Expressions of iron transport related proteins in the spinal cord of amyotrophic lateral sclerosis transgenic mice / 解剖学报

Objective To investigate the relationship between the expressions of iron transport related proteins and the dysregulation of iron homeostasis in the spinal cord of amyotrophic lateral sclerosis (ALS) transgenic mice. Methods The hSOD1

The establishment of atherosclerotic cell model and the preliminary study of intracellular iron metabolism disorder / 介入放射学杂志

Objective To establish the atherosclerosis (AS) cell model,to study the relationship between the iron metabolism and AS,and to explore the molecular mechanisms of iron metabolism disorders within the macrophages in AS plaques so as to provide theoretical basis for clinical intervention of iron metabolism and for the prevention and treatment of AS.Methods RAW264.3 cells were used to prepare foam cells through oxidized low density lipoprotein (ox-LDL) induced oxidation.In order to verify the foam cells,oil red staining and enzyme-linked immunosorbent were employed to assay the total lipids and free lipids (including total cholesterol and free cholesterol) in the foam cells.Western blotting was adopted to test the levels of iron metabolism-associated ferritin (FT) and ferroportin1 (FPN1);immunohistochemistry and immunofluorescence methods were used to determine and locate the expressions of FPN1 in foam cells.Results Large amounts of red lipid granules could be seen within the cytoplasm of ox-LDL-induced foam cells,and a great quantity of lipid fused into the form of large oil droplets,which was consistent with the morphological characteristics of foam cells.Massive lipid was accumulated in the foam cells,and the proportion of cholesterol ester (CE) in the foam cells was significantly higher than that in the normal cells (P＜ 0.05).Compared with the normal control group,FT content in the foam cells was obviously higher than that in the normal cells;the content of FPN1 was increased,and the increased content of FPN1 mainly existed in cytoplasm.Conclusion In the foam cells the iron metabolism is disordered and a great quantity of FPN1 is accumulated in cytoplasm.The non-cell-membrane localization of FPN1 can prevent iron from being effectively discharged from macrophages and can increase the accumulation of iron in foam cells,which may aggravate the formation and development of AS plaques.

Effects of obesity on the expression of hepcidin, lipocalin-2 and ferroportin-1 related with iron metabolism of mice’ s liver / 中国比较医学杂志

Objective We established the animal models of obesity induced by high-fat diet, in order to study the mRNA and protein expression of regulation molecules related with iron metabolism about hepcidin, lipocalin-2 ( LCN2 ) , ferroportin-1 (FPN1) in obese mice’ s liver and the molecular regulation mechanism.Methods C57BL/6J (4 ~6 weeks) mice were randomly divided into control group and obesity model group, each group of ten.The obesity group were fed with a high-fat diet and the control group were given the normal diet for lasting 15 weeks.After we successfully established the obesity animal model, the expression level of hepcidin, LCN2 and FPN1 mRNA in the liver were measured by Real-time fluorescent quantitative PCR method and the protein expression level of LCN2 and FPN1 were measured by Western-Blot.Results Compared with the control group, the expression level of hepcidin mRNA in the liver was increased in obesity group (P 0.05).Conclusion Obesity can increase the expression of hepcidin mRNA, however, there was no significantly effect on the expression of LCN2, FPN1.So, we can’t think that obesity can affect the expression of LCN2 and FPN1, lead to the ability of cells uptake and release iron abnormal, then appear iron metabolism disorders.As a result, leading to iron deficiency.Maybe obesity can affect other regulatory molecules related with iron metabolism through up-regulation the expression of Hepcidin or the more complex regulatory mechanisms.We still need further experimental research and exploration.This research also provides the basis of theoretical and experimental for the further study the effects of obesity on the expression of regulation molecules related with iron metabolism in obesity mice’ s liver and the mechanism of iron deficiency.

Effect of lipopolysaccharide on the iron metabolism in macrophages / 解剖学报

Objective To study the effect of lipopolysaccharide ( LPS ) on the activity of primary cultured macrophages and the distribution of divalent metal transporter 1 ( DMT1 ) and ferroportin 1 ( FPN1 ) .Methods Primary cell culture , MTT chromotest , cytochemistry chromotest and cell immunofluorescence techniques were used in this work . Results DMT1 was mainly distributed in the cytoplasm , which illuminates that DMT1 mediates the macrophage intracellular transit of iron from phagolysosome to cytoplasm .FPN1 was distributed in the cytoplasm and membrane , and the cytoplasm was the main site of FPN 1 distribution in macrophages .Conclusion Iron liberation from heme inside the phagolysosome occurs after erythrophagocytosis and it is possible that FPN 1 mediates intracellular transit of iron released by heme catabolism .The study found that LPS promoted the cell growth and this effect was reached to the peak in the 10 -5μg/L LPS group, but the cell growth was blocked with the increase of LPS concentration .

