Discussion Of The Molecular Mechanism Involved In Iron Accumulation In Liver Under Psychological Stress

Iron is one of the essential metal elements for human. When the harm of iron deficiency is well-known today, more and more attention has been paid to iron overload. It was reported that chronic hepatic iron overload could cause fibrosis and cirrhosis, porphyria cutanea tarda and hepatocellular carcinoma.Liver is the center organ of iron metabolism and is particularly susceptible to injury from iron overload. In order to maintain optimal health, there must be a mechanism tightly controlling the concentration and distribution of iron to provide enough to meet cellular requirements while avoiding excessive levels that are toxic. But our previous study has found that psychological stress(PS) can cause iron accumulation in liver, but the mechanism remains unclear.It is well-known that glucocorticoid(GC) is largely secreted under stress. It was shown that GC could regulate the intestinal heavy and light ferritin(Fn) expression in rats. Steroid hormones have also been shown to increase transferrin receptorl(TfR1) expression in the testes of hypophysectomized rats. Our previous study also found that the binding activity of glucocorticoid receptor and STAT5 with related DNA sequences in IRP1 gene promoter was enhanced under psychological stress. Taken together, it appears that largely secreted glucocorticoid may bring changes to the expression of iron metabolism controlling protein, then cause iron mal-metabolism under stress. The burden of social life and work is so heavy recently, it is important to interpret the mechanism of iron accumulation in liver under stress, and it may prevent harm to human.ObjectiveTo study the characteristic effects of largely secreted GC under psychological stress on liver iron metabolism and to establish a useful experimental basis for interpreting iron mal-metabolism and accumulation in liver under PS, providing medical clues for iron accumulation related hepatic disease.Method1. Effects of psychological stress on iron concentrations and iron metabolism controlling protein in the rat liverTo 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±10 g 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 7 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.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.Measurement of liver iron concentrations in ratsIron concentrations were determined using a Varian SpectrAA-220G graphite furnace atomic absorption spectrometer equipped with a GTA 110 atomizer, programmable sample dispenser, and deuterium background correction. Standard addition method was used for calibration. Standards and control samples were prepared in an identical manner to the experimental samples.Determination of Fn, TfRl, IRP1 and IRP2 mRNA levelsReal time PCR was performed using IQ5 Real-Time PCR Detection System. Two step RT-PCR method was performed using Real Time PCR Master Mix. Primers used to analyze all the transcripts have been reported else where. The RT-PCR data were analyzed by 2-ΔΔCT method as described.Determination of Fn, TfR1 and IRP1 protein levelsThe concentrations of Fn, TfR1 and IRP1 in the liver, samples were assessed by Western blot.2. Effects of injected glucocorticoid on iron concentrations and iron metabolism controlling protein in the rat liverTo divide experimental animals into groupsForty male Sprague-Dawley(SD) rats, weighting 200±10g (Shanghai-BK Co., Ltd. Shanghai, China), were housed individually in cages under standard laboratory conditions(24±1℃; humidity of 55±5%; 12:12-h light-dark cycle) and were given normal chow and free access to water. After 7 days'adaptation, the rats were evenly divided into four groups randomly:0.9% saline group,10-9 mol/L corticosterone group,10-6 mol/L corticosterone group (sigma,USA) and GR blocking group (RU486, sigma, USA).The agents were administrated through the rat caudal vein. After 7 days exposure, all rats were deeply anesthetized by i.p. injection of 10% chloral hydrate, and then perfused through the left cardiac ventricle with ice-cold phosphate buffered saline (PBS; pH7.4) to flush out the blood. The whole liver were quickly removed and snap frozen in liquid nitrogen, and kept in a-80℃freezer till use.3. Effects of molecular mechanisms of glucocorticoid on IRP1 expression in HL7702 cells Cell CultureThe human hepatic cell line HL7702 was obtained from Chinese academy of sciences shanghai branch. Cells were grown in RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS),2mM glutamine, 100U/mL penicillin and 0.1mg/mL streptomycin at 37℃in humidified air containing 5% CO2. The cells were plated into six-well plate at a density of about 1×106 cells/mL and were treated with hydrocortisone (sigma,USA) at 0,10-5,10-6,10-7,10-8,10-9 mol/L respectively. RU486(sigma,USA) was added into cells at 10-5 mol/L and 1 hour later, cells were treated with hydrocortisone at 10-6mol/L. Cells were also treated with STAT5 inhibitor (calbiochem,Germany), then with hydrocortisone. After each treatment, cells were collected and kept in a-80℃freezer till use.4. Statistical analysisAll results were expressed as mean±S.E. Statistical analysis was carried out by using SPSS 11.5. 