心理应激致肝铁蓄积的机制探讨
摘要
铁是人体必需微量元素中含量最多的一种,参与机体许多重要的生理功能,铁过多或过少对机体都会造成损害。铁缺乏对人体影响被了解的同时,铁过负荷对人体的危害也越来越受到重视。有资料显示,肝铁过负荷可导致肝纤维化、肝硬化；迟发性皮肤卟啉症和肝细胞癌等疾病。
肝脏是机体铁贮存的主要部位,在铁代谢中发挥着中心和枢纽的作用。为了维持铁平衡,肝细胞存在精细的铁调控机制,可以维持正常生命活动所需的铁量,同时又会避免铁过多蓄积而产生的毒性作用。然而课题组研究发现心理应激可以引起肝铁蓄积,肝细胞氧化应激损伤,但机制尚不明确。
众所周知,应激情况下糖皮质激素大量分泌,文献报道,糖皮质激素能够调控大鼠肠道内铁蛋白轻链重链的表达,增加垂体切除鼠体内的转铁蛋白受体1的含量,有意义的是,课题组前期研究结果发现应激情况下,糖皮质激素受体和STAT5与铁调节蛋白1基因启动子位点上的相应DNA序列结合活性增强,综合国内外相关研究,提示心理应激情况下的肝铁蓄积可能与糖皮质激素的大量分泌导致铁调控蛋白的表达改变有关。应激在现代社会无处不在,阐明应激肝铁紊乱的机制对防治应激损伤具有积极的意义。
研究目的
了解心理应激糖皮质激素大量分泌与肝铁蓄积的关系,及心理应激导致肝铁蓄积的机制,为阐明心理应激情况下肝铁代谢紊乱、肝铁蓄积的机制提供实验基础,并为防治与肝铁蓄积相关的疾病提供线索。
研究方法
1.心理应激对大鼠肝铁浓度及铁调控蛋白的影响
1.1实验动物分组
雄性SD大鼠(购自上海西普尔-必凯公司),体重(120±10)g。按体重随机分为空白对照组、心理应激组和足底电击组,每组10只。动物饲养实验室环境温度24℃±1℃,湿度50%～60%；使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
1.2大鼠心理应激模型的制作
采用Communication Box System制作大鼠心理应激模型。Communication Box System由透明丙烯酸板组成,一半小室(A室)底部铺板绝缘,另一半小室(B室)通电。在B室的实验大鼠接受30min/d,足底电击(电压90V,电流0.80mA)；电击组大鼠跳跃,尖叫,在A室的实验大鼠通过视觉听觉产生恐惧的心理反应,即为心理应激大鼠模型。
1.3大鼠肝组织铁含量的测定
采用原子吸收分光光度计(日本日立公司Z-2000型)火焰法测定。100μ/ml铁标准贮备液(GBW08616)由国家标准物质中心提供。
1.4大鼠肝脏Fn, TfR1, IRP1, IRP2 mRNA表达量的测定
采用实时定量荧光PCR法测定心理应激组和对照组大鼠肝脏内铁蛋白,转铁蛋白受体1,铁调节蛋白1及铁调节蛋白2 mRNA的表达水平。
1.5大鼠肝脏Fn,TfR1,IRP1蛋白含量的测定
Western blot测定实验组和对照组大鼠肝脏内铁蛋白,转铁蛋白受体1以及铁调节蛋白1的蛋白含量。
2.注射糖皮质激素对肝铁浓度及铁调控蛋白的影响
2.1实验动物分组
雄性SD大鼠(购白上海西普尔-必凯公司),体重(200±10)g。按体重随机分为0.9%生理盐水组,10-9 mol/L皮质酮组,10-6mol/L皮质酮组和RU486组,每组10只。动物饲养实验室环境温度24℃±1℃,湿度50%～60%；使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
2.2实验动物模型制作
大鼠尾静脉注射0.9%生理盐水,10-7mol/L皮质酮,10-4 mol/L皮质酮或10-2mol/L RU486(1h后再注射104 mol/L皮质酮),按照0.1ml/100g bw注射试剂,连续7天注射后腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗,取出大鼠肝脏存-80℃冰箱备用。
2.3大鼠肝组织铁含量的测定：同前。
2.4大鼠肝脏Fn,TfR1,IRP1mRNA和蛋白含量的测定：同前。
3.糖皮质激素调控肝细胞铁调节蛋白1表达的分子机制研究
3.1细胞培养
选用人正常肝细胞HL7702, RPMI1640培养液中加入10%胎牛血清、双抗,培养箱中生长。
3.2氢化可的松干预细胞
用精密电子天平称取3.6247mg的氢化可的松,用无水乙醇稀释溶解至1ml,配成10-2mol/L,用1640培养液稀释成10-5,10-6,10-7,10-8,10-9mol/L后加入HL7702细胞。
3.3 CCK-8法测定肝细胞活性
弃去培养HL7702细胞96孔板中的上清,PBS漂洗3次加入CCK-8反应液,于37℃孵育2h,使用酶标仪450 nm波长处测定吸光度(A)值。以实验组吸光度值与对照组的吸光度值之百分比作为细胞相对活性。
3.4阻断HL7702细胞内糖皮质激素受体或STAT5
