| BackgroundIron is one of the most important elements, widely participates in vivo metabolism process. Therefore the body needs to have a strict regulatory mechanism of iron, can provide enough iron to perform their physiological function, but also prevents production of iron toxicity due to overload in order to maintain iron homeostasis. The liver is the main iron storage site in the body, while the regulation of iron homeostasis play a central and pivotal role of hepcidin and other iron metabolism of many important proteins are secreted by the liver-specific synthesis. Iron homeostasis in cell is primarily through the IRE/IRP system of regulation, iron homeostasis in body is mainly through hepcidin regulation.Clinical data and epidemiological investigation found that serum iron levels in diabetic group were higher than that in the control group. Increased iron indices have been associated with the development of diabetes and its complications. While in vitro experiments, it showed that the H-ferritin mRNA of islets in mice treated with 20mmol/L glucose was 4-8 times higher than that treated with lmmol/L glucose. In vivo studies it showed that TfR expression increased and DMT1 expression decreased in the STZ-induced diabetic rat kidney. The main clinical features of diabetes is elevated blood glucose, which can stimulate cells to produce excess ROS. Excessive ROS play an important part not only in direct cytotoxicity, but also in the expression of the gene through regulation of cell signaling system.More and more evidence reveals that there is an interaction between iron metabolism and glucose metabolism,which is that iron affect glucose metabolism and long-term high blood glucose damage the iron metabolism pathway. But the exact mechanism between them has not been fully understood, which need further study and discussion.ObjectIn this study, we cultured normal human heptical cells LO2 in different concentrations of glucose environment in vitro, to investgate the relationship between cells treated with different concentrations of glucose and iron metabolism regulatory protein expression for the basic research. Method1.Effects of different concentrations of glucose on iron metabolism-related proteins of LO2 cells.LO2 cells was cultured in Dulbecco's Modified Eagle Medium (DMEM, glucose,5.5 mM) containing 10%(v/v) newborn calf bovine serum (CBS),100 U/ml penicillin, and 100μg/ml streptomycin in a humidified atmosphere with 5%CO2 at 37℃. Cells were exchanged medium and passaged every 2 days. For experiments,cells were detached with 0.25% trypsin plus 0.02% EDTA, passaged into 6-well culture dishes,96-well culture dishes and grown in DMEM containing 10% CBS until 70-80%.Then the medium was replaced with DMEM containing 2% CBS,and the cells were allowed to adjust to the medium conditions for 24 h. Cells were cultured in 5.5mM glucose DMEM,15mM glucose DMEM and 25mM glucose DMEM respectively for 0-,12-,24-,48h.ROS was detected by reactive oxygen species assay.MDA was detected by malondialdehyde assay. IRP1,TfR1,TfR2 and hepcidin-25 were detected by western blotting.2.Effects of H2O2 on iron metabolism-related proteins of LO2 cells.Cells were cultured in 5.5mM glucose DMEM with 2% CBS. The 100μmol/LH2O2 was added in to the groups for 60min.ROS was detected by reactive oxygen species assay.MDA was detected by malondialdehyde assay. IRP-1,TfR-1,TfR-2 and hepcidin-25 were detected by western blotting.3. Effects of Vit.E on iron metabolism-related proteins of LO2 cells induced different concentrations of glucose.Cells were treated with Vit-E (0.1mg/L,l mg/L,10 mg/L) in the medium that contained 5.5 mM/15 mM/25 mM glucose with 2% new-born calf serum for 24h.ROS was detected by reactive oxygen species assay.MDA was detected by malondialdehyde assay. IRP-1,TfR-1,TfR-2 and hepcidin-25 were detected by western blotting.Results are presented as mean±SEM. One-way analysis of variance (ANOVA) followed by LSD was used to compare the differences between the means in more than two groups. Data are presented as mean±SEM. P<0.05 was considered to be statistically significant.Result1.Effects of different concentrations of glucose on iron metabolism-related proteins of LO2 cells.1.1 Carboxy-H2DCFDA-stained cells were observed by fluorescent microscope12h,24h,48h, fluorescence intensity gradually increased