HCP1Expression And Mechanism Research In The Brain Of Phychologically Stressed Rats

BackgroundAs the society rhythm speeding up, people face all kinds of psychologicalstress.Fencing Stress has become a hot topic in current researches. Our department earlyfound that psychological stress can lead to some parts of brain iron accumulation, andbrain iron accumulation will induce cell oxidative stress,DNA or other damage.Existingstudies have shown that the brain iron overload is closely related to a variety of centralnervous system degenerative diseases, such as Parkinson’s disease, alzheimer’s disease.Butso far, regulatory mechanism of the brain iron absorption via psychological stress isunclear.There are two kinds of iron in organism，inorganic iron and bonding iron. bondingiron include protein bonding iron,enzyme iron and so on,but the major is heme bongingiron.According to report,it has been suggested that hameorrhage stroke can generate redblood cells to release hemin,which may be responsibleoxidative damage and death.Heme carrier protein1(HCP1) was found recently,it was confirmed to transportheme into cell.more research found that the rat primary astrocyte can express HCP1anduptake hemin,more hemin will cause cell damage and death.It is significant that our department early gene chiPresearch results show that thepsychological stress rats after liver heme carrier protein1(HCP1) higher abnormalexpression. iron accμMulation of rats brain whether involved in HCP1is unclear.Thisarticle is to illustrate the problems in the following research.ObjectiveTo observe the influence of psychological stress to HCP1expression of some brainregions and the iron in tissues,to confirm HT-22can uptake hemin via HCP1, On this basisto explore the metabolism of HCP1shifty expression by psychological stress.Method1. The HCP1expression and the iron content in some brain regions via Ps1.1copy psychological stress rat model1.1.1laboratory animal groupsAdult male SD rats, weight about120g.According to the weight, randomlydivided into blank control group, the psychological stress group, foot shock group.Animal breeding laboratory environment temperature is24℃or so, humidity is50%to60%;Using single cage, the free food and drink.1.1.2methodModel is composed to twenty area by transparent resin plate, divided into twodifferent area of the cross. one bottom is insular, another is the electricity. Two area arecrossed.psychological stress group in insular area, around are electric group.after thepower supply, electric group exclaimed, jump, the psychological stress group byobserving the electric shock group of rats.power supply at9am for30min,about80V,7days at all.1.1.3hormone levels in rats after psychological stressuse the radioimmunofocus assays kit.1.2iron content of some brain regions after psychological stressanesthesia immediately after the completion of the model, cut bilateral hippocampusand cortex and striatum tissues.Sample digestion with wet digestion method, and addwith deionized water extractly. using The air-acetylene flame atomic absorption methodto determin the content of Fe, calculate results according to the standard curve of themeasurement. Iron content in the tissue is showed by ng/mg tissue.1.3heme carrier protein HCP1expression of rats brain regionsAccording to the instruction using Trizol reagent extraction in the rat hippocampus,cortex, striate, total RNA.The control group and stress group rats hippocampus andcortex, striate HCP1mRNA expression levels in the body are tested by RT-PCR method(real-time quantitative polymerase chain reactin,real-time PCR). control and stress grouprats HCP1content in hippocampus and cortex are measured by Protein immunoblotmethod (Western blot).2. whether hippocampus nerve cell HT-22can transport heme via HCP12.1iron content of hippocampus nerve cell HT-22via hemeCells culture with DMEM containing10%fetal bovine serum nutrient solution.offerheme with different concentration for different time. Use CCK-8to detect the activity ofcell after given the heme of different concentration for different time.Cells cracking withNaOH and be measured total protein by BCA method, atomic absorptionspectrophotometer to measure total iron content in the cell.Iron content in the cell isshowed by nmol/mg protein. 2.2whether heme can enter into HT-22cell by using ZnPPIX insteadCells cultured as above. when cell growed to about80%,offer ZnPPIX for2h,thenfixed with formaldehyde for30min after cell washed,add anti-fluorescence perish,afterthat watch by autofluoresence.3. mechanism of HCP1expression by psychological stress3.1observe HCP1mRNA expression regulation of transcription factors inpsychological stress ratHCP1transcription factor content changes of hippocampus and cortex Using Trizolreagent on the extraction of total RNA following the reagent instruction, real-timefluorescent quantitative PCR method is used to determine the KLF4、NRF1、CDX2、YY1mRNA expression level of psychological stress rat hippocampus and cortex,striate.Western blot method to detect KLF4content of psychological stress rats’hippocampus and cortex.3.2CORT influence HCP1expression via KLF4in HT-223.2.1the influence of HCP1expression on HT-22by CORTCells culture with DMEM at37℃in cultivation box, containing10%fetal bovineserum.Cells are given CORT of different concentration (0μM、1μM、10μM、15μM、30μM)and culture36h. First，detect cell activity after given the heme of differentconcentration for different time.measure HCP1mRNA expression level by RT-PCR.Western blot method to detect HCP1protein content in cells after intervene.3.2.2the impact of KLF4by CORTCell culture as the same as above, the experimental group cells were joined CORT30μM for different time(0~24h), real-time fluorescent quantitative PCR method tosurvey KLF4mRNA expression levels in the control group and interventiongroup.Western blot method for KLF4protein content in the control