Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.     

模拟高原低氧环境对小鼠脾脏铁代谢的影响*

目的:探讨模拟海拔6 000 m高原低氧环境对小鼠脾脏铁代谢的影响。方法:将C57BL/6小鼠按体重随机分为常压常氧组(Nor)和低压低氧组(HH)。HH组小鼠放置于低压低氧动物实验舱内,模拟急性海拔6 000 m高原低氧环境,控制光照时间比大约12 h∶12 h。Nor组置于同等条件的常压常氧环境。HH组又分为低氧12 h组(HH-12 h)和3 d组(HH-3 d),对照组对应分为(Nor-12 h及Nor-3 d),每组9只小鼠。采用血常规检测、HE染色、组织铁染色、蛋白质免疫印迹(WB)、免疫组织化学(IHC)综合评价模拟高原低氧环境下小鼠脾脏铁代谢情况。结果:与相同时间点Nor组相比:①HH-12 h组小鼠红细胞数(RBC)、血红蛋白量(HGB)、红细胞压积(HCT)均无明显变化。HH-3 d组RBC、HGB及HCT均显著增加(P＜0.05),平均血红蛋白量(MCH)在HH-12 h和HH-3 d组均无显著变化。②与Nor-3 d相比,HH-3 d组小鼠脾脏明显增大,HE染色显示脾窦变窄,铁染色结果显示HH-3 d组脾脏红髓中铁含量明显增加。③WB结果显示,HH-3 d组低氧诱导因子1α(HIF-1α),转铁蛋白受体1(TfR1),铁输出蛋白(Fpn)表达均显著增加,而铁蛋白(Ft-L)的表达显著降低(P＜0.05);IHC结果也与WB结果一致,高原低氧暴露3 d后脾脏红髓TfR1、Fpn表达和分布均明显增多,Ft-L表达分布明显减少。结论:模拟海拔6 000 m高原低氧暴露3 d后小鼠脾脏截留处理RBC增多,脾索铁沉积,脾脏组织细胞内铁动员加速。高原低氧下脾脏铁代谢异常可能是引起高原低氧暴露下红细胞病理性增多甚至造成高原红细胞增多症的主要原因。     

Molecular analysis of increased iron status in moderately exercised rats

Although iron plays a critical role in exercise, the regulatory mechanism of iron metabolism remains poorly understood. The aims of the present study were to investigate the effects of different intensity exercise on body iron status and the regulatory mechanism of duodenal iron absorption. Thirty female Sprague-Dawley rats (90–100 g) were randomly divided into three groups: a control group (remained sedentary, CG), a moderately exercised group (swam 1.5 h/day, MG) and a strenuously exercised group (swam with different load, SG). Serum iron status, serum ferritin and Hct were examined after 10 weeks of swimming. Western blot was performed to detect the expression of iron transport proteins: divalent metal transporter1 (DMT1) and ferroportin 1 (FPN1) in duodenal epithelium. The expression of hepcidin mRNA in liver was examined by RT-PCR. The results showed: (1) the body iron status in MG was kept at a high level compared to that of CG and SG, (2) Western blot showed DMT1 with iron responsive element (IRE) and FPN1 in duodenal epithelium which were higher in MG than that of CG and (3) the expression of hepatic hepcidin mRNA was down regulated in MG (p < 0.05). The data suggested that moderate exercise improved iron status and that was likely regulated by increased DMT1 with IRE and FPN1 expression. Hepcidin signaling pathway may involve in the regulation of duodenal iron absorption proteins. Xiang Lin Duan and Yan Zhong Chang share Senior Authorship     

Hypobaric hypoxia down-regulated junctional protein complex: Implications to vascular leakage