Effect of obesity on the expression of DMT1 and FPN1 in the duodenal in mice / 中国比较医学杂志

Objective To study the expression of divalent metal transporter 1(DMT1)and ferroportin 1(FPN1)in obese mice’ s duodenal epithelium and investigate the mechanism of the effect of obesity on iron absorption in mice. Methods C57BL/6J mice were randomly divided into control group and obesity model group, each group of 6, To establish obese mice model by having a high-fat diet and the control group were fed with a normal diet for 12 weeks.After completion of modeling, The level of DMT1 and FPN1 mRNA expression in the duodenum were measured by real-time fluorescent quantitative PCR( Real-time PCR) method, the protein expression of FPN1 was measured by Western-Blot. Results Compared with the control group, the level of DMT1、FPN1 mRNA and FPN1 protein expression in the duodenum were decreased significantly in obese mice ( P <0.05 ) .Conclusion Obesity can decrease the expression levels of DMT1、FPN1 mRNA and FPN1 protein and induce iron deficiency,in order to provide experimental and theoretical basis for studying the mechanism of iron deficiency caused by obesity further.

Quantitative study of iron metabolism-related genes expression in rat / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the multiple iron metabolism-related genes expression, its regulation by iron and the expression correlation among the genes in rat tissues.</p><p><b>METHODS</b>Two groups (n=30) of Sprague-Dawley female weanling rats were fed with a control diet and an iron deficient diet respectively for 4 weeks. All rats were then sacrificed, and blood and tissue samples were collected. The routine blood examination was performed with a veterinary automatic blood cell analyzer. Elemental iron levels in liver, spleen and serum were determined by atomic absorption spectrophotometry. The mRNA expression of genes was detected by real-time fluorescence quantitative PCR.</p><p><b>RESULTS</b>After 4 weeks, the hemoglobin (Hb) level and red blood cell (RBC) count were significantly lower in the iron deficient group compared with those in the control group. The iron levels in liver, spleen and serum in the iron deficient group were significantly lower than those in the control group. In reference to small intestine, the relative expression of each iron-related gene varied in the different tissues. Under the iron deficiency, the expression of these genes changed in a tissue-specific manner. The expression of most of the genes significantly correlated in intestine, spleen and lung, but few correlated in liver, heart and kidney.</p><p><b>CONCLUSION</b>Findings from our study provides new understandings about the relative expression, regulation by iron and correlation among the mRNA expressions of transferrin receptors 1 and 2, divalent metal transporter 1, ferritin, iron regulation proteins 1 and 2, hereditary hemochromatosis protein, hepcidin, ferroportin 1 and hephaestin in intestine, liver, spleen, kidney, heart, and lung of rat.</p>

Effects of lipopolysaccharide on mice iron metabolism related genes mRNA expression / 解剖学报

ObjectiveTo investigate the effects of lipopolysaccharide (LPS) on mRNA expression of iron metabolism related genes. Methods Ten male mice (2 months) were injected intraperitoneally with lipopolysaccharide(0.5 μg/g). After 6 hours, mice were sacrificed and then sera, liver and spleen were collected. The mice blood routine was measured. The serum iron and total iron binding capacity (TIBC) were determined with reagent kit. The quasi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed for mRNA of hepatic hepcidin(HP), ferroportin1(Fpn1), transferrin receptor 1(TfR1) and spleenic HP, Fpn1 and interleukin-6(IL-6). Results The serum iron and TIBC were reduced in mice injected LPS, which exhibited mild anemia(P＜0.05) . LPS can increase the expression of hepatic hepcidin and decrease Fpn1 and TfR1 in liver after LPS administration 6 hours(P＜0.05). In spleen, IL-6 was upregulated and Fpn1 downregulated(P＜0.05). Conclusion LPS can influence serum iron through regulating the mRNA expression of hepatic and spleenic iron metabolism related genes, such as HP, Fpn1 and TfR1.

