Effects Of Psychological Stress On Hepatic Iron Metabolism And Its Mechanism

Iron is essential for DNA synthesis and key metabolic processes in living organisms. The levels of iron in cell must be delicately balanced, as iron loading leads to free radical damage by the Fenton reaction. Iron mal-regulation may be induced by infection and inflammation, the reasons of which need to be studied. A series of iron-regulatory proteins, including iron transporters and soluble mediators have been identified, which have greatly enhanced our understanding of iron metabolism. Hepcidin has been shown to play a central role in the regulation of iron metabolism. Hepcidin knockout mice develop iron overload in the liver, pancreas and heart, whereas mice overexpressing hepcidin display severe iron deficiency and anemia. Iron overload increases and iron deficiency decreases the expression of hepcidin in the liver. Hepcidin is also regulated by inflammatory signals. However, the role of hepcidin in psychological stress is unknown.Recently, the burden of social life and work is so heavy that people suffer psychological stress frequently, which is considered to be the causes of many disorders, such as hypertension, gastric ulcer, and hyperthermia. Our previous findings have demonstrated that repeated psychological stress exposure could decrease the serum iron level and inhibit erythropoiesis [6]. Because the liver plays a central role in iron metabolism, the hepatic transferrin receptors tend to be responsible for the systemic physiologic changes. The disturbance of iron metabolism is related with many diseases, such as iron deficiency anemia and chronic hepatitis. So it is important to investigate the effect of psychological stress on liver and to understand the molecular mechanisms under iron metabolism.Design and MethodsAnimalsSprague-Dawley (SD) rats, male, aged 10 weeks (Shanghai-BK Co., Ltd) were caged individually at room temperature and 55±5% humidity. They were feed on with a standard diet and tap water freely. After 7 d adaptation, the rats were randomized into two groups: the psychological stress group and the control group, each group of which was subdivided into 1 d group, 3 d group, 7 d group and 14 d group. Pieces from the right part of liver were rapidly dissected and snap-frozen. For anti-IL-6 antibody experiments, rats were either received normal sodium or anti-rat IL-6 monoclonal antibody(2μg /d, R&D Systems Inc, USA) by intraperitoneal injection daily before psychological stress exposure. All animal studies were in accordance with institutional animal care guidelines and were approved by the animal research committee at the Second Military Medicine University, Shanghai, China.Psychological stress exposureThe communication box paradigm was used equipped with a grid floor composed of stainless steel rods. Plastic plates were placed on half of the grid floors for electric insulation. The rats (foot shock group) in compartments without plastic plates received foot shock without a warning signal. The rats (psychological stress group) in compartments with plastic plates did not receive foot shock, but received emotional stimuli from the rats in the foot shock group. All rats were exposed to the psychological stress for 30 min every morning, at 10:00-10:30. Compared with the stressed rats, the control rats were placed individually in the compartments with plastic plates and without exposure to any stress.Iron analysisBlood was sampled by retro-orbital phlebotomy into heparinized tubes before the rats were humanely killed. Quantification of serum and liver iron content was measured using a Varian SpectrAA-220G graphite furnace atomic absorption spectrophotometer, equipped with a GTA 110 atomizer, programmable sample dispenser, and deuterium background correction.ELISA and Western blot analysisHepatic tissue was homogenized and lysed for ELISA and western blots. TFR1 and ferritin (R&D Systems Inc, USA) were analyzed using commercially available ELISA kits. Western blotting was performed with rabbit polyclonal anti-mouse TFR2 (Santa Cruz, Inc.). Identical samples were blotted with anti-β-actin (Sigma) polyclonal antibody to keep the amount of loading protein equal. Immunoreactive bands were detected by goat polyclonal anti-rabbit-HRP antibodies (Santa Cruz, Inc.).Measurement of superoxide dismutase (SOD) activity, malondialdehyde (MDA) and NTBI concentrationsThe thiobarbiruric acid (TBA) method was used to measure the liver MDA level with MDA-586 kit (Nanjing Jiancheng Bioengineering Institute). SOD activity was measured with kit-WST (Dojindo Laboratories). For NTBI determination, the liver tissue homogenates were analyzed using bathophenanthroline disulfonate (BPS) to chelate ferrous iron, thus forming a complex which could be analyzed with spectrophotometry. Commercially available BPS (4, 7-diphenyl-1,10-phenanthroline disulfonate) and ferrous ammonium sulfate [(NH4)2 Fe(SO4)2 ] of the highest purity (Sigma) were used in the measurement.Perl's stainingSamples were fixed in aqueous formaldehyde solution (buffered 4% vol/vol) and were embedded in paraffin. The liver sections were stained with Perls' Prussian blue to assess the non-heme iron deposition.Statistical AnalysisSPSS 12.0 software (SPSS institute, Chicago, IL, USA) was used for statistical analysis. Student's t-test was used to determine whether differences were statistically significant in groups. Data were expressed as (X|-)±SD. P <0.05 represents statistically significant difference.ResultsPsychological stress leads to hepatic iron accumulationPsychological stress exposure increased hepatic ferritin and decreased TFR1 expression as early as 7 days (p<0.05) in rats. No significant difference was detected on 3 day between control group and psychological stress group. On contrary, TFR2 expression was up-regulated by psychological stress exposure in liver as early as 3 days. As displayed in figure 2, we revealed that liver tissue SOD activity and MDA level in the psychological stress group were higher than that in control group (p<0.05); simultaneously, the hepatic NTBI levels in the psychological stress group were increased obviously compared to that in the control one (p<0.05). Control group and 3 days psychological stress group did not show iron overload, whereas at 7 and 14 days some iron deposition was observed, principally in hepatocytes and macrophages. Liver structure was normal.Cytokine and iron metabolism response to psychological stressIL-6 and TNF-αproduction increased since 3 days after psychological stress exposure in rat liver. There was no significant difference in levels of IL-10. Western blot analysis documented that rat liver displayed an up-regulation of hepcidin and down-regulation of ferroportin on 3 days and 7 days in response to psychological stress. Furthermore, rat duodenal displayed a down-regulation of ferroportin and apparent absorptivity of iron decreased on 3 days and 7 days in response to psychological stress.Anti-IL-6 antibody inhibits the effect of psychological stress on iron distribution and iron metabolismAfter rats received intraperitoneal injection of anti-rat IL-6 antibody, psychological stress exposure didn't significantly changed liver iron store or serum iron level on 3 days and 7 days compared with control group. Compared with NS-treated group, anti-rat IL-6 antibody significantly decreased liver iron store on 7 days and increased serum iron level of psychological stress rats on 3 days. Anti-rat IL-6 antibody down-regulated hepcidin and up-regulated ferroportin expression in liver induced by psychological stress on 3 days and 7 days.The present study suggests that psychological stress leads to hypoferremia, which is regulated by IL-6-hepcidin axis. Inhibition of iron absorption and increased liver iron store contribute to hypoferremia. The hepatic TFR2 was up-regulated by psychological stress in rats which may contribute to the increased hepatic iron accumulation as haemosiderin, ferritin and NTBI, and increasing products of lipid peroxidation. However, the mechanism that how the psychological stress induces iron mal-regulation still needs to be investigated.