Acute mountain sickness (AMS) can cause capillary hyper-permeability and vasogenic edema. However, its underlying mechanisms remained unclear and there is no previous in vitro study on AMS. We therefore conducted an in vitro study and examined whether continuous hypobaric hypoxia (CHH) could alter expression of junctional protein complex of vascular endothelial cells, causing hyper-permeabilization. EA.hy926 human endothelial cells were exposed to either CHH or normoxia for up to 24 h. Flow cytometry using annexin V/propidium iodide co-staining demonstrated that cell death had no significant difference at 12-h, but was increased by CHH at 24-h. Transendothelial resistance (TER) of endothelial cell monolayer was progressively decreased by CHH from 1-h to 24-h. Western blot analysis and immunofluorescence study demonstrated decreased expression levels of VE-cadherin, PECAM-1 and ZO-1 junctional proteins at both 12-h and 24-h exposure time-points. Interestingly, while the main form of ZO-1 (220 kDa) was decreased, its degraded form (100 kDa) was increased by 24-h CHH that might be linked to the increased cell death. Our data have demonstrated that CHH caused vascular endothelial hyper-permeability and defective junctional protein complex by reducing expression levels of VE-cadherin, PECAM-1, and ZO-1. Taken together, these data may explain pathophysiology underlying vascular hyper-permeability in AMS.     

间歇性低压低氧与连续性低压低氧对大鼠血液动力学作用的比较(英文)

本研究旨在探讨并比较慢性间歇性低压低氧（intermitten thypobaric hypoxia,IHH）和慢性连续性低压低氧（continuous hypobaric hypoxia,CHH）对大鼠血液动力学作用的影响。40只成年Sprague—Dawley大鼠随机分为5组：对照组（CON）,28天IHH处理组（IHH28）,42天IHH处理组（IHH42）,28天CHH组（CHH28）和42天CHH组（CHH42）。IHH火鼠于低压氧舱分别接受28或42天模拟5000m海拔高度低氧（11．1％O2）处理、每天6h。CHH处理大鼠生活在低压氧舱环境中,除每天半小时常氧供食、供水和清洁外,其余时间均分别接受时程为28或42天的模拟5000m海拔高度低氧（11．1％O2）处理。每周定时测定大鼠体重。通过导管法测定基础常氧和急性低氧状态下的血液动力学,包括半均动脉压（meanartery blood pressure,MAP）、心率（heartrate,HR）、左审收缩峰压（1eft ventricular systolic pressure,LVSP）、正负左率最人压力变化速率（maximum change rate of left ventricular pressure,&#177;LVdP／dtmax）。通过生物化学方法测定大鼠心肌超氧化物岐化酶活性和丙二醛含量。并分别测定全心、左心室和右心室重量。结果显示：（1）CHH42大鼠基础HR和MAP低于CON,IHH和CHH28大鼠（P〈0．05）。（2）IHH大鼠表现出明显的抗心肌缺氧／复氧损伤作用,表现为急性低氧状态下的HR、MAP、LVSP和＋LVdP/dtmax,改变明显低于CON大鼠（P〈0．05）;CHH大鼠表现出更为明显的抗急性低氧心脏保护作用,表现为急性低氧的HR、MAP、LVSP和&#177;LVdP/dtmax;改变明显低于CON和IHH火鼠（P〈0．05）,但出现复氧损伤作用,表现为复氧过程中血液动力学的恢复明显低于CON和IHH大鼠（P〈0．05）。（3）与CON大鼠相比较,IHH和CHH大鼠心肌抗氧化能力明显增强（P〈0．05,P〈0．01）。（4）与IHH和CON大鼠相比较,CHH大鼠表现明显的右心室肥厚（P〈0．01）。结果表明,IHH可诱导有效的心脏保护作用,而无明显的不良反应,因而具有潜在的实际应用价值。     

Intermittent hypobaric hypoxia applicability in myocardial infarction prevention and recovery

