Effects Of Psychological Stress On Mechanisms Of Iron Storage And Release In The Cell Of Rat Brain

Iron is essential trace metal for almost all living organism by participating in a wide variety of metabolic process.Adequate iron is of great importance for organism growth,cell differentiation and multiply.Iron homeostasis is critical for normal brain growth and physiologic function.But excess iron can be deleterious.Iron can catalyze the production of hydroxyl radical through Fenton -reaction(Fe²⁺+H₂O₂→Fe³⁺+OH +OH-),and the Haber-Weiss reaction(Fe³⁺+O₂→Fe²⁺+O₂;Fe²⁺+H₂O₂→Fe³⁺ +OH+OH-(iron catalayzed)) aggravating oxidative stress in the body and resulting in damage to tissues and cells,more and more researches indicate that excess iron accumulation in the body will bring a huge damage,especially iron overload in the central nervous system.Hence,the brain have elegant systems for mobilization and of iron sequestration.From serum iron crossing the blood-brain-barrier to neural cells uptaking iron,each process is strictly regulated.It has been demonstrated that in some neurodegenerative diseases such as Alzheimer disease(AD),Parkinson's disease(PD) and Huntington's chorea,iron was redistributed and accumulated in some regions of brain.It is believed that oxidative stress resulting from excessly high iron content and defects of anti-oxidation defense in the brain is the key point to death of neurons.But how iron is redistributed and accumulates in the brain is still not elucidated.With the progress of related research, more and more evidence to support the idea that iron efflux was also played a vital role in iron homeostasis regulation.Previous researches including some experiments results our office did showed that motion sickness,sports and fatigue can make iron redistribution in the rat body.The facts indicate that mental stress may be one of reasons for the brain iron metabolism disorders.Our previous study on psychological stress rat reveal that some brain region with iron overload was up-regulated in IRP1 by enhancing the transcriptional regulation of GR and STAT5 on IRP1 gene,resulting in higher expression of IRP1 and consequently higher expression of TfR.We presume that psychological stress not only changes the iron uptaking but also changes the iron storage and iron efflux.The procedure of this study was as followed. ObjectiveTo study the characteristic effects of psychological stress on brain iron storage proteins and efflux proteins and establish a useful experimental basis for further study involving how stress changes cell normal iron homeostasis and the consequent effects on physiological function of the human body.Method1.Effects of psychological stress on NHFâ…¡(nonheme ferrous iron )and NHFâ…¢(nonheme ferric iron) concentrations in the rat brain1.1 To divide experimental animals into groupsAll experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University. Guidelines and Policy on using and caring of the laboratory animals were followed at all time.Male SD rats(120±5g body weight) fed with a standard diet were purchased from the Shanghai-BK Ltd.Co,and were housed individually in a cage in a temperature-controlled room(24±1℃,55±5%humidity) with a 12-hour light and 12-hour dark cycle.After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),psychological stress group(PSG) and the control group(CG). Each rat was exposed to stress for 30 minutes every day,successive 7 days.1.2 To build psychological stress model of SD ratsUsing a communication box system,footshock stress(FS) and psychological stress(PS) were administered to the rats.The communication box was divided into two parts with a transparent acrylic board,i.e.,Part A including ten rooms with a plastic board-covered floor for electric insulation and part B including ten rooms with a metal grid-exposed floor.Rats in part B were administered an electrical shock through the floor(90 V,0.8 mA for 1 second) randomly for 30 min,90 times in total, and then exhibited a nociceptive stimulation-evoked response such as jumping up, defecation and crying.Thus they were exposed to systemic(physical) stress.Rats in part A were not directly administered the electrical shock,but were exposed to psychological stress in response to the actions of the rats in Room B. 1.3 Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry in rats brian1.3.1 Perfusion-Perls methods supplemented by DAB intensification for NHFâ…¢histochemistry in rats brianAfter successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH7.4) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferrocyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.1.3.2 Perfusion- Turnbull methods supplemented by DAB intensification for NHFâ…¡histochemistry in rats brianAfter successive 7 days ps treatment,The animals were anesthetized with intraperitoneal pentobarbital sodium(50 mg/kg) and transcardially perfused