Effects Of Various Physical Stresses On Brain Iron Metabolism And Its Mechanisms In Rats

Iron is one of the essential metal elements for human,which can be part of various iron-containing enzymes and participate in many physical activities.Adequate iron is of great importance for organisms to grow,differentiate and multiply.But excess iron can be deleterious.Iron can catalyze the production of hydroxyl radical through Fenton reaction(Fe²⁺+H₂O₂→Fe³⁺+·OH+OH^-),aggravating oxidative stress in the body and resulting in damage to tissues and cells.Brain iron metabolism is well controlled homeostasis.From serum iron crossing the blood-brain-barrier to neural cells uptaking iron,each process is strictly regulated. Brain iron homeostasis is mainly maintained through coordinating of expression and function of many iron metabolism related proteins.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 associated with death of neurons.But how iron is redistributed and accumulates in the brain is still not elucidated.Previous studies showed that brain iron content of animals was altered after some physical stresses such as exposure to high temperature,exercise and motion sickness. This is supported by experiments of our research group.All these results suggested that various physical stresses can alter brain iron homeostasis.To test this idea,we should determine the effects of various physical stresses on brain iron metabolism and explore the way in which physical stress affects brain iron metabolism.Results from these experiments will provide theoretical and experimental support for treatment of some neurodegenerative diseases characterized by misregulation of brain iron metabolism.The procedure of this study was as followed.Part 1:Effects of various physical stresses on iron metabolism in rat brainObjective:To determine the effects of various physical stresses on iron contents of whole rat brain and various regions of rat brain.Methods: 1.Establishing rat physical stress modelThree physical stress model:foot shock(FS),Water-immersion restraint(WRS) and trauma(TR) stress model.Foot shock stress model:the rats were administered 30 min's foot shock(90 V, 0.80mA) between 8:00AM and 10:00AM in the morning for successive 7 days(referring to Communication Box Stress Model).Water-immersion restraint stress model:the rats were bound to restrict motion of extremities and head.The rats were immerged into water bath(19℃±1℃) vertically with sternoxiphoid just above the water for 6 hours for successive 7 days.Trauma stress model:the rats were amputated of right hind limb after anesthesia by 10%Chloral Hydrate solution.2.GroupingHealthy adult SD rats(body weight:120±10g) were used as experimental animals and randomly assigned to control(CTL),foot shock(FS),water-immersion restraint(WRS) and trauma(TR) groups.Rats were raised in SPF-compliant breeding room with a temperature of(24±1)℃and humidity of 50%～60%.Rats were raised individually in stainless steel cages with access to feeds and water ad libitum and in an 12/12 hours circadian rhythm.Rats of three different physical stress group were treated for successive 7 days while rats from control group received no treatment.After the last stress treatment, rats of all four groups were anesthetized by 10%Chloral Hydrate solution.Blood were collected from the hearts and centrifuged.Rats were perfused with cold saline through the heart and brains were removed.Some of the brains were dissected into such regions as prefrontal cortex,hippocampus,striatum,brain stem and cerebellum.Tissue samples were immediately put into liquid nitrogen.3.Atomic absorption spectrophotometry determination of iron content of whole rat brain and various regions of brainWhole and various regions of rat brain were weighted,1:20(wt/v) diluted with 20mmol/l HEPES buffer and homogenated.30 uL homogenate was mixed with equal volume of ultra pure nitric acid and digested in a 50℃warm water bath for 48 hours. Iron standard solution(50mg/L) was diluted with 5%nitric acid and used in plotting of standard curve.Blank and sample tubes were successively measured for 3 times with atomic absorption flame spectrophotometry and absorbance at 248.3 nm was recorded for further analysis. Results:1.Compared to control group,all three physical stress groups showed no significant change in iron content of whole rat brains;there is on significant difference in iron content of whole rat brains between either two of these three physical stress groups.2.Compared to control group,FS,WRS and TR groups showed an increase of 37.1%,34.5%and 25.3%in iron content of prefrontal cortex,respectively(P＜0.05) with no significant difference in iron content of prefrontal cortex between either two of these three physical stress groups;Compared to control group,FS,WRS and TR groups showed an increase of 58.3%,77.4%and 95.2%in iron content of hippocampus,respectively(P＜0.05) with no significant difference in iron content of hippocampus