用精密电子天平称取4.2959mg的RU486,用无水乙醇稀释溶解至1ml,配成10-2mol/L的RU486,用1640培养液稀释为10-5mol/L后加入HL7702细胞,半小时后再加入10-6mol/L氢化可的松。
用精密电子天平称取适量STAT5抑制剂溶于DMSO,使终浓度达到10mg/ml。
3.5肝细胞IRP1和STAT5 mRNA及蛋白含量的测定
实时定量荧光PCR法测定mRNA的表达水平,Western blot测定蛋白含量的变化。
4.数据的统计与处理
采用quantity one图像分析系统对Western条带进行灰度分析,实验数据用(X±S)表示。应用SPSS11.5统计软件进行实验数据分析,两组间差异采用成组设计资料的t检验,多组间差异采用单因素方差分析。各组方差齐时,对照组与各实验组间比较采用Dunnett法,各组间两两比较采用LSD-t检验和SNK-q检验；方差不齐时采用Dunnett's C检验,P<0.05为显著性水平,P<0.01为非常显著性水平。
结果
1.心理应激对大鼠肝铁浓度及铁调控蛋白的影响
1.1心理应激对大鼠肝铁含量的影响
7天心理应激组大鼠肝铁含量与对照组比较升高幅度达到52.7%(P<0.01)；与1天和3天心理应激组比较分别升高了40.8%(P<0.01)和31.8%(P<0.05)而1天和3天心理应激组大鼠肝铁含量较对照组有升高趋势,但无统计学意义(P>0.05)。
1.2心理应激对大鼠肝脏Fn, TfR1, IRP1, IRP2 mRNA表达的影响
大鼠肝脏Fn mRNA表达在1天和3天心理应激后与对照组比较有降低趋势,在7天心理应激后较对照组明显降低(P<0.05)；大鼠肝脏TfR1 mRNA表达在1天和3天心理应激后与对照组比较有升高趋势,7天心理应激后较对照组明显升高(P<0.05)；而大鼠肝脏IRP1 mRNA表达在1,3,7天心理应激后较对照组均有显著升高,其中7天心理应激组升高幅度最大,达到36%(P<0.05)；大鼠肝脏IRP2mRNA表达各组间均无明显变化(P>0.05)。
1.3心理应激对大鼠肝脏Fn, TfR1, IRP1蛋白含量的影响
心理应激1天和3天时,大鼠肝脏Fn, TfR1蛋白表达与对照组相比无显著性差异,7天心理应激时与对照组相比,大鼠肝脏Fn蛋白表达降低,而TfRl蛋白表达升高(P<0.05)；1,3,7天心理应激组大鼠肝脏IRP1蛋白表达较对照组均有增加,分别增加1.5倍(P<0.05),1.8倍(P<0.05)和2.6倍(P<0.01)
2.注射糖皮质激素对大鼠肝铁浓度及铁调控蛋白的影响
2.1注射皮质酮对大鼠肝铁含量的影响
10-9mol/L皮质酮组大鼠肝铁含量与0.9%生理盐水组相比无显著差异,但连续7天每天注射10-6mol/L皮质酮后,大鼠肝铁含量与0.9%生理盐水组比较有显著上升,升高39.5%(P<0.05),与连续7天注射10-9mol/L皮质酮组比较也升高了39.1%(P<0.05)2.2注射皮质酮对大鼠肝脏Fn, TfR1, IRP1 mRNA表达的影响
10-9mol/L皮质酮组Fn, TfR1 mRNA的表达与0.9%生理盐水组相比无显著性差异,而10-6 mol/L皮质酮连续注射7天后Fn mRNA表达较0.9%生理盐水组降低,TfR1 mRNA表达较生理盐水组升高,大鼠肝脏IRP1 mRNA表达在注射10-9和10-6。mol/L皮质酮后较0.9%生理盐水组均有升高,分别较对照组增高1.2倍和1.5倍(P<0.05)。
2.3注射皮质酮对大鼠肝脏Fn,TfR1,IRP1蛋白含量的影响
Western blot显示大鼠肝脏Fn,TfR1蛋白表达在连续7天给予10-9mol/L皮质酮后与0.9%生理盐水组相比无显著性差异,而连续注射10-6mol/L皮质酮7天后,与0.9%生理盐水组比较,Fn蛋白表达降低,TfR1蛋白表达升高(P<0.05),10-9mol/L和10-6mol/L皮质酮组的IRP1蛋白表达较0.9%生理盐水组均升高(P<0.05)
3.糖皮质激素调控肝细胞铁调节蛋白1表达的分子机制研究
3.1氢化可的松对HL7702细胞IRP1mRNA表达的影响
细胞内加入10-6,10-7,10-8,10-9。mol/L氢化可的松24h后HL7702细胞IRP1mRNA表达较对照组均增高,分别增高1.65倍,1.38倍,1.37倍和1.38倍(P<0.05),10-6mol/L氢化可的松组的IRP1 mRNA表达较其他剂量氢化可的松组均增高(P<0.05),而10-5mol/L糖皮质激素组IRP1mRNA表达与对照组无显著差异。
3.2氢化可的松对HL7702细胞IRP1蛋白含量的影响
Western blot结果显示细胞内加入10-6,10-7,10-8,10-9mol/L氢化可的松24h后HL7702细胞IRP1蛋白含量较对照组均增高(P<0.05),其中10-6mol/L氢化可的松组IRP1蛋白含量与10-7mol/L组比差异不显著(P>0.05),与10-8,10-9mol/L组比较IRP1蛋白含量均增高(P<0.05),而10-Smol/L组IRP1含量与对照组无显著差异,CCK-8检测显示,10-5mol/L组的肝细胞活性与对照组相比显著降低(P<0.01)。
