高浓度葡萄糖下铁的神经毒性作用.docx

1、高浓度葡萄糖下铁的神经毒性作用Neurotoxic effects of iron overload under high glucose concentration*Shi Zhao1, Lin Zhang1, Zihui Xu1, Weiqun Chen21 Department of Endocrinology, Wuhan Central Hospital, Wuhan 430014, Hubei Province, China2 Central Laboratory, Wuhan Central Hospital, Wuhan 430014, Hubei Province, Chin

2、aShi Zhao and Lin Zhang contributed equally to this study.Corresponding author: Shi Zhao, Professor, Masters supervisor, Chief physician, Department of Endocrinology, Wuhan Central Hospital, Wuhan 430014, Hubei Province, China, zhaoshiwuhan .Received: 2013-08-29Accepted: 2013-11-02(N201304023)Acknow

3、ledgments: The authors would like to thank Xu ZH from the Department of Endocrinology, Wuhan Central Hospital, Wuhan, Hubei Province, China for assisting with paper editing and data discussion.Funding: The study was supported by the Natural Science Foundation of Hubei Province, No. 2010CDB09001.Grap

4、hical AbstractAbstract Iron overload can lead to cytotoxicity, and it is a risk factor for diabetic peripheral neuropathy. However, the underlying mechanism remains unclear. We conjectured that iron overload-induced neurotoxicity might be associated with oxidative stress and the NF-E2-related factor

5、 2 (Nrf2)/ARE signaling pathway. As an in vitro cellular model of diabetic peripheral neuropathy, PC12 cells exposed to high glucose concentration were used in this study. PC12 cells were cultured with ferric ammonium citrate at different concentrations to create iron overload. PC12 cells cultured i

6、n ferric ammonium citrate under high glucose concentration had significantly low cell viability, a high rate of apoptosis, and elevated reactive oxygen species and malondialdehyde levels. These changes were dependent on ferric ammonium citrate concentration. Nrf2 mRNA and protein expression in the f

7、erric ammonium citrate groups were inhibited markedly in a dose-dependent manner. All changes could be inhibited by addition of deferoxamine. These results indicate that iron overload aggravates oxidative stress injury in neural cells under high glucose concentration and that the Nrf2/ARE signaling

8、pathway might play an important role in this process.Key Wordsneural regeneration; peripheral nerve injury; iron overload; oxidative stress; diabetic peripheral neuropathy; reactive oxygen species; high glucose; PC12 cells; Nrf2/ARE; grants-supported paper; neuroregeneration Author contributions: Zh

9、ao S conceived and directed the study, revised the manuscript. Zhang L designed the study, conducted the experiments, analyzed the data, and drafted the manuscript. Xu ZH revised the paper. Chen WQ guided the study and provided technical support. All authors approved the final version of the paper.C

10、onflicts of interest: None declared.Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential informati

11、on or authorship/patent application/funding source disputations.INTRODUCTION Diabetic peripheral neuropathy is one of most common chronic complications induced by diabetic hyperglycemia, and is associated with axonal atrophy, blunted regenerative potential, demyelination, and loss of peripheral nerv

12、e fibers1. Although numerous factors contribute to diabetic peripheral neuropathy, including insulin-induced resistance to neuronal trophic support2, decreased (Na/K)-ATP-ase activity3 and Schwann cell dysfunction4, increased oxidative stress and mitochondrial dysfunction seem intimately associated

13、with nerve dysfunction and diminished regenerative capacity. Oxidative stress and apoptosis have been found to play crucial roles in diabetic peripheral neuropathy5-6. Under hyperglycemia, large amounts of reactive oxygen species are produced by the mitochondrial respiratory chain, and neuronal apop

14、tosis is increased7. Despite advances in understanding the etiology of diabetic peripheral neuropathy, few approved therapies exist for the pharmacological management of the disease. Therefore, identifying novel therapeutic strategies remains paramount.Iron is ubiquitous in cells and is essential fo

15、r biological functioning. Normal iron balance is maintained by meticulous regulation of its absorption from the intestine and release from macrophages. It is modulated in response to requirement from body iron stores and demand from erythropoiesis to prevent deleterious extremes of iron deficiency o

16、r excess8. However, without adequate management, excess amounts of free iron may cause progressive damage. In recent years, there has been increasing interest in brain iron metabolism during normal ageing, particularly as excessive iron deposition has been found in neurological disorders9. Iron over

17、load is also a risk factor for diabetes. The link between iron and diabetes was first recognized in pathologic conditions (hereditary hemochromatosis and thalassemia), but high levels of dietary iron also confer diabetes risk. Iron plays a direct and causal role in diabetes pathogenesis, which invol