Intermittent hypobaric hypoxia (IHH) has been the focus of important research in cardioprotection, and it has been associated with several mechanisms. Intermittent hypobaric hypoxia inhibits prolyl hydroxylases (PHD) activity, increasing the stabilization of hypoxia-inducible factor-1 (HIF-1) and activating crucial adaptative genes. It has been hence suggested that IHH might be a simple intervention, which may offer a thoughtful benefits to patients with acute myocardial infarction and no complications. Nevertheless, several doubts exist as to whether IHH is a really safe technique, with little to no complications in post-myocardial infarction patients. Intermittent hypobaric hypoxia might produce instead unfavourable changes such as impairment of vascular hemodynamics and hypertensive response, increased risk of hemoconcentration and thrombosis, cardiac rhythm perturbations, coronary artery disease and heart failure, insulin resistance, steatohepatitis and even high-altitude pulmonary oedema in susceptible or nonacclimatized patients. Although intermittent and chronic exposures seem effective in cardioprotection, IHH safety issues have been mostly overlooked, so that assorted concerns should be raised about the opportunity to use IHH in the post-myocardial infarction period. Several IHH protocols used in some studies were also aggressive, which would hamper their widespread introduction within the clinical practice. As such, further research is needed before IHH can be widely advocated in myocardial infarction prevention and recovery.     

低压低氧大鼠摄氧量测定装置

目的：设计并制作一种用于测定模拟不同海拔低压低氧环境中大鼠摄氧量的装置。方法：该装置容积约为0．01m3。它包括动物舱、参照舱、高度表、高度升降速率表、动物舱与参照舱间气压差感应和氧气自动补偿装置、低范围气压计、钠石灰和氯化钙、小风扇、温度湿度表、循环水温度控制装置、真空泵。利用该装置检测大鼠在平原（50m）、4000m和6000m模拟海拔环境中的摄氧量。结果：大鼠在平原（50m,北京）、4000m和6000m模拟海拔环境中的摄氧量分别为（24．4±2．1）、（10．8±2．0）和（8．8±1．6）ml／（kg·min）（x±s,n=10）,随着海拔高度的升高其摄氧量逐渐降低。结论：本文制作的低压低氧动物摄氧量测定装置可用于测定不同海拔环境中大鼠的摄氧量。     

低氧暴露对大鼠外周血T淋巴细胞活化的影响

目的：探讨低氧暴露后大鼠外周血T淋巴细胞亚群及活化共刺激分子的变化,为干预措施的研究提供科学依据。方法：采用三色免疫荧光标记流式细胞仪分析法,观察在低氧暴露前和模拟海拔8 000 m低氧暴露8 h、3d6、d和10 d后,大鼠外周血T淋巴细胞亚群和T淋巴细胞活化共刺激分子的变化。结果：模拟海拔8 000 m低氧暴露8 h后,大鼠CD3＋、CD8＋、CD8＋CD28-细胞数较低氧前均显著降低（P均〈0.01）;低氧暴露3 d后,除上述变化外,CD4＋CD28＋细胞数也显著降低（P〈0.01）,CD4＋CD28-细胞数显著增加（P〈0.01）;低氧暴露6 d和10 d后,CD3＋、CD4＋呈现进一步降低趋势,CD8＋CD28＋细胞数显著增加（P〈0.01）。结论：说明模拟海拔8 000 m低氧暴露8 h和3 d后,大鼠外周血CD8＋、CD4＋T淋巴细胞活化水平均显著下降,随着低氧暴露时间的延长,CD8＋T淋巴细胞活化水平由降低变为增加。     

The effect of hypoxia on red blood cells of flounder: a morphologic and autoradiographic study

Flounders were maintained in water with low oxygen content (2.6–2.7 mg 1 ^{- 1}, 30% saturation) for 2, 6 and 24 h. Hypoxia enhanced the proliferation and differentiation process among juvenile red blood cells (RBCs) in the pronephros. It was accompanied by an increase in RBCs number in blood by up to 37% (P<0.01) 6 h after adaptation. The number of dividing mature RBCs in blood during the initial period of adaptation (2 h) was 2.5 times greater (P<0.001) than in the control group. It was shown ( in vitro ) that the serum contained factors stimulating division of mature RBCs in blood during adaptation to hypoxia.     

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.     

