Mechanisms Of Iron Overload Induced Injury In Human Hepatocytes

Myelodysplastic syndrome(MDS) is a group of heterogeneous clonal disorders derived from hematopoietic myeloid directional stem cells or multipotent stem cells. The incidence trend of MDS rises year by year in China, which has reached three over one hundred thousand. Furthermore, onset age of MDS patients in China is ten years younger than western countries. Current therapeutic strategies for the treatment of MDS mainly comprise hematopoietic stem cell transplantation(HSCT) and immunosuppression. More and more evidence indicates that the patient’s liver shows typical symptoms of iron overload, which leads to liver damage after HSCT. In addition, iron overload also has a negative impact on survival after allogeneic transplantation. Although clinical use of iron chelator such as desferrioxamine, deferiprone, deferasirox and other drugs can lower iron levels in the parenchymal cells and improve the prognosis of patients, the appropriate use of iron chelation therapy in patients is an area of considerable controversy and drug therapy is a very long and expensive process. A good understanding of molecular mechanism of iron overload-induced damage is therefore of paramount importance for treatment of iron overload in patients.Making use of human hepatocyte cell line HH4 as research models, our group examined iron overload-indued apoptosis, the effects of NF-κB on human Hepcidin expression, and the variations of the proteomics indued by iron overload in more detail. The results in this study were summerized as follows:(1) Research on iron overload-induced apoptosis. Hepatic HH4 cells were exposed to FAC(Ferric ammonium citrate) to force iron uptake, and cellular responses were determined. Incubation with 5 m M FAC resulted in increased intracellular iron content in a time-dependent manner. High concentration of FAC impaired cell viability and increased level of ROS(Reactive oxygen species); addition of antioxidant reagent such as GSH(Glutathione) or NAC(N-acetylcysteine) dramatically reduced FAC-induced intracellular ROS generation. FAC overload significantly increased the phosphorylation of IκB-α, p38 MAPK and NF-κB p65, and promoted the nuclear translocation of NF-κB p65. After 12-72 hr incubation with 5 mM FAC, expression levels of apoptosis-related genes in HH4 cells were significantly increased and subsequently apoptosis was confirmed by flow cytometric analysis. Knockdown of Fas and Bid expression by siRNA in iron-treated HH4 cells resulted in restoration of cell viability. Reduction of apoptosis by GSH was involved in downregulation of p-IκB-α, p-p38 MAPK, p-NF-κB, Caspase-8, Cytc and Caspase-3, which are key molecules for oxidative stress-activated signaling pathways and apoptosis signaling pathways respectively. Experimental results in this section showed that iron overload triggered reactive oxygen species-mediated apoptosis via both extrinsic and intrinsic pathways in human hepatic cells.(2) Research on the effects of NF-κB on human Hepcidin expression. Non-transformed HH4 cells were exposed to FAC(0.1, 1, 5 and 10 mmol/L) for 48 hr, expression of iron regulatory genes Hepcidin were determined by semi-quantitative RT-PCR; The effects of NF-κB on Hepcidin activity were detected using Ch IP(Chromatin immunoprecipitation), EMSA(Electrophoretic mobility shift assays) and dual luciferase reporter system, combined with inhibition experiments of intracellular NF-κB activity. Results showed that 5 mmol/L and 10 mmol/L FAC treatment significantly enhanced expression of Hepcidin; ChIP and EMSA showed binding of NF-κB on upstream of the Hepcidin promoter. The luciferase activity of recombinant luciferase reporter plasmid was obviously higher than that of control group; Treatment with the NF-κB inhibitor BAY 11-7082 attenuated Hepcidin expression. Experimental results in this section showed that NF-κB played an important role in upregulation of human Hepcidin expression induced by iron overload.(3) Proteomics research of hepatic HH4 cells after iron overload treatment. To gain insight into the effect of iron overload on HH4 cells, 2D-LC-MS were carried out to compare the protein profiles of HH4 cells stimulated by FAC solution at concentrations of 0 and 5 mM, respectively. The identification results showed that compared to control group, 58 proteins were up-regulated(superior to 1.5 folds) and 35 proteins were down-regulated(inferior to 0.67 fold) in FAC-treatment group. Then we analyzed the main properties of these identified differentially expressed proteins via Gene Ontology(GO) analysis. The results showed that these proteins are involved in various biological process including endocytosis, di-, tri-valent inorganic cation homeostasis, response to wounding, inflammatory response, positive regulation of cytokine production, regulation of growth, anti-apoptosis, apoptotic mitochondrial changes and other processes. In addition, the experiments results of proteomics showed that protein expression level of TLR2 and IL6 ST had a statistically significant increase in iron overload group, respectively 7 times and 2.9 times. Subsequent western blot experiments and TLR2 gene interference experiments confirmed that iron overload activated TLR2-MyD88-p-NF-κB-IL-6 pathway in HH4 cells. On another face, phosphoproteomics experiments showed that iron overload caused 335 differentially expressed phosphorylated proteins, 11 percent of which were related to cell cycle progression. Meanwhile, the experiments results of phosphoproteomics also showed that iron overload treatment resulted in an increase of approximately 10.9 times on Thr 14/Tyr 15-phosphorylated CDK1. Further flow cytometry detection revealed that FAC treatment induced a significant G2/M phase arrest in HH4 and NMH(the non-transformed murine hepatocyte cell line) cells. While subsequent RT-PCR and western blot experiments indicated that FAC-induced G2/M phase arrest may be related to activation of p53-p21-CDK1, p53-14-3-3 sigma-CDK1 or 14-3-3 gamma pathway. These experimental results in this section showed that iron overload caused TLR2-mediated inflammatory response and G2/M phase arrest in human hepatic cells.