18、ves both cell failure and insulin resistance. Iron also regulates metabolism in most tissues involved in energy homeostasis, with the adipocyte in particular having an iron- sensing role. The molecular mechanisms underlying these processes are numerous and incompletely understood, but include oxidat

19、ive stress and the modulation of adipokine and intracellular signal transduction pathways10.A large body of evidence shows that iron overload is closely related to diabetes mellitus as well as its chronic complications11-15; oxidative stress and inflammatory factors may play a pivotal role in this r

20、elationship16. However, there is no direct evidence on whether abnormal iron metabolism is related to diabetic neuropathy.In this study, we made use of a cellular model of diabetic peripheral neuropathy using PC12 cells exposed to high glucose concentration, and examined cell viability and apoptosis

21、 under iron overload. We measured the levels of reactive oxygen species and malondialdehyde, and the expression of the transcriptional activator NF-E2-related factor 2 (Nrf2). RESULTSIron overload aggravated high glucose concentration-induced neurotoxicity in PC12 cellsHyperglycemia was recently sho

22、wn to induce oxidative stress and generate reactive oxygen species in neurons, resulting in neuronal damage and dysfunction17. In addition, high glucose induced oxidative damage in PC12 cells18. Thus, we generated a cell culture model of diabetic peripheral neuropathy by culturing PC12 cells in high

23、 glucose (25 mmol/L). Iron overload was created by exposure to ferric ammonium citrate19. To determine the appropriate experimental concentration of ferric ammonium citrate, PC12 cells cultured under high glucose (25 mmol/L) were exposed to 12 different concentrations of the compound (0, 12.5, 25, 5

24、0, 100, 200, 300, 400, 500, 600, 700, 800 mol/L) for 24 hours.The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay was used to assess PC12 cell growth inhibition. Ferric ammonium citrate inhibited the growth of PC12 cells in a concentration-dependent manner (Figure 1A). The d

25、egree of growth inhibition could be divided into three phases according to ferric ammonium citrate concentrationthe initial phase (less than 50 mol/L), the rapid rising phase (50200 mol/L), and the plateau phase (more than 200 mol/L). The growth inhibitory effect was statistically significant when t

26、he 25, 100 and 400 mol/L treatment doses were compared with each other (P 0.05 or P 0.01). Hence, we chose these three concentrations of ferric ammonium citrate for use in the subsequent experiments.Deferoxamine is a chelating agent used to remove excess free iron from the body. Therefore, we examin

27、ed the effect of deferoxamine on our cell culture model of diabetic peripheral neuropathy. After PC12 cells were exposed to high glucose (25 mmol/L) for 24 hours, ferric ammonium citrate and/or deferoxamine were added and the cells were cultured for an additional 24, 48 or 72 hours, and cell viabili

28、ty was assessed (Figure 1BD). BACDEFigure 1 PC12 cell growth and viability were inhibited by ferric ammonium citrate (FAC) and rescued by deferoxamine (DFO) (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay).(A) The growth inhibition ratio of PC12 cells after FAC treatment. Cells w

29、ere exposed to 12 different concentrations of FAC (0, 12.5, 25, 50, 100, 200, 300, 400, 500, 600, 700 and 800 mol/L) under high glucose (25 mol/L) for 24 hours. The cell viability of PC12 cells after 24 hours (B), 48 hours (C) and 72 hours (D) of culture. Cell morphology of PC12 cells at 48 hours sh

30、owing that cells subjected to high glucose and/or FAC treatment failed to extend long neurites compared with the normal glucose concentration group. Deferoxamine protected PC12 cells by promoting neurite growth and cell proliferation(E).(AD) All data are the percentage to control PC12 cells without

31、supplementation with glucose, FAC or DFO. Data are shown as mean SD from triplicate experiments. One-way analysis of variance was adopted for multiple-group comparison; two-tailed Students t-test was used for intergroup comparison. aP 0.05, vs. HGG; bP 0.01, vs. 25 mol/L FAC group; cP 0.01, vs. 100

32、mol/L FAC group; dP 0.01, vs. 400 mol/L FAC group.NGG: Normal glucose concentration group; HGG: high glucose concentration group; FAC25: 25 mol/L FAC group; FAC100: 100 mol/L FAC group; FAC400: 400 mol/L FAC group; FAC + DFO: 400 mol/L FAC + 200 mol/L DFO group.Interestingly, our data showed that cell viability in the high glucose concentration group was significantly higher than in the normal glucose concentration group 24 hours after adding the drug (P 0.05), but there was no significan