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The Expression of Sfxnl in Psychological Stress Rat Liver and the Influence on Iron-related Molecules

Author: XuManNi
Tutor: LiMin
School: Second Military Medical University
Course: Nutrition and Food Hygiene
Keywords: psychological stress liver iron gene chip sfxn1 TfR1
CLC: R151.2
Type: PhD thesis
Year: 2013
Downloads: 46
Quote: 0
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BackgroundIron is the most abundant transition metal in the body that is mainly present inprotein-bound forms such as heme and non-heme proteins, playing a major role in electrontransfer and oxygen utilization. Iron deficiency and iron overload can occur and may haveserious clinical consequences. The most common disorder associated with iron depletion isiron deficiency anemia,which affects more than30%of the world’s population.At the otherend of the spectrum,iron overload can occur.This transition metal promotes free radicalgeneration through Fenton and/or Haber–Weiss reactions, thus triggering secondary chainreactions in the oxidative modification of lipids, proteins, and DNA in different organs. Soiron metabolism is tightly regulated.The liver is the major storage site for iron.But excessive iron deposition in liver willlead to further injury such as hepatocellular necrosis, inflammation, fibrosis and in somecases even to carcinoma. Hepatic iron overload could contribute to the development ofNAFLD or lipotoxicity.Epidemiological studies have shown that psychological stress may lead to a variety ofdiseases, such as hypertension, ulcer disease, cerebrovascular accident, myocardialinfarction, diabetes, cancer, etc.Our previous studies have shown that psychological stress can increase the liver irondeposition, but the exact mechanism is still not very clear. In this study, The technique ofmicroarray was used to analysis the liver gene expression profiles of psychological stressrat and to screen out iron related genes.We find out that psychological stress can increasesfxn1expression. Sfxn1also known as tricarboxylate carrier protein.It’s a member of thesideroflexin family. It’s located in the mitochondrial membrane. Sfxn1function might beinvolved in the transport of a component required for iron utilization into or out of themitochondria. But it is still unclear the exact role of the sfxn1. We study the sfxn1if it caneffect iron metabolism related molecules, in order to provide new ideas about thepsychological stress cause liver iron overload.ObjectiveIn this study, we use gene chip screen liver differentially expressed genes in PS rats.On this basis to find genes may be involved in iron metabolism of psychological stress, provide a new idea about the psychological stress cause liver iron overload. To clarify thepsychological stress how to lead to liver iron overload.Methods1. Effects of psychological stress on the rat serum iron and hepatic iron concentration1.1Experimental animals48male Sprague Dawley rat(s120±10g)were purchased from Shanghai-BK Ltd. Afteradaptation for5days, the rats were randomized into three groups: control group(n=8),psychological stress groups(futher divided into three subgroup as PS-1,PS-3,PS-7,with8rats in each subgroup),food-shocked group. Psychological stress rats were exposed tostress for30minutes every day. Animal were housed in a temperature-controlled room at24℃under a12-hour light/dark cycle. All animals used in this study received anutritionally-balanced rodent diet with “normal” iron level (35mg iron/kg diet) and waterad libitum. All animal procedures are carried out in accordance with establishedprocedures, Second Military Medical University Animal Care and Use Committee.1.2Build psychological stress model of SD ratsThe communication box was selected as the psychological stress apparatus. It dividedinto20compartments by transparent acrylic board with several small hole. The boardprevented each rat from physical contact but allowed them to receive cues such as visual,auditory and olfactory sensations from the neighboring animals. The compartments weredivided into two parts. Half of all small rooms spread insulation panels (A Room), and theother half do not shop insulated panels (B room). An electric generator with80voltageswas connected to the grid floor to generate electric foot shock.Rats in part B after theshock,exhibited a nociceptive stimulation-evoked response such as screaming,jumping,defecation. Rats in part A were not directly administered the foot electrical shock, butproduced psychological stress.1.3Measurement of serum ACTH、CORT and NE concentrationsSerum ACTH concentration was measured by the enzyme-linked immunoassays(ELISAs) kit; the CORT and NE levels were measured by the radioimmunofocus assays(RI) kit. 1.4Measurement of liver iron concentrationSpecimens were weighed and digested with concentrated nitric-perchloric acid mixture(4to1ratio). Iron estimated by atomic absorption spectrophotometry.1.5Determination of serum ironSerum iron concentration was determined in nonhemolyzed serum samples by atomicabsorption spectrophotometer.1.6Determination of serum Transferrin SaturationAccording to the manufacturer’s protocol,serum iron and total iron bindingcapacity(TIBC) were determined.TS(%)=serum iron/TIBC×100%2. Gene expression analysis of iron related genes in PS rat2.1Experimental animals and treatmentSame as in part1.2.2Gene expression profile analysisThe technique of cDNA microarray was used to screen out iron related genes onpsychological stress rats.Before and1,3,7day psychological stress,total liver RNA wereextracted and