）,resulting in enhanced lipid peroxidation,thus promoting ferroptosis and exerting neurotoxic effects.This study will provide new research data for the prevention and treatment of neurotoxic damage caused by La and other neurological diseases.Methods:Using the experimental clean-grade healthy adult Wistar rats purchased by the experimental animal center,16 female and male rats were each with a weight of 245±10g,and the ratio of male and female was 1:1.Female clean healthy adult Wistar rats were completely randomly divided into control group,low,medium and high dose LaCl₃groups.From the 0th day of pregnancy,the rats in the control group drank distilled water,and the pregnant rats in the low-dose,medium-dose and high-dose LaCl₃groups drank distilled water solutions containing 0.125%,0.25%and 0.5%LaCl₃,respectively,until the rats were weaned and the infection was terminated.The Morris water maze test was used to detect the learning and memory ability.Ultrastructural changes of rat hippocampal neurons were observed by electron microscopy.Ferritin staining was used to observe the ferritin of rat hippocampal neurons.The expression of ATF3 protein in rat hippocampal neurons was observed by immunofluorescence assay.The expressions of ROS,MDA,GSH/GSSG and Fe in the hippocampus were detected by the kit.And the Western blot test was used to detect the expressions of endoplasmic reticulum stress-related proteins（PERK,GRP78,ATF4）,ATF3,NOX4,SLC7A11,GPX4 and mitochondrial electron transport chain complex proteins（NDUFB8,SDHB,UQCRC2and ATP5A）in the hippocampus of offspring rats.Wistar rats within 24 h of neonatal were cultured in vitro primary neuronal cells,and after maturity,medium containing 0,0.025,0.05 and 0.1m M LaCl₃was given for 24h,and the relevant indexes were detected.Neuron-specific enolase（NSE）was used to identify neurons.The expression of Fe and MDA in neurons was detected by the kit.Western blot was used to detect the expressions of PERK,GRP78,ATF4,ATF3,NOX4,SLC7A11,GPX4,NDUFB8,SDHB,UQCRC2 and ATP5A proteins in primary neurons.Results:1.In the positioning navigation test of Morris water maze,the evasion latency period and swimming distance of offspring rats increased with the increase of LaCl₃exposure dose.In the results of space exploration experiments,the number of offspring rats crossing the target platform and the residence time of the target quadrant decreased with the increase of LaCl₃exposure dose.2.With the increase of LaCl₃dose,the morphological changes of neurons,nuclear membrane shrinkage,mitochondrial swelling and vacuolation were observed by electron microscopy,and mitochondrial morphological changes were obvious.3.With the increase of LaCl₃dose,ferritin staining results showed an increase of ferritin in the hippocampus of neonatal rats.4.With the increase of LaCl₃dose,the kit results showed that the expressions of ROS,MDA and Fe increased in the hippocampus,while the expressions of GSH/GSSG decreased in the hippocampus.5.With the increase of LaCl₃dose,immunofluorescence results showed that the expression of ATF3 in CA1,CA3 and DG regions of hippocampus increased.6.LaCl₃exposure can up-regulate the expression levels of PERK,GRP78,ATF4,ATF3 and NOX4 proteins in hippocampus,and down-regulate the expression levels of SLC7A11,GPX4,NDUFB8,SDHB,UQCRC2 and ATP5A in hippocampus.7.LaCl₃exposure up-regulated the expression levels of MDA and Fe in primary cultured neurons.8.LaCl₃exposure can up-regulate the expression levels of PERK,GRP78,ATF4,ATF3 and NOX4 proteins in primary cultured neurons,and down-regulate the expression levels of SLC7A11,GPX4,NDUFB8,SDHB,UQCRC2 and ATP5A proteins in primary cultured neurons.Conclusion:1.LaCl₃exposure during pregnancy in female rats caused iron death of neurons in the hippocampus,resulting in impaired learning and memory ability of offspring rats.2.The ferroptosis induced by LaCl₃exposure may be related to the endoplasmic reticulum stress and upregulation of ATF3 signaling pathways.