between either two of these three physical stress groups;Compared to control group,FS,WRS and TR groups showed an increase of 70.7%,75.2%and 61.2%in iron content of striatum,respectively(P＜0.05) with no significant difference in iron content of striatum between either two of these three physical stress groups;there was no significant difference in iron content of cerebellum or brain stem between either two of all four groups.Part 2.Effects of physical stress on important iron metabolism related proteins in rat brainObjective:To determine the effects of physical stress on important iron metabolism related proteins in rat brain and give a primary explanation of how brain iron homeostasis was disrupted.Methods:1.Animal model:Foot shock stress model(see Part 1).2.ELISA determination of ferritin and transferring receptor in rat hippocampus:Hippocampus from both control and FS groups(n=10) was weighted,added to RIPA lysis buffer containing protease inhibitors,phosphotase inhibitors and PMSF and homogenated at 4℃.Homogenates was centrifuged at 10000 g for 30 min.and supernatant was collected and stored at -70℃.Protein concentration was determined with BCA protein quantification kit.ELISA tests were carried out according to instruction of test kits to determine the content of ferritin and transferring receptor.3.Western blot analysis of ferritin,transferring receptor,DMT1 and Lf in rat hippocampus:Hippocampus extracts 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: 3000 diluted mouse anti-human TfR antibody,1:3000 diluted rabbit anti-rat DMT1(with IRE) antibody,1:3000 diluted rabbit anti-rat DMT1(without IRE) antibody,1:1000 rabbit anti-rat Lf antibody and 1:1000 mouse anti-humanβ-actin antibody for 12 hours at 4℃. Blots were washed 3 times with TBST 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 Image J software.Results:1.Results of ELISA tests showed a decrease of 57.4%in content of ferritin and an increase of 75.0%in content of TfR respectively in rat hippocampus of FS group compared with control group(P＜0.05).2.Results of Western blot analysis showed decrease in ferritin expression and increase in TfR expression and no change in expression of DMT1(with or without IRE) and Lf in rat hippocampus of FS group compared with control group.Part 3:Effects of physical stress on iron regulatory mechanism in rat hippocampusObjective:To determine the expression of iron regulatory proteins at transcriptional and translational levels and give a primary explanation of how iron metabolism was affected by physical stress in rat hippocampus.Methods:1.Animal model:Foot shock stress model(see Part 1).2.Real-time PCR determination of IRP1å’ŒIRP2 mRNA:RNA was extracted from rat hippocampus of both control and FS groups(n=3) with Trizol reagents according to the instructions of manufacturer.20ug RNA was treated with 10 units of DNase I(TAKARA) for 30min at 37℃.cDNA was synthesized with Oligo dT primers after purification of total RNA.1 ul cDNA was added to a PCR system of 30ul containing 27.5ul Real Time PCR Master Mix,15pmol primers and 7.5pmol TaqMan probe(primers and probes were designed with Primer Premier5.0 software).3.Western blot analysis of IRP1 in rat hippocampus:Hippocampus extracts 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 rabbit anti-rat IRP1 antibody and 1:1000 mouse anti-humanβ-actin antibody for 12 hours at 4℃.Blots were washed 3 times with TBST 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 Image J software.Results:1.Results of Real-time PCR analysis showed an increase of 57.0%(P＜0.05) in IRP1 mRNA level and no change in IRP2 mRNA level in rat hippocampus of FS group compared with control group.2.Results of Western blot analysis showed increase in IRP1 expression in rat hippocampus of FS group compared with control group(P＜0.05).Part 4:Study on transcriptional regulation of IRP1 gene in rat hippocampus after physical stressObjective:To determine the transcriptional regulation of IRP1 gene in rat hippocampus after physical stress through bioinformatic analysis of rat IRP1 gene promoter and determination of activities of some transcription factor related to IRP1 gene expression.Methods:1.Animal model:Foot shock stress model(see Part 1).2.Prediction of potential transcription factors binding sites in IRP1 gene promoter:Human,mouse and rat IRP1 genes were retrieved in PubMed website. Promoter region of IRP1 gene(-2000 to +100 bp) was analyzed with TESS(Transcription Element Search System) software(http://www.cbil.upenn.edu/tess/) and TRANSFAC database(http://www.gene-regulation.com/pub/databases.html#transfac) for potential transcription factors binding sites and conserved sites were picked out.3.Array test of activity of transcription factors:Tests were carried out with Panomics protein/DNA array kit according to the manufacturer's instruction.Briefly, nuclear proteins were extracted from rat hippocampus with Panomics Nuclear Extraction Kit according to manufacture's