3.3阻断糖皮质激素受体对大鼠肝脏IRP1含量的影响
大鼠尾静脉注射10-4mol/L糖皮质激素受体阻断剂RU486 1小时后注射10-6mol/L皮质酮,发现IRP1 mRNA表达较10-6mol/L皮质酮组明显降低(P<0.05)。IRP1蛋白含量变化与mRNA一致,RU48+皮质酮组大鼠肝脏IRP1蛋白含量较单纯加皮质酮组明显降低(P<0.05)。
3.4阻断糖皮质激素受体对HL7702细胞IRP1含量的影响
RT-PCR结果显示HL7702细胞内加入10-5 mol/L RU486后可以降低氢化可的松诱导的IRP1的生成,与10-6mol/L氢化可的松组对比降低达3倍(P<0.05)。Western blot结果同样显示RU486可以降低氢化可的松诱导的IRP1蛋白生成。
3.5氢化可的松对STAT5表达的影响
HL7702细胞内加入10-6mol/L氢化可的松后,STAT5的mRNA及蛋白表达较对照组均无显著差异(P>0.05),Western blot结果显示,在给予10-6mol/L氢化可的松后,HL7702细胞内P-STAT5蛋白含量较对照组明显升高(P<0.05)。
3.6抑制STAT5对HL7702细胞IRP1生成的影响
抑制STAT5后,HL7702细胞IRP1蛋白表达较10-6mol/L氢化可的松组明显降低(P<0.05)。
3.7同时阻断糖皮质激素受体和STAT5对HL7702细胞IRP1生成的影响
当同时阻断糖皮质激素受体和STAT5后再给予氢化可的松,HL7702细胞中IRP1蛋白几乎不表达,与10-6mol/L糖皮质激素组比较差异非常显著(P<0.01)。
结论
1.连续7天心理应激可以引起大鼠肝脏铁蛋白表达减少,而转铁蛋白受体1表达增加,肝细胞摄入铁增加引起肝铁蓄积；肝脏铁调节蛋白1含量随着应激天数增加而增加,通过与IRE结合转录后调控铁蛋白,转铁蛋白受体1的表达可能是心理应激情况下肝铁蓄积的原因。
2.连续7天每天注射10-6mol/L的皮质酮可以引起大鼠肝脏铁调节蛋白1表达上调、转铁蛋白受体1含量增加、铁蛋白减少、肝脏铁含量增加,与7天心理应激后大鼠肝脏铁代谢变化一致,结合以往研究结果提示心理应激时糖皮质激素的大量分泌可能是导致大鼠肝脏铁调节蛋白1表达增加的重要原因。
3.通过整体动物和细胞实验发现,糖皮质激素可以上调铁调节蛋白1的表达。分别采用特异性糖皮质激素受体抑制剂RU486或STAT5抑制剂后,铁调节蛋白1的表达有不同程度的降低,而在培养基中同时添加上述两种抑制剂几乎可完全抑制铁调节蛋白1的表达,表明糖皮质激素可通过增强GR和STAT5信号通路上调铁调节蛋白1的表达。
综上所述,心理应激时糖皮质激素大量释放,通过GR和STAT5与铁调节蛋白1基因位点上的相应DNA结合位点结合,上调铁调节蛋白1基因的表达,随着铁调节蛋白1含量的逐渐增加,与IRE结合,通过转录后调控引起转铁蛋白受体1表达增加,铁蛋白表达减少,肝细胞摄入铁增加,导致肝铁蓄积肝脏损伤。
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.
Objective
To 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.
Method
1. Effects of psychological stress on iron concentrations and iron metabolism controlling protein in the rat liver
To divide experimental animals into groups
All 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 rats
Using 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 rats
Iron 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 levels
Real 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 levels
The 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 liver
To divide experimental animals into groups
Forty 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 Culture
