Effects Study Of ATF3 Inhibiting Xc~- System In MeHg-induced Ferroptosis In Brain Nerve Cell

Objective:Methylmercury（MeHg）is an environmental accumulation pollutant mainly present in the aquatic environment with strong neurotoxicity.MeHg accumulates in brain tissue upon entry into the body and causes damage to the central nervous system（CNS）.Ferroptosis,an iron-dependent form of non-apoptotic cell death,is associated with the abnormal expression of ATF3 and p53 and the dysfunction of Xc^-system,and Xc^-system is regulated by p53 and ATF3.MeHg was found to induce ferroptosis.However,the mechanism of ATF3,p53,and Xc^-systems in MeHg-induced ferroptosis remains unclear.In this study,the molecular mechanism of iron death induced by MeHg in brain nerve cells was studied through animal in vivo animal experiments and in vitro cellular experiments,providing an experimental and theoretical basis for the prevention and control of MeHg poisoning.Methods:Animal model of MeHg exposure was established using Kunming mice.Seventy-two eight-week-old mice were randomly divided into four groups:control group,4μmol/kg MeHg group,8μmol/kg MeHg group,and 12μmol/kg MeHg group,with 18 males and females in each group.Gavage was administered once a day at 10:00am for 4 weeks.The body weight was weighed and recorded every other day during the course of contamination;the neurobehavioral damage of mice was detected by open field test and paw grip test;pathological damage of mouse cerebral cortex and neurons observed by HE and Nissl staining;the ROS content was detected by flow cytometry;the kits measured the tissue iron,Glu,GSH,GSSG,GSH/GSSG and MDA contents were measured by flow cytometry,and RT-qPCR and Western blotting were used to detect the m RNA and protein expression levels of ferroptosis and ATF3/p53 pathway related indicators.Cell models of MeHg exposure was established using HT22 cell.HT22 cells were cultured at 37°C in a constant temperature incubator with 5%CO₂.The first part of the experiment included 4 groups:control,Fer-1 control,MeHg,MeHg+Fer-1.Cellular activity was measured by CCK-8,cellular ROS level by flow cytometry,and ferroptosis-related protein expression level in cells by Western blotting.The second part of the experiment included 4 groups:control,control si RNA,MeHg,ATF3 si RNA+MeHg.Western blotting detected the expression levels of ATF3,p53and SLC7A11 proteins in the cells,and double immunofluorescence staining detected the co-localization of ATF3 and p53 in the cells.Results:1.MeHg exposure caused abnormal nervous system function and pathological brain damage in mice.After MeHg exposure,mice showed slowed body weight gain;decreased spontaneous mobility and active exploration;and decreased limb strength.Histopathological changes were observed in the mouse cerebral cortex with HE staining and neuronal pathological damage was observed with Nissl staining.2.MeHg induced ferroptosis in neuronal cells.After MeHg exposure,mice showed increased iron content in cerebral cortex and increased iron ion deposition in tissues;increased MDA content;increased ROS levels;significantly lower protein and m RNA expression levels of ferritin,GPX4 and SLC7A11.In HT22 cells,ROS levels were increased in the methylmercury group compared with the control group;however,protein expression of cell viability,ferritin,GPX4,and SLC7A11 were decreased.Cell viability and ROS levels as well as protein expression of ferritin,GPX4 and SLC7A11were reversed in the methylmercury+Fer-1 group compared with the methylmercury group.3.ATF3 inhibits the expression of the Xc^-system through the p53 pathway during MeHg-induced ferroptosis.After MeHg exposure,the expression levels of ATF3,p53protein and m RNA were increased in mouse cerebral cortex;Glu content was increased;GSH,GSSG and GSH/GSSG content were significantly decreased.In HT22 cells,compared with the control group,the expression levels of ATF3,p53 protein were increased in the MeHg group;SLC7A11 protein expression level was decreased;the co-localization area of ATF3 and p53 was increased in the HT22 cells.p53 protein expression levels were elevated;SLC7A11 protein expression levels were decreased;co-localization of ATF3 and p53 in cells was increased.Compared with the MeHg group,ATF3 si RNA+MeHg group showed decreased ATF3 and p53 protein expression levels;SLC7A11 protein expression level was increased;co-localization of ATF3 and p53 in cells was decreased.Conclusion:The mechanism of ferroptosis induced by MeHg exposure is related to the increased expression of ATF3.Increased ATF3 expression inhibits SLC7A11expression through a p53-dependent pathway,thereby inhibiting the Xc-system.Finally,ferroptosis was induced in brain nerve cells...