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

Construction of Ndfip1 plasmid and its expression in neuroblastoma SH-SY5Y cells / 吉林大学学报(医学版)

Objective::To observe the expression of divalent metal transporter 1 (DMT1) in neurons under the condition of Ndfip1 overexpression, and to explore the regulation effect of Ndfip1 on DMT1. Methods:The Ndfip1 eukaryotic expression plasmid was constructed, and the human neuroblastoma SH-SY5Y cells were transfected with Ndfip1 plasmid; the transfection efficiency was detected. The SH-SY5Y cells transfected with Ndfip1 plasmid were used as experimental group,and the SH-SY5Y cells transfected with empty plasmid were used as control group. The expression intensities of DMT1 protein in the SH-SY5Y cells in two groups were observed by immunofluorescence method,and the expression levels of DMT1 protein in the SH-SY5Y cells in two groups were detected by Western blotting method. Results:The plasmid was amplified, electrophoretically separated and sequenced, and the results proved that the plasmid was successfully constructed. After transfection of SH-SY5Y cells with Ndfip1 plasmid for 48 h, the expression level of Ndfip1 was significantly increased compared with before transfection (P<0.01). The immunofluorescence results showed that the fluorescence intensity of DMT1 in the cells in experimental group was significantly decreased compared with control group. The Western blotting results showed that the expression level of DMT1 in the cells in experimental group was decreased (P<0.05) compared with control group. Conclusion:The overexpression of Ndfip1 can down-regulate the expression of DMT1 protein in the nerve cells, and Ndfip1 nerve has a negatively regulatory effect.

Sulfasalazine induces ferroptosis of breast cancer ZR-75-1 cells and its mechanism / 肿瘤

Objective: To observe the activation effect of sulfasalazine on ferroptosis of breast cancer ZR-75-1 cells, and to explore its possible mechanism. Methods: The different concentrations of sulfasalazine (0, 1.0, 0.5, 1.0, and 2.0 mmol/L) were used to treat ZR-75-1 cells. The morphological change of ZR-75-1 cells was observed under an inverted microscope. The proliferation inhibitory rate of ZR-75-1 cells was detected by CCK-8 method. The expression levels of glutathione peroxidase 4 (GPX4) and cysteine-glutamate antiporter subunit (xCT) proteins in ZR-75-1 cells were detected by Western blotting. The fluorescent probe was used to label the ZR-75-1 cells treated with different concentrations of sulfasalazine, then the change of reactive oxygen specie (ROS) level (as indicated by fluorescence intensity) was detected by FCM method. The expression level of divalent metal transporter 1 (DMT1) mRNA was detected by real-time fluorescent quantitative PCR. Results: Sulfasalazine caused the morphological changes and death of ZR-75-1 cells. The low doses of sulfasalazine had little effect on the proliferation of ZR-75-1 cells (P > 0.05), but the high concentrations of sulfasalazine inhibited cell proliferation significantly (P < 0.05). The high concentrations of sulfasalazine inhibited the expressions of GPX4 and xCT proteins in ZR-75-1 cells (both P < 0.05), and also promoted the accumulation of intracellular ROS (P < 0.05). Sulfasalazine increased the expression of DMT1 mRNA in ZR-75-1 cells (P < 0.05). Conclusion: Sulfasalazine can increase the accumulation of ROS by inhibiting the expressions of GPX4 and xCT proteins to induce the ferroptosis of ZR-75-1 cells. In which, the activation of DMT1 expression may be one of the mechanism.

Elevation of Divalent Metal Transporter 1 Protein in the Cerebellar Cortex of the APP/PS1 Transgenic Mouse / 中国医科大学学报

Objective To investigate the distribution of divalent metal transporter 1 (DMT1) in the cerebellum of APP/PS1 transgenic mouse. Methods Immunohistochemistry, immunofluorescence, and confocal laser scanning microscopy were used to analyze the relationship between DMTl and amyloid beta (Aβ) and their distribution in senile plaques. Western blotting was used to analyze DMT1 protein level in the APP/PS1 transgenic mouse cerebellum. Results DMTl and Aβ were mainly located in the amyloid plaques, which were predominately located in the molecular layer of the cerebellar cortex of the transgenic mouse. Only a few plaques could be seen in the Purkinje cell layer and granular layer. Confocal laser microscopy revealed the DMTl and Aβ were co-localized in senile plaques. Conclusion The abundant expression of DMTl protein suggests that DMTl and the divalent metal ions that it transports might be involved in the formation of Aβ senile plaques and other pathological processes in the cerebellum in Alzheimer' s disease.