The 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 analysis
All 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.
Results
1. PS exposure increased the iron concentrations in rat liver
1.1 Effects of psychological stress on iron concentrations
We 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 expression
Real 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 levels
TfR1 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 liver
2.1 Effects of GC on iron concentrations
Hepatic 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 expression
We 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 levels
The 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 GC
Hydrocortisone 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 RU486
When 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 IRP1
When 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 IRP1
when RU486 and STAT5 inhibitor were added at the same time, hydrocortisone could hardly induce the expression of IRP1 in HL7702 cells(P<0.01).
Conclusions
1.7 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.
肝脏是机体铁贮存的主要部位,在铁代谢中发挥着中心和枢纽的作用。为了维持铁平衡,肝细胞存在精细的铁调控机制,可以维持正常生命活动所需的铁量,同时又会避免铁过多蓄积而产生的毒性作用。然而课题组研究发现心理应激可以引起肝铁蓄积,肝细胞氧化应激损伤,但机制尚不明确。
众所周知,应激情况下糖皮质激素大量分泌,文献报道,糖皮质激素能够调控大鼠肠道内铁蛋白轻链重链的表达,增加垂体切除鼠体内的转铁蛋白受体1的含量,有意义的是,课题组前期研究结果发现应激情况下,糖皮质激素受体和STAT5与铁调节蛋白1基因启动子位点上的相应DNA序列结合活性增强,综合国内外相关研究,提示心理应激情况下的肝铁蓄积可能与糖皮质激素的大量分泌导致铁调控蛋白的表达改变有关。应激在现代社会无处不在,阐明应激肝铁紊乱的机制对防治应激损伤具有积极的意义。
研究目的
了解心理应激糖皮质激素大量分泌与肝铁蓄积的关系,及心理应激导致肝铁蓄积的机制,为阐明心理应激情况下肝铁代谢紊乱、肝铁蓄积的机制提供实验基础,并为防治与肝铁蓄积相关的疾病提供线索。
研究方法
1.心理应激对大鼠肝铁浓度及铁调控蛋白的影响
1.1实验动物分组
雄性SD大鼠(购自上海西普尔-必凯公司),体重(120±10)g。按体重随机分为空白对照组、心理应激组和足底电击组,每组10只。动物饲养实验室环境温度24℃±1℃,湿度50%～60%；使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
1.2大鼠心理应激模型的制作
采用Communication Box System制作大鼠心理应激模型。Communication Box System由透明丙烯酸板组成,一半小室(A室)底部铺板绝缘,另一半小室(B室)通电。在B室的实验大鼠接受30min/d,足底电击(电压90V,电流0.80mA)；电击组大鼠跳跃,尖叫,在A室的实验大鼠通过视觉听觉产生恐惧的心理反应,即为心理应激大鼠模型。
1.3大鼠肝组织铁含量的测定
采用原子吸收分光光度计(日本日立公司Z-2000型)火焰法测定。100μ/ml铁标准贮备液(GBW08616)由国家标准物质中心提供。
1.4大鼠肝脏Fn, TfR1, IRP1, IRP2 mRNA表达量的测定
