| Objective: Stress is the combined response of the body to risk through the participation and regulation of nerves,endocrinology and other systems.By properly adjusting the metabolic level of each system,the body can increase its ability to stabilize its internal state,thereby effectively reducing the influence of risk factors on the body,but excessive stress can cause damage to the body and affect the body’s normal psychological and physiological functions.Iron death is one of the hotspots of research in recent years at home and abroad.The pre-research of our team shows that restraint stress can cause pathological damage in multiple brain regions,but whether iron death plays a role in stress brain injury is unknown.Studies to date have shown that stress causes a loss of equilibrium of iron homeostasis in the brain.Iron is a necessary trace element in the human body and participates in a variety of biological processes.In 2012,Dr.Brent Stockwell’s laboratory formally defined iron death as a novel cellular programmatic death caused by iron-dependent oxidative damage,distinguished from apoptotic cell autophagy.Iron death has the characteristics of reduced mitochondrial membrane area and reduced ridges,increased iron content in cells,and accumulation of lipid ROS in cells.Mitochondrial changes and intracellular lipid ROS accumulation can also be observed in stress brain injury.Therefore,we speculate that iron death is involved in the occurrence of stress brain injury.Therefore,the purpose of this experiment is to investigate the change of expression of iron death-related signaling pathway protein in the process of stress brain injury and provide morphological basis for the study of stress brain injury mechanism.Methods:1.The 30 C57BL/6 mice were divided into acute group,chronic group and control group according to random numerical table.Mice in acute group were bound for 16 hours a day for 3 days.The chronic group was bound 6-8 h a day for 3 weeks;The control group did nothing.The stress model was successfully established by observing three indexes,namely,the behavioural changes in the mice after stress,the expression of glucocorticoid receptors in the hippocampus,hypothalamus and cortex,and the weight changes in mice before and after stress.Biochemical determination of GSH,MDA and iron ions in the serum of mice.HE staining observed routine pathological changes in the hippocampus,cortex and hypothalamus of mice.The morphological changes of NIS in three cerebral nerve cells of hippocampus,cortex and hypothalamus were observed in mice with toluidine blue staining.The pathological changes of ultrastructure of hippocampal tissue in mice were observed by transmission electron microscopy.IHC staining observed the expression changes of iron death-related proteins TF,TFR,DMT1,SLC7A11 and GPX4 in the hippocampus,cortex and hypothalamus of mice,and the semi-quantitative analysis of protein expression using Aperio.Scan Scope.CS2 panoramic scanner.The expression of iron death-related proteins TF,TFR,DMT1,SLC7A11 and GPX4 in the hippocampus and hypothalamus of mice was detected by Western-blast method.RT-qPCR was used to detect the expression of TF,DMT1 and GPX4 in hippocampal tissue in mice.2.Results Processing and Statistical Analysis: Data collation in this experiment was done by Excel 2016,and statistical analysis was performed by SPSS 23.0 statistical software.The data of glutathione,malondialdehyde,and iron ions in the blood of biochemical assays conforming to the normality test and chi-square test were expressed as mean ± standard error(mean ± SEM),and the comparison of the means of each group was performed by one-way ANOVA with the least significant difference(LSD).The two-way ANOVA was used to compare the mean values of immunohistochemical staining intensity between the experimental group and the control group in the hippocampus,hypothalamus and cortex,and the t-test of two independent samples was used to compare the positive intensity of immunohistochemical staining between the three brain regions.The above statistical results were considered statistically significant at P < 0.05.Results:1.Results of Weight Change and Behavioral Experiments:Weight statistics showed that the final weight was lower than the initial weight in the acute and chronic bondage group and higher than the initial weight in the control group.The weight loss in the acute and chronic bondage group was significantly greater than that in the control group.The results showed that the total moving distance of mice in acute and chronic bondage group was significantly lower than that in control group.The cumulative duration of the acute group decreased significantly compared with that of the control group,and there was no significant difference between the chronic group and the control group.2.Biochemical test results:In the experimental group,the content of glutathione(GSH)in hippocampus and hypothalamus decreased significantly compared with the control group,while the content of malonaldehyde(MDA)increased.The content of iron ions in mice serum was significantly lower than that in control group.3.HE staining results:In the acute group,the nerve cells in hippocampus were obviously edema,the gap between tooth gyrus and peripheral tissues was enlarged,the contours of cells were not clear,some cells were concentrated,a small amount of intracellular chromatin was missing and the staining became lighter.Part of the cortical nerve cell edema,cytoplasmic red-stained red neurons number more,clear nucleus structure disappears;Part of the hypothalamus nerve cells are mildly edema and immobilized.All three brain regions in the chronic group showed mild nerve cell edema,and a few red neurons appeared in the cortex.No significant pathological changes were seen in control group mice.4.Tissue-specific(toluidine blue stain)Results:The