模拟高原低压低氧环境对大鼠心脏结构和功能影响*

目的: 探讨模拟海拔7 000 m低压低氧环境对大鼠心脏结构和功能的影响。方法: 96只雄性SD大鼠随机分为常压常氧对照组(对照组)和高原低压低氧组(低氧组)。低氧组大鼠放置于大型多因素复合环境模拟实验舱内,模拟海拔7 000 m高原环境饲养。实验舱运行时间23 h/d,控制昼夜比大约12 h∶12 h;对照组置于相同条件的常压常氧环境下饲养。低氧组又根据低氧时间不同分为3 d组、7 d组、14 d组和28 d组,同时设置与各低氧组相对应的对照组,每组均12只大鼠。应用超声心动图、心电图、血常规、血生化综合评价高原低压低氧环境下大鼠心脏结构和功能变化,心肌组织HE染色分析心肌组织病理变化。结果: 与相同时间点对照组比较①随着低压低氧暴露时间延长,大鼠体质量增长明显缓慢,动脉血氧饱和度14 d和28 d显著降低(P＜0.05)。②低氧组大鼠左心室舒张末期前壁厚度(LVAWD)及左心室舒张末期后壁厚度(LVPWD)于28 d时显著升高(P＜0.05)。舒张末期左心室腔直径(LVIDD)及收缩末期左心室腔直径(LVIDS)于28 d时明显降低(P＜0.05,P＜0.01)。左心室射血分数(EF%)、左室短轴缩短率(FS%)、肺静脉血流峰值速度(PV peak velocity)及肺静脉血流峰梯度(PV peak gradient)于低氧7 d 下降明显(P＜0.05,P＜0.01),低氧14 d 及低氧28 d 恢复。③低氧组大鼠心电图QRS间期与QT间期在14 d 及28 d 显著延长(P＜0.05,P＜0.01)。ST段3 d和7 d显著压低(P＜0.05,P＜0.01)。R波振幅于 7 d、14 d 及28 d 显著降低(P＜0.05,P＜0.01)。④低氧各组大鼠红细胞计数(RBC)、血红蛋白(HGB)、红细胞分布宽度(RDW)均明显升高(P＜0.01)。血小板计数(PLT)于14 d 及28 d 明显下降(P＜0.01)。血肌酐(CR)于14 d及28 d显著升高(P＜0.05)。⑤心肌病理提示,低氧3 d 和7 d 可见心肌水肿、肌浆凝聚,横纹不清,灶状变性和坏死伴炎性细胞浸润。低氧14 d 和28 d 心肌组织炎症性病理损伤逐渐减少。心肌细胞逐渐肥大,成纤维细胞逐渐增生。心肌间质胶原纤维逐渐增多等心肌代偿修复性病理变化显著。结论: 暴露于模拟海拔7 000 m低压低氧环境下3 d大鼠心功能明显降低,7 d最为显著。     

Hypobaric hypoxia induces oxidative stress in rat brain

High altitude exposure results in decreased partial pressure of oxygen and an increased formation of reactive oxygen and nitrogen species (RONS), which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is very less in brain tissue and is very much susceptible to hypoxic stress. The aim of the present study was to investigate the time dependent and region specific changes in cortex, hippocampus and striatum on oxidative stress markers on chronic exposure to hypobaric hypoxia. The rats were exposed to simulated high altitude equivalent to 6100 m in animal decompression chamber for 3 and 7 days. Results indicate an increase in oxidative stress as seen by increase in free radical production, nitric oxide level, lipid peroxidation and lactate dehydrogenase levels. The magnitude of increase in oxidative stress was more in 7 days exposure group as compared to 3 days exposure group. The antioxidant defence system such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and reduced/oxidized glutathione (GSH/GSSG) levels were significantly decreased in all the three regions. The observation suggests that the hippocampus is more susceptible to hypoxia than the cortex and striatum. It may be concluded that hypoxia differentially affects the antioxidant status in the cortex, hippocampus and striatum.     