with the 130 ml of phosphate buffered saline(PBS,pH 7.4 ) containing heparin(5 units/ml)for 5 min,to flush out the blood,then 800 ml of the solution(pH 1.0) containing 1% potassium ferricyanide and 4%PFA in distilled water was perfused for tissue fixation and insute stain.After the perfusion,the brain was excised and cut off.To prepare 5 um thick sections,paraffin embedding and tissue cutting were done by the conventional methods.The sections were mounted on silan-coated slides,dewaxed and hydrated for further treatments.2.Effects of psychological stress on brain iron storage protein and iron release protein2.1 Determination of Ferroportin and Hephaestin mRNA levelsReal-time PCR determination of Ferroportin and Hephaestin mRNA:RNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase 1 for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1 ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg²⁺ 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).2.2 Western blotting analysis of Ferroportin and Ferritin expressionDissected tissues from the cortex,hippocampus and striatum from both control and PS groups(n=3) were homogenized separately by a dounce homogenizer in lysis buffer.Each group extractions containing 50ug protein was separated with 10% nondenaturing polyacrylamide gel electrophoresis(SDS-PAGE) and electrically transferred to nitrocellulose membrane.Blots were dyed with ponceau red solution to show whether protein had been transferred to the membrane.After blocking with skimmed milk for 2 hours,blots were incubated with 1:1000 diluted mouse anti-human ferritin antibody,1:2000 diluted rabbit anti-rat Ferroportin antibody and 1:2000 mouse anti-humanβ-actin antibody for 1 hours at room temperature.Blots were washed 3 times with PBST before incubated with HRP-labeled secondary antibody for 1 hour at room temperature.Blots were detected with ECL reagents and gel images were analyzed with quantity one software.3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux proteinDetermination of Hepc mRNA and IL-6 mRNA levelsRNA was extracted from rat cortex,hippocampus and striatum of both control and PS groups(n=5) with Trizol reagents according to the instructions of manufacturer. 20ug RNA was treated with 10 units of DNase I for 30min at 37℃.First strand cDNA was synthesized with M-MuLV Revers transcriptase after the purification of total RNA being incubated with Oligo dT primers.1ul cDNA was added to a PCR system of 25ul containing 10×PCR buffer 2.5ul,25mM Mg²⁺ 3.0ul,10mM dNTP mixture 0.5ul,5uM Taq DNA polymerase 0.5ul,10×SYBR-green I fluorescence dye 0.7ul,5uM forword primer and reverse primer 2.0ul and DNAase free water 12.8ul.(primers were designed with Primer Premier5.0 software).4.Statistical analysisPerfusion-Perls and -Turnbull methods for nonheme iron histochemistry image were analyzed by image-pro plus 6.0 software.Microscopic examination in ervry section and choose site as follows:Brain sub-district for cortex,Polymorphic cell layer for hippocampus and CPu region for striatum.Eight visual fields were selected by random.Accouding to the mean object gray scale and object area density,PU(positive units )were defined.The quantity of PU represent stained iron ion.Western-blot data were analyzed by Quantity One software.Real-time PCR data were analyzed by rotor-gene 6.0.14 software.All results were expressed as mean±S.D.Statistical analysis was carried out by using SPSS 13.0 in independent sample T test method.All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses.A P value less than 0.05 was considered statistically significant.Results1.Psychological stress exposure increased both NHFâ…¢and NHFâ…¡stained positive units levels in some brain regions1.1 Effects of psychological stress on NHFâ…¢concentrationsWe found that the NHFâ…¢stained positive units levels in the frontal cortex, hippocampus and were significantly higher in the PS exposure group than in the control group(P＜0.05);however,no significant higher was observed in NHFâ…¢stained positive units levels striatum between the two groups.(P＞0.05) The neurons of CA1,CA2 region in PS exposure group hippocampus were significantly stained stronger than in the control group,a great quantity of NHFâ…¢stained positive units were deposit in the area between anterior horn of lateral ventricle and frontal lobe,where in exposure group is significant higher than in the control group.1.2 Effects of psychological stress on NHFâ…¡concentrations We found that the NHFâ…¡stained positive units levels in the frontal cortex, hippocampus and striatum were significantly higher in the PS exposure group than in the control group(P＜0.05).The CA1,CA2 region in PS exposure group hippocampus were stained stronger than that in the control group,the structure of pyramidal neuron cell boby were demolished can be observed,as well as the quantity of pyramidal neuron were diminished.2.Effects of