results of toluidine blue staining showed that the nerve cells in the three brain regions of the experimental group had different degrees of edema,Nitidite disappeared,and the pathological changes of cell cement concentration were observed.5.Immunohistochemical staining results:5.1.Immunohistochemical results of stress indicators in the hippocampus,hypothalamus and cortexThe overall positive expression of mice in acute and chronic bondage group was significantly higher than that of control group in slices containing hippocampus,hypothalamus and cortex.5.2.Immunohistochemical results of iron death-related protein indexes in hippocampus,hypothalamus and cortex5.2.1.Expression of TF in hippocampus,hypothalamus,cortexThe immunohistochemical results of TF in hippocampus of mice showed that the expression level of acute and chronic restraint group was significantly higher than that of control group.The results of hypothalamus showed that the expression level of acute and chronic bondage group was significantly higher than that of control group.The cerebral cortex showed that the expression level of acute bound group was significantly higher than that of control group.5.2.2.Expression of TFR in hippocampus,hypothalamus,cortexThe immunohistochemical results of TFR in the hippocampus and cortex of mice showed that the expression level of acute and chronic binding group was significantly higher than that of control group.The immunohistochemical results of hypothalamus showed that the expression level of acute bondage group was significantly higher than that of control group.5.2.3.DMT1 Expression in hippocampus,hypothalamus,cortexDMT1 Immunohistochemistry in the hippocampus,cortex and hypothalamus of mice showed that the expression levels of acute and chronic bondage were significantly higher than those of control groups.5.2.4.SLC7A11 Expression in hippocampus,hypothalamus,cortexSLC7A11 immunohistochemical results in hippocampus of mice showed that there was no significant difference in expression level of acute and chronic restraint group compared with control group.Results from cerebral cortex and hypothalamus showed that the expression level of acute and chronic bondage group decreased significantly compared with control group.5.2.5.GPX4 Expression in hippocampus,hypothalamus,cortexGPX4 immunohistochemical results in hippocampus of mice showed that the expression level of acute and chronic restraint group was not significantly different compared with control group.The results of cerebral cortex and hypothalamus showed that the expression level of acute and chronic restraint group decreased significantly compared with control group.6.Western-Blot Results:6.1.Expression of TF in hippocampus and hypothalamusThe WB results of TF in hippocampus of mouse brain showed that the expression level of acute and chronic bondage group was significantly higher than that of control group.WB in hypothalamus showed that the expression level of chronic group was higher than that of control group.6.2.Expression of TFR in hippocampus and hypothalamusThe WB results of TFR in the hippocampus and hypothalamus of mice showed that the expression level of chronic group was significantly higher than that of control group.There was no significant difference in expression levels in the acute group.6.3.DMT1 Expression in hippocampus,hypothalamusDMT1,the expression level of chronic group was higher than that of control group,and there was no significant difference between acute group and control group.The WB results of hypothalamus showed that the expression level of acute and chronic bondage group was significantly higher than that of control group.6.4.SLC7A11 Expression in hippocampus and hypothalamusSLC7A11 WB results in hippocampus of mice showed that the expression level of acute and chronic restraint group was significantly lower than that of control group.The results of hypothalamus showed that the expression level of chronic group was significantly lower than that of control group.6.5.GPX4 Expression in hippocampus and hypothalamusGPX4 results of WB in hippocampus of mice showed that the expression level of acute and chronic restraint group decreased significantly compared with control group.The results of hypothalamus showed that the expression level of chronic group was lower than that of control group,and there was no significant difference between acute group and control group.7.RT-qPCR results:The expression of TF mRNA did not differ between the experimental group and the control group.mRNA expression increased DMT1 the three-day group,and there was no difference between the three-week group and the control group.The three-day and three-week groups of GPX4 mRNA were lower than those of the control group.8.Transmission Electron Microscopy Results:The ultrastructures showed that intracellular mitochondrial swelling occurred in the three-day group and the hippocampal tissue in the three-week group compared with the control group.Conclusions:Based on the successful establishment of the C57BL/6 mouse bondage stress model,the pathological changes of hippocampus,cortex and hypothalamus and the molecular mechanisms of pathological changes were investigated.1.Bind stress mice lost weight,behavioral activity decreased,the hippocampus,hypothalamus,corticosteroid receptor content increased,suggesting the success of the stress model.2.HE staining,toluaniline blue staining and transmission electron microscopy of the hippocampus,hypothalamus and cortex of the bondage stress group showed that the pathological changes of neural cells were obvious.3.Binding stress can lead to increased mRNA and protein expression of TF,TFR,DMT and decreased antioxidant reaction GPX4 and SLC7A11.It is suggested that iron death is involved in stress-induced nerve cell damage. |
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