instruction.After quantification of protein concentration, 10ug nuclear extract was incubated with 10ul TranSignal probe mix.Transcription factors attached to biotin-labeled oligonucleotide were separated and collected with Panomics spin columns.Oligonucletides attached specifically to transcription factors were eluted and incubated with TranSignal array membranes at 42℃.The membranes were washed and incubated with 20μl HRP-labeled streptavidin.Throw off excess water and image with X-ray films.Scan the films,save the images as pictures and analyze them with ScanAlyze software.4.EMSA tests:EMSA tests of GR and STAT5 were carried out to verify the results of Protein/DNA array tests.Nuclear extracts were separated with non-denaturing polyacrylamide gel electrophoresis(SDS-PAGE) and electrically transferred to nylon membranes.The membranes were incubated with specific GR or STAT5 DNA probes. After cross-linking with 254nm ultraviolet light,blocking and washing,the membranes were incubated with chemiluminescent reagents.After exposure,development and fixation, X-ray films were scanned and analyzed with Image J software.Results:1.Prediction of potential transcription factors binding sites in IRP1 gene promoter:After analysis of the promoter with TESS software and TRANSFAC database, we found some well-matched conserved binding sites including ones responding to GR and STAT5.2.Panomics Protein/DNA array tests:results of array tests showed 32 transcription factors including GR and STAT5 with binding activities 2 times higher in FS group than that in control group and 124 transcription factors with binding activities 2 times lower in FS group than that in control group.3.EMSA tests:Comparing the results of Protein/DNA array tests with the results of analysis of potential transcription factors binding sites in IRP1 gene promoter,we found two transcription factors(GR and STAT5 ) with 2 times higher binding activities in FS group than that in control group responding to respective conserved binding sites in IRP1 gene promoter.EMSA tests showed binding activities of GR and STAT5 with specific DNA sequences were enhanced in FS group than that in control group,consistent with results of array tests.Conclusion:This study was designed to(1) determine effects of various physical stresses on iron metabolism in rat brain,(2)find out effects of physical stress on important iron metabolism related proteins in rat brain,(3)understand effects of physical stress on iron regulatory mechanism in rat hippocampus and(4)study the transcriptional regulation of IRP1 gene in rat hippocampus after physical stress.The results showed:1.Compared to control group,FS,WRS and TR groups showed a significant increase in iron content of prefrontal cortex,hippocampus and striatum(P＜0.05) with no significant difference in iron content of prefrontal cortex,hippocampus and striatum between either two of these three physical stress groups;there was no significant difference in iron content of cerebellum or brain stem between either two of all four groups, indicating that various physical stresses can alter iron homeostasis in rat brain,resulting in accumulation of excess iron in such regions as prefrontal cortex,hippocampus and striatum;2.Physical stress resulted in a significant decrease in ferritin expression and a significant increase in TfR expression while no significant change in Lf and DMT1(with or without IRE) expression in rat hippocampus,indicating that physical stress induced up-regulation of TfR and down-regulation of ferritin expression resulting in increased iron uptake and decreased capacity of iron storage was the reason why iron accumulated in some regions including hippocampus in rat brain;3.Physical stress resulted in a significant increase of IRP1 gene expression at both transcriptional and translational levels while no significant change in IRP2 gene expression, indicating that post-transcriptional regulation of IRP1 maybe was reason of increased expression of TfR and decreased expression of ferritin in rat hippocampus after physical stress;4.Physical stress resulted in enhanced DNA binding activities of two transcription factors(GR and STAT5) which can specifically bind to conserved sequences in IRP1 promoter,indicating that activation of binding activity of GR and STAT5 to specific DNA sequences may be the reason of enhanced gene transcription of IRP1 in rat hippocampus after physical stress.The results of this study suggested that physical stress may enhance the transcriptional regulation of GR and STAT5 on IRP1 gene,resulting in higher expression of IRP1 and consequently higher expression of TfR and lower expression of ferritin.Finally,this may lead to disturbed iron homeostasis featuring iron accumulating in some regions of rat brain including prefrontal cortex,hippocampus and striatum.However,further study is needed to explore the exact signaling pathways leading to disrupted iron homeostasis after physical stress.