Expression of DMT1 and its significance in hepatocellular carcinoma / 临床肝胆病杂志

ObjectiveTo measure the expression of divalent metal transporter 1 (DMT1) in hepatocellular carcinoma (HCC) and to explore its clinical and pathological significance. MethodsFifty-seven liver cancer specimens collected from patients with HCC and preserved in paraffin in Second Affiliated Hospital of Shantou University Medical College from July 1999 to July 2011 were established as HCC tissue group, and 7 specimens of normal liver tissues from autopsy and 8 specimens of liver tissues from patients with intrahepatic bile duct stones and preserved in paraffin were established as normal control group. SP immunohistochemistry was applied to measure the expression of DMT1 in 15 specimens of normal liver tissues and 57 specimens of HCC tissues. One-way analysis of variance was applied to analyze the difference in expression of DMT1 in HCC tissues with varying degrees of differentiation, and χ2 test was applied for the comparison of rates; the correlation between expression of DMT1 and tumor size was analyzed by Pearson correlation analysis. ResultsDMT1 in the liver tissues in normal control group was negative or weakly positive, with a positive rate of 20% (3/5); in HCC tissue group, the positive rate was 789% (45/57), with a strong positive rate of 15.8% (9/57); the two groups showed a significant difference in positive rate (P＜0.05). The expression of DMT1 varied significantly between patients with varying degrees of differentiation in HCC tissues (F=4.011, P＜0.05), while the positive rate of DMT1 showed no significant differences between patients with different clinical stages and with or without blood vessel infiltration (all P＞0.05). Correlation analysis did not find the correlation between expression of DMT1 and tumor size (r=-0.047, P＞005). ConclusionThe positive expression rate of DMT1 increases significantly in HCC tissues and is closely related to the degree of tumor differentiation, which is potentially valuable for clinical and pathological grading of liver cancer.

Effects of developmental iron deficiency and post-weaning iron repletion on the levels of iron transporter proteins in rats

BACKGROUND/OBJECTIVES: Iron deficiency in early life is associated with developmental problems, which may persist until later in life. The question of whether iron repletion after developmental iron deficiency could restore iron homeostasis is not well characterized. In the present study, we investigated the changes of iron transporters after iron depletion during the gestational-neonatal period and iron repletion during the post-weaning period. MATERIALS/METHODS: Pregnant rats were provided iron-deficient (< 6 ppm Fe) or control (36 ppm Fe) diets from gestational day 2. At weaning, pups from iron-deficient dams were fed either iron-deficient (ID group) or control (IDR group) diets for 4 week. Pups from control dams were continued to be fed with the control diet throughout the study period (CON). RESULTS: Compared to the CON, ID rats had significantly lower hemoglobin and hematocrits in the blood and significantly lower tissue iron in the liver and spleen. Hepatic hepcidin and BMP6 mRNA levels were also strongly down-regulated in the ID group. Developmental iron deficiency significantly increased iron transporters divalent metal transporter 1 (DMT1) and ferroportin (FPN) in the duodenum, but decreased DMT1 in the liver. Dietary iron repletion restored the levels of hemoglobin and hematocrit to a normal range, but the tissue iron levels and hepatic hepcidin mRNA levels were significantly lower than those in the CON group. Both FPN and DMT1 protein levels in the liver and in the duodenum were not different between the IDR and the CON. By contrast, DMT1 in the spleen was significantly lower in the IDR, compared to the CON. The splenic FPN was also decreased in the IDR more than in the CON, although the difference did not reach statistical significance. CONCLUSIONS: Our findings demonstrate that iron transporter proteins in the duodenum, liver and spleen are differentially regulated during developmental iron deficiency. Also, post-weaning iron repletion efficiently restores iron transporters in the duodenum and the liver but not in the spleen, which suggests that early-life iron deficiency may cause long term abnormalities in iron recycling from the spleen.

Influence of Iron Supplementation on DMT1 (IRE)-induced Transport of Lead by Brain Barrier Systems in vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the potential involvement of DMT1 (IRE) protein in the brain vascular system in vivo during Pb exposure.</p><p><b>METHODS</b>Three groups of male Sprague-Dawley rats were exposed to Pb in drinking water, among which two groups were concurrently administered by oral gavage once every other day as the low and high Fe treatment group, respectively, for 6 weeks. At the same time, the group only supplied with high Fe was also set as a reference. The animals were decapitated, then brain capillary-rich fraction was isolate from cerebral cortex. Western blot method was used to identify protein expression, and RT-PCR to detect the change of the mRNA.</p><p><b>RESULTS</b>Pb exposure significantly increased Pb concentrations in cerebral cortex. Low Fe dose significantly reduced the cortex Pb levels, However, high Fe dose increased the cortex Pb levels. Interestingly, changes of DMT1 (IRE) protein in brain capillary-rich fraction were highly related to the Pb level, but those of DMT1 (IRE) mRNA were not significantly different. Moreover, the consistent changes in the levels of p-ERK1/2 or IRP1 with the changes in the levels of DMT1 (IRE).</p><p><b>CONCLUSION</b>These results suggest that Pb is transported into the brain through DMT1 (IRE), and the ERK MAPK pathway is involved in DMT1 (IRE)-mediated transport regulation in brain vascular system in vivo.</p>

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.