with the rise of glucose concentration.Fluorescence intensity of each glucose group were prolonged with a gradual increase with time, fluorescence intensity in 15mmol/L and 25mmol/L glucose group were more obvious than that in 5.5mmol/L.1.2 The expression level of ROS and MDA in LO2 cells induced by different concentrations of glucoseCompared with 5.5mmol/L group:12h, ROS and MDA levels in 15mmol/L group were increased by 44.8% and 96.5%, ROS and MDA levels in 25mmol/L group were increased by 76.3%and 227.8%(P<0.01);48h, ROS and MDA levels in 15mmol/L group were increased by 56.8% and 145.3%, ROS and MDA levels in 25mmol/L group were increased by 92.9% and 208.7%(P<0.01).Compared with Oh:ROS and MDA levels in 5.5mmol/L group were increased by 26.3% and 17.3% at 24h,37.5% and 34.4% at 48h (P<0.05); ROS and MDA levels in 15mmol/L group were increased by 72.6% and 98.4% at 12h,111.8% and 177.3% at 24h,155.2% and 236% at 48h (P<0.001). ROS and MDA levels in 25mmol/L group were increased by 101.5% and 262.1% at 12h,145.3% and 310.6% at 24h,171.9% and 324.3% at 48h (P<0.001).1.3 The expression of iron metabolism-related proteins in LO2 cells induced by different concentrations of glucose1.3.1 The expression of IRP1 in LO2 cells induced by different concentrations of glucoseCompared with 5.5mmol/L group:12h, the expression of IRP1 in 15mmol/L group and 25mmol/L group were increased by 23.5% and 40.7%(P<0.01); 24h, the expression of IRP1 in 15mmol/L group and 25mmol/L group were increased by 85% and 116.8% (P<0.001);48h, the expression of IRP1 in 15mmol/L group and 25mmol/L group were increased by 118.4% and 166.9%(P<0.001).Compared with Oh:There was no significant change in the expression of IRP1 in 5.5mmol/L glucose groups (P>0.05).The expression of IRP1 in 15mmol/L group were increased by 37.9% at 12h,77% at 24h,109% at 48h(P<0.001); The expression of IRP1 in 25mmol/L group were increased by 45.3% at 12h,91.9% at 24h,136.3% at 48h(P<0.001).1.3.2 The expression of TfRl in LO2 cells induced by different concentrations of glucoseCompared with 5.5mmol/L group:12h, the expression of TfRl in 15mmol/L group and 25mmol/L group were increased by 23.8% and 45.9%(P<0.01); 24h, the expression of TfR1 in 15mmol/L group and 25mmol/L group were increased by 52.2% and 78.5% (P<0.001);48h, the expression of TfR1 in 15mmol/L group and 25mmol/L group were increased by 100.1% and 111.2%(P<0.001)Compared with Oh:There was no significant change in the expression of TfR1 in 5.5mmol/L glucose groups (P>0.05).The expression of TfR1 in 15mmol/L group were increased by 31.6% at 12h,56.4% at 24h,100.4% at 48h (P<0.001); The expression of TfR1 in 25mmol/L group were increased by 51% at 12h,78.6% at 24h,106% at 48h (P<0.001).1.3.3 The expression of TfR2 in LO2 cells induced by different concentrations of glucoseCompared with 5.5mmol/L group:12h, the expression of TfR2 in 15mmol/L group and 25mmol/L group were increased by 90.4% and 123.1%(P<0.01); 24h, the expression of TfR2 in 15mmol/L group and 25mmol/L group were increased by 141.2% and 159.2% (P<0.001);48h, the expression of TfR2 in 15mmol/L group and 25mmol/L group were increased by 190.6%and 207.5%(P<0.001).Compared with Oh:There was no significant change in the expression of TfR2 in 5.5mmol/L glucose groups (P>0.05).The expression of TfR2 in 15mmol/L group were increased by 88.4% at 12h,133.6% at 24h,183% at 48h (P<0.001); The expression of TfR2 in 25mmol/L group were increased by 111.5% at 12h,140.5% at 24h,187% at 48h (P<0.001).1.3.4 The expression of hepcidin in LO2 cells induced by different concentrations of glucoseCompared with 5.5mmol/L group:There was no significant change in the expression of hepcidin in 5.5/15/25 mmol/L glucose groups at 12h (P>0.05); 24h, the expression of hepcidin in 15mmol/L group and 25mmol/L group were increased by 85.9% and 84% (P<0.001);48h, the expression of hepcidin in 15mmol/L group and 25mmol/L group were increased by 126.1% and 136.3% (P<0.001)Compared with Oh:There was no significant change in the expression of hepcidin in 5.5mmol/L glucose groups (P>0.05).The expression of hepcidin in 15mmol/L group were increased by 105.4% at 24h,134% at 48h (P<0.001); The expression of hepcidin in 25mmol/L group were increased by 91.5% at 24h,130.4% at 48h (P<0.001)2.Effects of H2O2 on iron metabolism-related proteins of LO2 cells.2.1 