采用实时定量荧光PCR法测定心理应激组和对照组大鼠肝脏内铁蛋白,转铁蛋白受体1,铁调节蛋白1及铁调节蛋白2 mRNA的表达水平。
1.5大鼠肝脏Fn,TfR1,IRP1蛋白含量的测定
Western blot测定实验组和对照组大鼠肝脏内铁蛋白,转铁蛋白受体1以及铁调节蛋白1的蛋白含量。
2.注射糖皮质激素对肝铁浓度及铁调控蛋白的影响
2.1实验动物分组
雄性SD大鼠(购白上海西普尔-必凯公司),体重(200±10)g。按体重随机分为0.9%生理盐水组,10-9 mol/L皮质酮组,10-6mol/L皮质酮组和RU486组,每组10只。动物饲养实验室环境温度24℃±1℃,湿度50%～60%；使用不锈钢笼具单笼饲养,自由饮食,自然昼夜节律变化光照。
2.2实验动物模型制作
大鼠尾静脉注射0.9%生理盐水,10-7mol/L皮质酮,10-4 mol/L皮质酮或10-2mol/L RU486(1h后再注射104 mol/L皮质酮),按照0.1ml/100g bw注射试剂,连续7天注射后腹腔注射10%水合氯醛进行麻醉,暴露胸腔,剪开右心耳,然后迅速经心脏冷PBS冲洗,取出大鼠肝脏存-80℃冰箱备用。
2.3大鼠肝组织铁含量的测定：同前。
2.4大鼠肝脏Fn,TfR1,IRP1mRNA和蛋白含量的测定：同前。
3.糖皮质激素调控肝细胞铁调节蛋白1表达的分子机制研究
3.1细胞培养
选用人正常肝细胞HL7702, RPMI1640培养液中加入10%胎牛血清、双抗,培养箱中生长。
3.2氢化可的松干预细胞
用精密电子天平称取3.6247mg的氢化可的松,用无水乙醇稀释溶解至1ml,配成10-2mol/L,用1640培养液稀释成10-5,10-6,10-7,10-8,10-9mol/L后加入HL7702细胞。
3.3 CCK-8法测定肝细胞活性
弃去培养HL7702细胞96孔板中的上清,PBS漂洗3次加入CCK-8反应液,于37℃孵育2h,使用酶标仪450 nm波长处测定吸光度(A)值。以实验组吸光度值与对照组的吸光度值之百分比作为细胞相对活性。
3.4阻断HL7702细胞内糖皮质激素受体或STAT5
用精密电子天平称取4.2959mg的RU486,用无水乙醇稀释溶解至1ml,配成10-2mol/L的RU486,用1640培养液稀释为10-5mol/L后加入HL7702细胞,半小时后再加入10-6mol/L氢化可的松。
用精密电子天平称取适量STAT5抑制剂溶于DMSO,使终浓度达到10mg/ml。
3.5肝细胞IRP1和STAT5 mRNA及蛋白含量的测定
实时定量荧光PCR法测定mRNA的表达水平,Western blot测定蛋白含量的变化。
4.数据的统计与处理
采用quantity one图像分析系统对Western条带进行灰度分析,实验数据用(X±S)表示。应用SPSS11.5统计软件进行实验数据分析,两组间差异采用成组设计资料的t检验,多组间差异采用单因素方差分析。各组方差齐时,对照组与各实验组间比较采用Dunnett法,各组间两两比较采用LSD-t检验和SNK-q检验；方差不齐时采用Dunnett's C检验,P<0.05为显著性水平,P<0.01为非常显著性水平。
结果
1.心理应激对大鼠肝铁浓度及铁调控蛋白的影响
1.1心理应激对大鼠肝铁含量的影响
7天心理应激组大鼠肝铁含量与对照组比较升高幅度达到52.7%(P<0.01)；与1天和3天心理应激组比较分别升高了40.8%(P<0.01)和31.8%(P<0.05)而1天和3天心理应激组大鼠肝铁含量较对照组有升高趋势,但无统计学意义(P>0.05)。
1.2心理应激对大鼠肝脏Fn, TfR1, IRP1, IRP2 mRNA表达的影响
大鼠肝脏Fn mRNA表达在1天和3天心理应激后与对照组比较有降低趋势,在7天心理应激后较对照组明显降低(P<0.05)；大鼠肝脏TfR1 mRNA表达在1天和3天心理应激后与对照组比较有升高趋势,7天心理应激后较对照组明显升高(P<0.05)；而大鼠肝脏IRP1 mRNA表达在1,3,7天心理应激后较对照组均有显著升高,其中7天心理应激组升高幅度最大,达到36%(P<0.05)；大鼠肝脏IRP2mRNA表达各组间均无明显变化(P>0.05)。
1.3心理应激对大鼠肝脏Fn, TfR1, IRP1蛋白含量的影响
心理应激1天和3天时,大鼠肝脏Fn, TfR1蛋白表达与对照组相比无显著性差异,7天心理应激时与对照组相比,大鼠肝脏Fn蛋白表达降低,而TfRl蛋白表达升高(P<0.05)；1,3,7天心理应激组大鼠肝脏IRP1蛋白表达较对照组均有增加,分别增加1.5倍(P<0.05),1.8倍(P<0.05)和2.6倍(P<0.01)
2.注射糖皮质激素对大鼠肝铁浓度及铁调控蛋白的影响
2.1注射皮质酮对大鼠肝铁含量的影响