group andintervention group.3.2.3whether CORT impact HCP1after add siKLF4Design three siRNA by jima company,901,1331,1379and Nc as control.then eddthis three siRNA and Nc to cell,then extract the total RNA,use RT-PCR to detectKLF4mRNA,1331was the best one, which is the lowest down to10percent. After usingsiRNA1331interference KLF4intervention group for24h, add30μM CORT in intervention group and control group and continue incubation for24h. Real-timefluorescent quantitative PCR method for HCP1mRNA expression level. Westernblotting analysis of HCP1protein expression.3.2.4whether CORT impact HCP1after blocking the glucocorticoid receptor in HT-22cellsHT-22cells use concentration10μM RU486incubation, join30μM CORT1h laterfor24h incubation, Western blot method to detect HCP1protein content in the cell.3.3whether CORT effects iron content via KLF43.3.1the effects of iron content on hippocampus nerve cells HT-22a.Cells culture as before,then incubation with30μM CORT for24h, after that add30μM heme to the intervention group, then after cracking cells with NaOH solution,BCA method measuring the total protein in the cell, air-acetylene flame atomicabsorption spectrophotometer to measure total iron content in the cell.b. using ZnPPIX instead to wacth the iron content of HT-22cell after covered withCORT.Cells culture as before,then incubation with30μM CORT for24h, after that add30μM ZnPPIX to the intervention group for2h.then fixed with4%formaldehyde for30min,after cell washed,add anti-fluorescence perish,after that watch by autofluoresence.3.3.2the effects of iron content by CORT after interference of KLF4Intervention group cells use siRNA interference KLF4after24h, all cells are joinedCORT for24h, intervention group and control group continue incubation after joinedheme30μM incubat for0.5or1h.After the completion,using the BCA method tomeasure cell total protein, atomic absorption method to detect iron content in the cell.Statistical analysisQuantity one analysis system analyse the gray level image of Westernbelt.Differences between the two groups using the t-test method of group design data,multiple sets of difference between the One-way ANOVA namely single factor analysisof variance. P <0.05was considered significant for all tests, P <0.01was very significantdifference. P <0.001instead extremely significant difference.Result1. The expression of HCP1in Psychological stress rats 1.1hormone levels in ratsCompared with control group, CORT increased significantly in PS rats (P <0.05).1.2iron content in Psychological stress ratsCompared with control group, the iron content extremely significant increased in therat hippocampus (P <0.001), cortex iron content also increased significantly (P <0.01),striate body iron content also has increased, but the difference was not significant (P <0.05)1.3HCP1expression in some brain regions of Psychological stress ratsReal-time fluorescent quantitative PCR results showed a significant rise ofHCP1mRNA expression levels in Ps rats hippocampus about3times (P <0.001). in thecortex HCP1mRNA expression level significantly increased about seven times (P<0.001);Western blot results show that psychological stress group rats hippocampus HCP1content increased very significantly (P <0.01).cortex HCP1content increasedsignificantly (P <0.05).2.whether HCP1can transport heme in HT-22Total iron content in the cell increases with heme concentration and time (P <0.01).ZnPPIN is accumulated in cell.3. expression change mechanism of HCP1by Psychological stress3.1observe results of HCP1mRNA transcription factor regulationReal-time fluorescent quantitative PCR results show that KLF4mRNA significantlyhigher in the rat hippocampus and cortex (P <0.01);Western blot showed KLF4proteincontent of Ps rats was significantly higher than the control group in hippocampus andcortex of (P<0.01).Other related transcription factor has no obvious difference.3.2whether CORT can affect HCP1via KLF43.2.1After incubation with CORT, cell activity has no change after givenCORT(P>0.05) and the increasing trend of total iron content in cells (P<0.05).Intracellular HCP1mRNA expression level increased significantly with CORTconcentration and the extension of time (P <0.01).3.2.2Real-time fluorescent quantitative PCR results showed that after intervention of CORT,intracellular KLF4mRNA expression levels were significantly increased (P <0.01).Western blot showed intracellular HCP1and the content of KLF4interventiongrouPwas obviously increased (P <0.01).3.2.3After interference of KLF4, HCP1mRNA raised effect was cancelled (P>0.05)3.2.4after block glucocorticoid receptor, corticosterone on the hippocampal nervecells HCP1expression had no obvious effect (P>0.05).3.3CORT affect iron content via KLF4in HT-22after given CORT,iron content increased obviously（P<0.05）,but after interferenceof KLF4, iron content in cell had no obvious change (P>0.05).Conclusions1、The Psychological stress can cause rat hippocampus, cortex, striate obvious irondeposit; Along with HCP1expression significantly enhanced.2、the iron content of HT-22increase with the concentration and time,combain therhodamine picture,it means HT-22can transport hemin via HCP1. HT-22may increaseiron content in the cell through the intake of heme iron.3、the expression of KLF4increased overtly in rat hippocampus, cortex, striate,othertranscription factors have no change,cell experiment confirmed that CORT can impactHCP1via KLF4. After incubation of CORT, the iron content obviously increased in thecell,but without KLF4, the iron content has no change.means CORT can affect the ironcontent of hippocampal nerve cells HT-22via KLF4.synthesize the result above,we can conjecture that the reseaon of rat some brainregion obvious iron deposit after Psychological stress is CORT may enhanced the HCP1via factor KLF4,then heme iron content in cells increased,at last iron deposit in someparts of brain.