Carboxy-H2DCFDA-stained cells were observed by fluorescent microscopeThe fluorescence in H2O2 groups was significantly enhanced compared with the control groups.2.2 The expression level of ROS and MDA in LO2 cells induced by H2O2The expression level of ROS and MDA in H2O2 groups were significantly increased by 132.9% and 233.3% compared with the control groups (P<0.001)2.3 The expression of iron metabolism-related proteins in LO2 cells induced by H2O2The expression of IRPl,TfRl,TfR2 in H2O2 groups were increased by 95.4%,61.4% and 144.7% compared with the control groups (P<0.001).There was no significantly different but a downward trend in the expression of hepcidin in H2O2 groups comparing with the control groups (P>0.05)3 Effects of Vit.E on iron metabolism-related proteins of LO2 cells induced different concentrations of glucose.3.1 Carboxy-H2DCFDA-stained cells were observed by fluorescent microscopeThere were no significant changes in the fluorescence intensity between 5.5mmol/L glucose groups.The fluorescence intensity in 15mmol/L and 25mmol/L glucose groups with 1 mg/L Vit.E,decreased more significantly than the control groups.3.2 The effect of Vit.E on the expression level of ROS and MDA in LO2 cellsCompared with the control groups:There was no significant change in the expression level of ROS and MDA in 5.5mmol/L glucose groups.15mmol/L, in lmg/L Vit.E and 10 mg/L Vit.E,ROS were decreased by 17.5% and 7.4%, MDA were decreased by 44.2% and 29%(P<0.01); 25mmol/L, in lmg/L Vit.E and 10 mg/L Vit.E groups,ROS were decreased by 20.6% and 16%, MDA were decreased by 49% and 34.5%(P<0.01); There were no significant changes in the expression level of ROS and MDA in 0.1 mg/L Vit.E groups compared with the control group (P>0.05)3.3 The effect of Vit.E on the expression of IRP1,TFR1 and TfR2 in LO2 cellsCompared with the control groups:There was no significant change in the expression of IRP1,TFR1 and TfR2 proteins in 5.5mmol/L groups(P>0.05).15mmol/L,the expression of IRP1 in lmg/L Vit.E and 10 mg/L Vit.E were decreased by 33.2% and 23.2%, TfRl were decreased by 18.5% and 13.8%, TfR2 were decreased by 22.9% and 21.9%(P<0.01). 25mmol/L, the expression of IRP1 in lmg/L Vit.E and 10 mg/L Vit.E were decreased by 21.9% and 14.3%, TFR1 were decreased by 22.9% and 17.7%, TfR2 were decreased by 14.2% and 11.1%(P<0.01).There was no significant change in the expression of TfRl and TfR2 in lmg/L Vit.E and 10 mg/L Vit.E groups(P>0.05).3.4 The effect of Vit.E on the expression of hepcidin in LO2 cellsCompared with the control groups:There was no significant change in the expression of hepcidin in each concentrations of groups(P>0.05). Compared with 5.5mmol/L groups, the expression of hepcidin in 15mmol/L and 25mmol/L groups were significantly increased (P<0.001). There was no significant change in the expression of hepcidin between 15mmol/L and 25mmol/L groups (P>0.05).ConclusionsIn this study, we cultured normal human heptical cells LO2 in different concentrations of glucose environment in vitro, to investgate the relationship between cells treated with different concentrations of glucose and iron metabolism regulatory protein expression.Conclusion were made as follows:1. The expression levels of ROS,MDA, IRP1,TfR1 and TfR2 were increased significantly with increased concentration of glucose and cultrued time.2. The expression of IRP1,TfR1 and TfR2 were increased significantly with increased concentration of glucose.3. The expression of IRP1, TfR1, TfR2 was significantly increased in LO2 cells induced by H2O2.4. Vitamin E, a potent intracellular ROS scavenger could partially abolish ROS formation and upregulation of IRP1,TfR1,TfR2.5. The expression of hepcidin cultured in high glucose increased significantly. Whereas, there was no significantly different but a downward trend in the expression of hepcidin in LO2 cells treated with H2O2. The expression of hepcidin did not reduce when the level of reactive oxygen species decreased after Vit.E treatment.We can conclude that the excessive ROS formation induced by high glucose may be an important reason for the increased expression of IRP1, TfR1, TfR2.It indicated that the increased expression of hepcidin may be not induced by excessive ROS in high glucose condition,which may be affected by other factors.
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