慢性间歇低压低氧暴露对小鼠空间记忆及海马谷氨酸受体磷酸化 水平的影响

目的:观察慢性间歇低压低氧暴露对成年C57小鼠认知功能、海马区p-Glu R-831、845位点蛋白表达以及海马区突触囊泡释放的影响。方法:雄性C57小鼠,随机分为对照组(n=16)与暴露组(n=16)。暴露组给予每天6 h 5000 m低压低氧暴露,持续4w;对照组无低压低氧暴露。两组小鼠其他饲养条件一致。利用Morris水迷宫实验检测每组小鼠空间记忆能力;免疫印迹实验检测Glu R1蛋白ser831和ser845位点磷酸化水平变化;透射电镜实验观察低氧对突触囊泡的影响。结果:(1)水迷宫结果显示慢性间歇低压低氧暴露后,暴露组平均逃脱潜伏期(17.6±1.69 s)显著低于对照组(27.3±1.45 s),暴露组小鼠平台搜索能力提升;(2)免疫印迹结果显示,暴露组小鼠海马Glu R1蛋白ser831和ser845位点磷酸化水平显著高于对照组小鼠;(3)透射电镜结果显示,暴露组小鼠海马区突触囊泡数目显著多于对照组,且差异有统计学意义。结论:慢性间歇低压低氧暴露可以显著提升C57小鼠空间认知功能,其机制可能是通过增加Glu R1蛋白ser831和ser845位点磷酸化水平,并增加突触结构内囊泡数目。     

急性低压低氧对大鼠空间学习记忆的影响及与海马内孤啡肽的关系

目的:研究暴露在不同海拔高度的急性低压低氧环境中,大鼠空间学习记忆能力的变化及与海马内孤啡肽的关系.方法:采用低压舱模拟4 500 m(中度)和7 500 m(重度)两种海拨高度,Morris水迷宫训练方法和反转录多聚酶链式反应(RT-PCR)技术.结果:①海马内孤啡肽mRNA表达在急性重度低压低氧(8 h/d,连续6 d)后明显增加,然而在Morris 水迷宫训练(6 counts/d,连续6 d,定位航行潜伏期逐渐缩短)后则显著降低.②急性低压低氧后,定位航行潜伏期明显延长,而海马内孤啡肽mRNA表达较学习记忆训练组明显升高.结论:海马内孤啡肽参与急性低压低氧降低大鼠空间学习记忆的机制.     

Comparisons of serum testosterone and corticosterone between exercise training during normoxia and hypobaric hypoxia in rats

The purpose of this study was to compare the effects of continuous and intermittent exercise training on serum testosterone [T] and corticosterone concentrations [Cort] during normoxia and hypobaric hypoxia. Male rats swam with loads of 3% (normoxia) or 2.25% (462 mmHg) body mass for 60 min in the continuous training groups, and 15 min separated by a 7-min rest  × 4, with 60-min total exercise duration in the intermittent training groups, 5␣days · week⁻¹ for 6 weeks. Serum [T] were measured at␣rest and following exercise after 6 weeks of training. Serum [Cort] were measured immediately after an acute period of exercise or after 6 weeks of training at rest and following exercise. Continuous exercise induced decreases in [T] under both conditions. Intermittent exercise showed a tendency to increase [T] during normoxia, but caused a suppression during hypobaric hypoxia. The [Cort] was elevated by a similar margin after an acute period of exercise during both conditions. After 6 weeks of training, however, [Cort] increased slightly after exercise during normoxia. A lower resting [Cort], which was increased after exercise, was found in the training groups during hypoxia. No relevant relationship was found between the behaviours of [T] and [Cort] after exercise during either conditions. Accepted: 20 April 1998     

间歇性低氧对小白鼠营养代谢的影响

目的：探讨间歇性低氧对营养代谢功能的影响。方法：健康小白鼠随机分为正常、正常低氧、高脂、高脂低氧。通过17d的喂养和低氧训练,测量动物的体重、血糖、血胆固醇含量的变化及肝脏组织切片。结果：经间歇性低氧处理明显抑制高脂高糖饲养导致的体重、血糖、血胆固醇增高,肝脏脂肪细胞分布的密度和范围均比单纯高脂组有所降低。结论：适度的间歇性低氧可以降低血糖以及血液中胆固醇的水平,减轻体重,并可以有效防止肝细胞脂肪变性。