Psychological stress on brain iron storage protein and iron efflux protein2.1 Psychological stress exposure caused changes in Ferroportin,Hephaestin mRNAReal time-PCR analysis showed that PS exposure decreased Ferroportin mRNA levels in the cortex,hippocampus(P＜0.05) but increase Ferroportin mRNA in the striatum significantly(P＜0.05).The Hephaestin mRNA level of PS exposure group was significantly lower than that in the control group(P＜0.05),though the decrease was not significant in the striatum.2.2 Psychological stress exposure caused changes in Ferroportin and Ferritin levelsWestern blot analysis showed that Ferroportin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but a little higher in striatum(P＞0.05);Ferritin concentrations levels in the cortex and hippocampus were significantly lower than those in the control group (P＜0.05),but little higher in striatum(P＞0.05)3.Effects of molecular mechanisms of psychological stress on brain iron storage protein and iron efflux protein3.1 Effects of psychological stress on brain Hepcidin mRNA expressionReal time-PCR analysis showed that the level of cortex Hepcidin mRNA expression both in PS exposure group and control group were extremely higher than that in the hippocampus and striatum.PS exposure increased Hepcidin mRNA levels in the cortex significantly(P＜0.05),decreased in hippocampus(P＞0.05 )and no obvious changed in striatum. 3.2 Effects of psychological stress on brain IL-6 mRNA expressionReal time-PCR analysis manifested that Psychological stress exposure increased IL-6 mRNA levels in the cortex,hippocampus and striatum.significantly(P＜0.05)Conclusions1.Our study was built the psychological stress rats model by means of the Communication Box System.Take the advantage of the perfusion-Perls and -Turnbull methods supplemented by DAB intensification(+ DAB),we demonstrated visualization of nonheme ferric and ferrous iron in situ sensitively.We found that the NHFâ…¢stained positive units levels in the frontal cortex,hippocampus and were significantly higher in the PS exposure group than that in the control group,as well as the NHFâ…¡stained positive units levels.Furthermore,the structure of pyramidal neuron cell body were demolished can be observed in CA1 and CA2 region.All of these suggested psychological stress on rat brain lead to iron overload in specific encephalic region including cerebral cortex and hippocampus.The increasing of oxidative stress by NHFâ…¡may be the reason to resulting the changes of hippocampus CA1,CA2 region.2.We found that Ferritin concentrations levels in the cortex and hippocampus of psychological stress group were significantly lower while the Hephaestin mRNA levels and the Ferroportin concentrations levels and mRNA likewise.According to our previous research,psychological stress can evaluate iron intaking in cortex and hippocampus.Considerable iron ion was imported via DMT1,keeping themselves in divalence state.In cytosol,ferrous ion is considered to loosely complex with low-molecular weight organic bases forming the so-called low-molecular weight Iron pool(LIP),which contains some ferric iron as due to cytosolic oxidation.Because of its reactivity and cytotoxicity,the LIP size is maintained in very small amount.We speculate that LIP was expanded,meanwhile,ferritin(which has a ferroxidase core in H chain) and hephaestin(ferroxidase)were reduced,ferrous iron could not be oxidized into ferric iron immediately for storage in ferritin or final use in mitochondria and other organelles.Finally,ferrous iron was auto-oxidized to trivalence state and in a abnormal deposit form in cell.3.Ferroportin is the only iron efflux protein can export the iron out of cell,which process need hepaestin facilitating.We found both of them were decreased in cortex and hippocampus in the psychological stress rat brain.The iron export was blocked.As a result,iron was sequestered in neuron and glia cell.4.Our data showed that the psychological stress induced interleukin-6 in rat brian, interleukin-6(IL-6) appears to stimulate the Hepcidin,which is the important upstream regulator of ferroportin.Hepcidin binds to ferroportin,inducing its internalization and degradation,iron export is blocked and iron is trapped within.We believe that interleukin-6 was the initial factor contribute to brain iron overloaded.In conclusion,we found in the present study that the contents of iron and ferrous iron were increased in the cerebral cortex,hippocampus,and partial in striatum of rats exposed to psychological stress,accompanied by intense oxidative stress response and disorder of brain iron storage and efflux.We believe that as a systemic nerves -internal hormone—immunological network,psychological stress was against to stimulant,also influence the organism homeostasis,including iron metabolism.It may be the important reason resulting in brian dysfunction after excessive psychological stress.