10-9mol/L皮质酮组大鼠肝铁含量与0.9%生理盐水组相比无显著差异,但连续7天每天注射10-6mol/L皮质酮后,大鼠肝铁含量与0.9%生理盐水组比较有显著上升,升高39.5%(P<0.05),与连续7天注射10-9mol/L皮质酮组比较也升高了39.1%(P<0.05)2.2注射皮质酮对大鼠肝脏Fn, TfR1, IRP1 mRNA表达的影响
10-9mol/L皮质酮组Fn, TfR1 mRNA的表达与0.9%生理盐水组相比无显著性差异,而10-6 mol/L皮质酮连续注射7天后Fn mRNA表达较0.9%生理盐水组降低,TfR1 mRNA表达较生理盐水组升高,大鼠肝脏IRP1 mRNA表达在注射10-9和10-6。mol/L皮质酮后较0.9%生理盐水组均有升高,分别较对照组增高1.2倍和1.5倍(P<0.05)。
2.3注射皮质酮对大鼠肝脏Fn,TfR1,IRP1蛋白含量的影响
Western blot显示大鼠肝脏Fn,TfR1蛋白表达在连续7天给予10-9mol/L皮质酮后与0.9%生理盐水组相比无显著性差异,而连续注射10-6mol/L皮质酮7天后,与0.9%生理盐水组比较,Fn蛋白表达降低,TfR1蛋白表达升高(P<0.05),10-9mol/L和10-6mol/L皮质酮组的IRP1蛋白表达较0.9%生理盐水组均升高(P<0.05)
3.糖皮质激素调控肝细胞铁调节蛋白1表达的分子机制研究
3.1氢化可的松对HL7702细胞IRP1mRNA表达的影响
细胞内加入10-6,10-7,10-8,10-9。mol/L氢化可的松24h后HL7702细胞IRP1mRNA表达较对照组均增高,分别增高1.65倍,1.38倍,1.37倍和1.38倍(P<0.05),10-6mol/L氢化可的松组的IRP1 mRNA表达较其他剂量氢化可的松组均增高(P<0.05),而10-5mol/L糖皮质激素组IRP1mRNA表达与对照组无显著差异。
3.2氢化可的松对HL7702细胞IRP1蛋白含量的影响
Western blot结果显示细胞内加入10-6,10-7,10-8,10-9mol/L氢化可的松24h后HL7702细胞IRP1蛋白含量较对照组均增高(P<0.05),其中10-6mol/L氢化可的松组IRP1蛋白含量与10-7mol/L组比差异不显著(P>0.05),与10-8,10-9mol/L组比较IRP1蛋白含量均增高(P<0.05),而10-Smol/L组IRP1含量与对照组无显著差异,CCK-8检测显示,10-5mol/L组的肝细胞活性与对照组相比显著降低(P<0.01)。
3.3阻断糖皮质激素受体对大鼠肝脏IRP1含量的影响
大鼠尾静脉注射10-4mol/L糖皮质激素受体阻断剂RU486 1小时后注射10-6mol/L皮质酮,发现IRP1 mRNA表达较10-6mol/L皮质酮组明显降低(P<0.05)。IRP1蛋白含量变化与mRNA一致,RU48+皮质酮组大鼠肝脏IRP1蛋白含量较单纯加皮质酮组明显降低(P<0.05)。
3.4阻断糖皮质激素受体对HL7702细胞IRP1含量的影响
RT-PCR结果显示HL7702细胞内加入10-5 mol/L RU486后可以降低氢化可的松诱导的IRP1的生成,与10-6mol/L氢化可的松组对比降低达3倍(P<0.05)。Western blot结果同样显示RU486可以降低氢化可的松诱导的IRP1蛋白生成。
3.5氢化可的松对STAT5表达的影响
HL7702细胞内加入10-6mol/L氢化可的松后,STAT5的mRNA及蛋白表达较对照组均无显著差异(P>0.05),Western blot结果显示,在给予10-6mol/L氢化可的松后,HL7702细胞内P-STAT5蛋白含量较对照组明显升高(P<0.05)。
3.6抑制STAT5对HL7702细胞IRP1生成的影响
抑制STAT5后,HL7702细胞IRP1蛋白表达较10-6mol/L氢化可的松组明显降低(P<0.05)。
3.7同时阻断糖皮质激素受体和STAT5对HL7702细胞IRP1生成的影响
当同时阻断糖皮质激素受体和STAT5后再给予氢化可的松,HL7702细胞中IRP1蛋白几乎不表达,与10-6mol/L糖皮质激素组比较差异非常显著(P<0.01)。
结论
1.连续7天心理应激可以引起大鼠肝脏铁蛋白表达减少,而转铁蛋白受体1表达增加,肝细胞摄入铁增加引起肝铁蓄积；肝脏铁调节蛋白1含量随着应激天数增加而增加,通过与IRE结合转录后调控铁蛋白,转铁蛋白受体1的表达可能是心理应激情况下肝铁蓄积的原因。
2.连续7天每天注射10-6mol/L的皮质酮可以引起大鼠肝脏铁调节蛋白1表达上调、转铁蛋白受体1含量增加、铁蛋白减少、肝脏铁含量增加,与7天心理应激后大鼠肝脏铁代谢变化一致,结合以往研究结果提示心理应激时糖皮质激素的大量分泌可能是导致大鼠肝脏铁调节蛋白1表达增加的重要原因。
3.通过整体动物和细胞实验发现,糖皮质激素可以上调铁调节蛋白1的表达。分别采用特异性糖皮质激素受体抑制剂RU486或STAT5抑制剂后,铁调节蛋白1的表达有不同程度的降低,而在培养基中同时添加上述两种抑制剂几乎可完全抑制铁调节蛋白1的表达,表明糖皮质激素可通过增强GR和STAT5信号通路上调铁调节蛋白1的表达。