Cadmium increases ferroportin-1 gene expression in J774 macrophage cells via the production of reactive oxygen species

Cadmium intoxication has been associated with the dysregulation of iron homeostasis. In the present study, we investigated the effect of cadmium on the expression of ferroportin 1 (FPN1), an important iron transporter protein that is involved in iron release from macrophages. When we incubated cadmium with J774 mouse macrophage cells, FPN1 mRNA levels were significantly increased in a dose- and time-dependent manner. Furthermore, the cadmium-induced FPN1 mRNA expression was associated with increased levels of FPN1 protein. On the other hand, cadmium-mediated FPN1 mRNA induction in J774 cells was completely blocked when cells were co-treated with a transcription inhibitor, acitomycin D. Also, cadmium directly stimulated the activity of the FPN1-promoter driven luciferase reporter, suggesting that the cadmium up-regulates FPN1 gene expression in a transcription-dependent manner. Finally, cadmium exposure to J774 macrophages increased intracellular reactive oxygen species (ROS) levels by ~ 2-fold, compared to untreated controls. When J774 cells were co-treated with antioxidant N-acetylcystein, the cadmium-induced FPN1 mRNA induction was significantly attenuated. In summary, the results of this study clearly demonstrated that cadmium increased FPN1 expression in macrophages through a mechanism that involves ROS production, and suggests another important interaction between iron and cadmium metabolism.

Effect of Copper on the Regulation of Ferroportin-1 Gene Expression

Ferroportin-1 (FPN) is a transporter protein that is known to mediate iron export from macrophages. The purpose of this study was to investigate the effect of copper on the regulation of FPN gene expression in J774 mouse macrophage cells. J774 cells were treated with various concentrations of CuSO4 and RT-PCR analyses were performed to measure the steady-state levels of mRNAs for FPN and divalent metal transporter 1 (DMT1, an iron importer). Copper treatment significantly increased FPN mRNAs in a dose-dependent manner, but didn't change the levels of DMT1 mRNA. Experiments with transcriptional inhibitor actinomycin D (0.5 microgram/mL) revealed that copper treatment did not affect the half-life of FPN mRNAs in J774 cells. On the other hand, results from luciferase reporter assays showed that copper directly stimulated the promoter activity of FPN. In summary, our data showed copper induced FPN mRNA of macrophages via a transcriptional rather than post-transcriptional mechanisms.

Effects of various metal ions on the gene expression of iron exporter ferroportin-1 in J774 macrophages

Macrophages play a key role in iron metabolism by recycling iron through erythrophagocytosis. Ferroportin-1 (FPN1) is a transporter protein that is known to mediate iron export from macrophages. Since divalent metals often interact with iron metabolism, we examined if divalent metals could regulate the expression of FPN1 in macrophages. J774 macrophage cells were treated with copper, manganese, zinc, or cobalt at 10, 50, or 100 microM for 16 to 24 h. Then, FPN1 mRNA and protein levels were determined by quantitative real-time PCR and Western blot analyses, respectively. In addition, effects of divalent metals on FPN1 promoter activity were examined by luciferase reporter assays. Results showed that copper significantly increased FPN1 mRNA levels in a dose-dependent manner. The copper-induced expression of FPN1 mRNA was associated with a corresponding increase in FPN1 protein levels. Also, copper directly stimulated the activity of FPN1 promoter-driven reporter construct. In contrast, manganese and zinc had no effect on the FPN1 gene expression in J774 cells. Interestingly, cobalt treatment in J774 cells decreased FPN1 protein levels without affecting FPN1 mRNA levels. In conclusion, our study results demonstrate that divalent metals differentially regulate FPN1 expression in macrophages and indicate a potential interaction of divalent metals with the FPN1-mediated iron export in macrophages.

The effect of burns on iron-related proteins in liver and spleen of different sex mice / 西安交通大学学报(医学版)

Objective To investigate the changes of iron-related proteins in different sex mice before and after burns.Methods The burn model in mice were prepared with 90 ℃ vapor.Both liver and spleen iron contents were measured by colorimetric method.Hepatic hepcidin and Fpn1 mRNA content were determined with RT-PCR.The Fpn1 protein was determined with Western blot.Results After burns,the content of spleen iron was increased and the expression of hepatic hepcidin was decreased in male mice,and at the same time the Fpn1 of liver and spleen was reduced.The content of iron homeostasis abnormality in female mice was less serious.Conclusion The change of iron-related proteins after burns may be one cause of iron homeostasis abnormality.The differences of iron-related protein expression in different sex after burns suggest that estrogen has a potentially protective function.