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. PS exposure increased the iron concentrations in rat liver1.1 Effects of psychological stress on iron concentrationsWe found that the iron levels in rat liver were significantly higher in 7 days PS exposure group than in the control group (P<0.05); however, no significant differences were observed in 1 day and 3 days PS exposure group compared with the control group.1.2 PS exposure caused changes in Fn, TfR1, IRP1 and IRP2 mRNA expressionReal time-PCR analysis showed that 7 days PS exposure increased TfR1 mRNA levels and decreased Fn mRNA levels in rat liver than that in the control group (P<0.05), but no significant changes were seen in 1day and 3 days PS exposure, while IRP1 mRNA levels were increased in 1 day,3 days and 7 days PS exposure compared with the control group, but 7 days PS exposure group showed the most significant changes(P<0.05), IRP2 mRNA showed no significant differences in all groups.1.3 PS exposure caused changes in Fn, TfR1 and IRP1 protein levelsTfR1 protein levels in rat liver after 7 days PS exposure were significantly higher than those in the control group (P<0.05); Fn concentrations in rat liver were significantly lower in 7 days PS exposure group than in the control group (P<0.05); but no significant changes were seen in 1 day and 3 days PS exposure; IRP1 levels were increased in 1 day,3 days and 7 days PS exposure compared with the control group, but 7 days PS exposure group showed the most significant changes(P<0.05).2. GC increased the iron concentrations in rat liver2.1 Effects of GC on iron concentrationsHepatic iron levels were increased obviously by 7 days injection of 10-6 mol/L GC in rats compared with that in the other 2 groups. No significant difference was detected between control group and 7 days injection of 10-9 mol/L GC group.2.2 GC caused changes in Fn, TfRl and IRP1 mRNA expressionWe found that administration of corticosterone at 10-6 mol/L for 7 days could lead to decrease of Fn mRNA expression and increase of TfRl mRNA expression in rat liver (P<0.05), while IRP1 mRNA levels were both increased in 10-9mol/L and 10-6 mol/L GC group compared with the control group, but the changes in latter group were more significant(P<0.05).2.3 GC caused changes in Fn, TfRl and IRP1 protein levelsThe protein levels paralleled the change in mRNA levels,10-6mol/L GC decreased the expression of Fn protein and increase the expression of TfR1(P<0.05), while the expression of IRP1 protein was both increased in 10-9mol/L and 10-6mol/L GC group compared with the control group (P<0.05).3. Molecular mechanisms of GC regulates the expression of IRP1 3.1 Induction of IRP1 protein and mRNA in HL7702 cells by GCHydrocortisone readily induced IRP1 protein in HL7702 cells. The promotion of IRP1 protein was detected at 10-6,10-7,10-8,10-9mol/L after treatment for 24h, the promoted effect at 10-6mol/L was most significant comparing with other groups (P<0.05). When analyzing the mRNA expression, we found that hydrocortisone treatment with HL7702 cells at 10-6mol/L led to a 1.65-fold induction of IRP1 mRNA, while 10'-,10-8 and 10-9 mol/L led to 1.38-fold,1.37-fold and 1.38-fold respectively, and the highest induction was at 10-6mol/L(P<0.05).3.2 Inhibition of IRP1 protein and mRNA by RU486When RU486 was administrated in advance, the induction of IRP1 protein and mRNA by GC was dropped. The expression of both IRP1 protein and mRNA in liver tissue and the cells decreased significantly(P<0.05). The results showed that 10-5mol/L RU486 could lead to 3-fold decrease of IRP1 mRNA induced by 10-6mol/LGC.3.3 The role of STATS in induction of IRP1When 10-6mol/L hydrocortisone was added in the HL7702cells, the expression of STAT5 protein and mRNA showed no obvious changes compared with control group, but P-STAT5 protein was expressed considerably(P<0.05). STAT5 inhibitor could make the expression of IRP1 by hydrocortisone decrease.3.4 The effect of RU486 and STAT5 inhibitor administrated at the same time on the expression IRP1when RU486 and STAT5 inhibitor were added at the same time, hydrocortisone could hardly induce the expression of IRP1 in HL7702 cells(P<0.01).Conclusions1.7 days PS could cause the expression of Fn decreased and the expression of TfRl increased in rat liver, the increased expression of TfRl might be the reason of iron accumulation in liver under PS. Meanwhile, the expression of IRP1 in rat liver also increased under PS, which might bring post-transcriptional regulation to the expression of Fn and TfR1.2.7 days injection of 10-6mol/L glucocorticoid could cause decreased expression of Fn, increased expression of TfRl and IRP1, paralled the changes in the expression under PS, suggesting that the large secreted GC under PS might be the main reason of iron accumulation in rat liver.3. Animal and cell experiments both showed that GC could increase the expression of IRP1 considerably. The expression of IRP1 could decrease when RU486 and STAT5 inhibitor were administrated separately, but the expression could be almost completely blocked when the two inhibitors were added at the same time, suggesting that GR and STAT5 were both involved in the up-regulation of IRP1 by GC.Taken together, we found in our experiment that PS could cause large secretion of glucocorticoid, which could up-regulate the expression of IRP1 through GR and STAT5 by binding with the related DNA sequences in the promoter of IRP1 gene. The increased expression of TfRl and decreased expression of Fn caused by the post-transcriptional regulation of IRP1 then made iron accumulation in liver and even damage to liver.