综上所述,心理应激时糖皮质激素大量释放,通过GR和STAT5与铁调节蛋白1基因位点上的相应DNA结合位点结合,上调铁调节蛋白1基因的表达,随着铁调节蛋白1含量的逐渐增加,与IRE结合,通过转录后调控引起转铁蛋白受体1表达增加,铁蛋白表达减少,肝细胞摄入铁增加,导致肝铁蓄积肝脏损伤。
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.
Objective
To 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.
Method
1. Effects of psychological stress on iron concentrations and iron metabolism controlling protein in the rat liver
To divide experimental animals into groups
All 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 rats
Using 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 rats
Iron 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 levels
Real 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 levels
The 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 liver
To divide experimental animals into groups
Forty 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 Culture
The 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 analysis
All 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.
Results
1. PS exposure increased the iron concentrations in rat liver
1.1 Effects of psychological stress on iron concentrations
We 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 expression
Real 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 levels
TfR1 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 liver
2.1 Effects of GC on iron concentrations
Hepatic 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 expression
We 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 levels
The 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 GC
Hydrocortisone 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 RU486
When 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 IRP1
When 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 IRP1
when RU486 and STAT5 inhibitor were added at the same time, hydrocortisone could hardly induce the expression of IRP1 in HL7702 cells(P<0.01).
Conclusions
1.7 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.
引文
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