detected by Rat Exon1.0ST Array Gene Chip.We analysised the liver geneexpression profiles and then searched the iron related genes use gene ontology description.2.3Real time PCR analysis for hepcidin、TfR1、sfxn1mRNA expressionTotal RNA was extracted from liver tissue using TRIzol reagent and cDNA wasprepared. Real-Time PCR was performed for individual genes using SYBR green detectionsystem. The RT-PCR data were analyzed by standard curve method as described.2.4Western blot analysis for hepcidin、TfR1、sfxn1protein levelsLivers were homogenized with lysis buffer. Proteins were quantified using the kanjiprotein assay kit based on the Bradford method. Aliquots of protein were loaded on10%sodium dodecyl sulfate polyarylamid gel electrophoresis under reducing conditions andsubsequently transferred to a PVDF membrane. The blot were blocked and probed withantibodies.The intensity of the specific bands was detected and analyzed.To ensure evenloading of the samples,the same membrane was proed with rabbit anti rat GAPDH polyclonal antibody.3. The effect of sfxn1on hepatic cell iron-related molecules3.1Cell CultureHepG2(human liver hepatocellular carcinoma cell line) was purchased from TypeCulture Collection of the Chinese Academy of Sciences, Shanghai. HepG2cells weregrown at37°C with5%CO2in DMEM medium supplemented with10%heat-inactivatedfetal bovine serum (FBS),100U/ml penicillin and100ug/ml streptomycin.3.2Sfxn1siRNA transfectionHepG2cells were transfected with sfxn1siRNA as protocol described.3.3Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA levels in HepG2cellsSame as in part2.3.3.4Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2protein level in HepG2cellsExperimental methods see part2.4.4. Statistical analysisStatistical analyses were performed using SPSS software for Windows (version16.0)(SPSS, Inc., Chicago, IL, USA). Data were presented as mean±S.D. All the experimentaldatas were analyzed by independent sample T test method or one-way ANOVA.Aprobability value of P <0.05was taken to be statistically significant.Results1. Effects of psychological stress on the rat serum iron and hepatic iron concentration1.1Determination of the PS model establishmentAfter psychological stress exposure the levels of serum ACTH, corticosterone andNE were significantly higher than those in the control animals (P<0.05),indicating thePS model is establishment successfully.1.2Effect of psychological stress on liver iron concentrationsWe found that rat liver iron concentrations of7day PS group than the control groupincreased significantly (P <0.05), but1day、3day PS exposure groups no differencecompared with the control group. 1.3Effect of psychological stress on serum iron concentrationsCompared with the control group,1d-,3d-and7d psychological stress group serumiron decreased by11.32%,25.53%and29.21%respectivlely, the serum iron levels in7days PS exposure group significantly lower than the control group (P<0.05).1.4Effect of psychological stress on serum Transferrin SaturationCompared with control group,7days psychological stress group serum transferrinsaturation decreased32.2%(P<0.05),1and3days psychological stress group Serumtransferrin saturation fell7.87%,19.85%(P>0.05).2. Gene expression analysis of iron related genes in PS rat2.1Gene expression profile analysisIn the psychological stress rat liver, The significant difference genes number is2246.There are seven genes related with iron metabolism is changed.2.2PS exposure caused changes in hepcidin、TfR1mRNA levelsAfter seven days of psychological stress, rat liver hepcidin mRNA expression levelswere significantly increased(P<0.01), but compared with the control group,1day and3days PS exposure did not change (P>0.05). TfR1mRNA levels were gradully increased in1day,3days and7days PS groups, but7days PS exposure group changed significantly(P<0.01).2.3PS exposure changed hepcidin、TfR1protein expressionAfter7days PS exposure, rat liver hepcidin and TfR1protein levels were significantlyhigher than the control group(P<0.05)..2.4PS exposure increase sfxn1expression in rat liverSfxn1mRNA levels were increased in3days、7days PS exposure compared with thecontrol group (P<0.01). After7days PS exposure, Sfxn1protein levels weresignificantly higher than the control group (P<0.05).3. The effect of sfxn1on hepatic cell iron-related molecules3.1Effect of sfxn1siRNA on sfxn1expression levels in HepG2cellsThe mRNA expression of sfxn1was significantly decreased in siRNA transfection HepG2cell (P<0.05). Sfxn1protein level was significantly decreased in siRNAtransfection group (P<0.01).3.2Sfxn1interference decrease mRNA expression of TfR1、IRP1The TfR1、IRP1mRNA levels decreased in siRNA interference groups compared tocontrol group (P<0.05).But hepcidin、IRP2mRNA showed no difference in siRNAinterference group compared with control group (P>0.05).3.3Effects of sfxn1interference on TfR1、IRP1protein levelsThe TfR1、IRP1protein levels decreased in siRNA interference groups compared tocontrol group (P<0.05).Conclusions1.7days PS could cause iron accumulation in liver.2. The gene chip found Hamp, Tfrc, sfxn1, GDF2,IREB2, NUBP1, NUBP2genes relatediron homeostasis is changed in the process of psychological stress.3. Hepcidin,TfR1and sfxn1expression is rising with the increase in the number of stressdays.4. sfxn1can affect the expression of TfR1Taken together, we found psychological stress can increase transferrin receptor1expression may be by sfxn1,Result in liver cells intake of iron increased, made ironaccumulation in liver and even damage to liver.

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