The Role Of Hepcidin In Regulating Plaque Stability In Atherosclerosis And The Underlying Mechanisms

BackgroundAtherosclerosis formation and plaque rupture are the common pathological basis of most of cardio-cerebrovascular diseases. It has been documented for decades that a state of sustained iron depletion or mild iron deficiency protects against atherosclerosis. Ferritin are highly expressed in human and rabbit atherosclerotic plaques and iron chelation is benefitial to the endothelial function in patients with coronary artery disease. Accumulation of ferritin and redox-active iron may contribute to the oxidative stress in atherogenesis. In macrophage, in the presence of iron, the oxidants can be activated to form ROS via the iron-catalyzed Haber-Weiss reaction or Fenton reaction. ROS are cytotoxic because of its ability to initiate lipid peroxidation, damage membranes,oxidize sulfhydryl compounds, and inactivate enzymes and transporters. Oxidative reactions associated with the over-loaded iron and lipids facilitate macrophage apoptosis with the release of cellular contents into the lesion, which further enhances inflammatory responses such as recruitment of more monocytes to amply this process.Recently, hepcidin has been demonstrated to be a key peptide in the regulation of iron homeostasis. Hepcidin binds to the iron transporter ferroportin1(Fp1) on the cell surface, and induces Fpn internalization and degradation. As a result, the intracellular iron level is elevated. Hepcidin is produced by a wide variety of cells including macrophages and hepcidin expression is increased in response to inflammation. Some inflammatory cytokines, including IL-6, LPS and IL-1were proven to stimulate the expression of hepcidin.Notably, hepcidin is a major determinant of the amount of iron retained in macrophages. Therefore, it has been proposed in2007by Sullivan, et al. that hepcidin promoted progression of atherosclerotic plaque by slowing or preventing the mobilization of iron from macrophages within atherosclerotic plaque. Hepcidin and metabolic syndrome are positively correlated. Recent research found that about15%of the metabolic syndrome patients were with iron overload, while about50%of the15%patients got NAFLD at the same time. Another report demonstrated recently that there was a positive correlation between hepcidin, macrophage iron, MCP-1and vascular damage in patients with metabolic syndrome.In summary, it has been proven that iron is associated with atherosclerosis and hepcidin is a key hormone in the regulation of iron balance and iron recycling, however, a direct, clear, and causal relationship between hepcidin and atherosclerotic lesion formation or plaque stability has not yet been established. In the present study, we studied the potential role of hepcidin in atherogenesis and plaque stability by hepcidin gain-and loss-of-function approaches in a mouse model of accelerated atherosclerosis and explored the underlying mechanisms.Objectives1. To observe the expression of hepcidin in the ApoE-/-mice atherosclerotic model.2. To explore the potential role of hepcidin in atherogenesis and plaque stability by hepcidin gain-and loss-of-function approaches in a ApoE-/-mice atherosclerotic model.3. To explore the underlying mechanisms of hepcidin on the development and progreesion of atherosclerosis.Methods1. Preparation of Adenoviral VectorsThe murine hepcidin cDNA was amplified by RT-PCR, cloned into pMD18-T vector, and then sub-cloned into pIRES2-EGFP vector using the EcoRI and BamH I sites. The hepcidin cDNA sequence was confirmed by sequencing. A shRNA sequence that is used to target hepcidin was cloned into the pcDNATM6.2-GW/EmGFPmiR vector and confirmed by sequencing. Both hepcidin and its shRNA IRES2-EGFP cassettes were cloned into the adenoviral expression vector pAd/CMV/V5-DEST using the Gateway Technology. Recombinant viruses were packaged and amplified in HEK293cells and purified by anion chromatography. The titer of the viral vectors was determined by TCID50method. The adenoviral vectors expressing EGFP alone (Ad-EGFP) was used as control.2. Animal Model and Gene TransferOur study in animal models consisted of two parts. In the first part of the in vivo study, forty male apoE-/-mice (6weeks of age) were randomly divided into a control group (n=20) and a model group (n=20). Mice in the control group were given a normal diet for13weeks and then euthanized. Mice in the model group received a high-fat diet for2weeks and then a constrictive silastic tube was placed around the left common carotid artery near its bifurcation. These mice were maintained on a high-fat diet for additional11weeks. The left common carotid arteries in both groups of mice were collected for histological and mRNA analysis.In the second part of the in vivo study, seventy-five male apoE-/-mice (6weeks of age) were given a high-fat diet for2weeks. Then a constrictive silastic tube was placed around the left common carotid artery near its bifurcation. Eight weeks after the collar placement, mice were randomly divided into three groups (n=25, each) for adenoviral gene delivery of EGFP, EGFP-hepcidin, and EGFP-hepcidin shRNA. These mice were maintained on a high-fat diet for additional3weeks, and then euthanized.3. Serum Index measurementAt the end point of the second part of the in vivo study, blood was collected from the inferior vena cava before perfusion-fixation. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein cholesterol (HDL), glucose, iron and hepcidin concentrations were measured.4. Histopathological and immunohistochemical measurementSections were stained with hematoxylin and eosin. Collagen was visualized by sirius red staining. Lipid deposition was identified by Oil-red O staining. Corresponding sections on separate slides were immunostained with anti-mouse monocyte/macrophage monoclonal antibodies, anti-a-smooth muscle (SM) actin monoclonal antibodies, anti-human interleukin-6(IL-6) polyclonal antibodies, anti-human tumor necrosis factor-a (TNF-a) monoclonal antibodies, anti-mouse monocyte chemoattractant protein-1(MCP-1) antibodies, anti-human matrix metalloproteinase (MMP-2) monoclonal antibodies, anti-mouse hepcidin monoclonal antibodies and anti-mouse ferritin antibodies. Positive staining areas of smooth muscle cells (SMCs), macrophages, lipids, collagen, IL-6, MCP-1, TNF-a, MMP-2, hepcidin, H-Ferritin and L-Ferritin were quantified by computer-assisted color-gated measurement, and the ratios of the positive staining area to the arterial cross sectional area (part1in vivo study) or plaque area (part2in vivo study) were calculated. The vulnerable index was calculated by the following formula:the relative positive staining area of (macrophages%+lipid%)/the relative positive staining area of (a-SMCs%+collagen%).5. ImmunofluorescenceTissue sections of the carotid arteries were incubated with double primary antibodies, including those against macrophages and hepcidin, SMCs and hepcidin, macrophages and ox-LDL as well as SMCs and ox-LDL. Fluorescent images were obtained by a laser scanning confocal microscopy.6. Measurement of non-heme iron by Atomic absorption spectrometryThe content of non-heme iron in atherosclerotic plaques were measured by flame atomic absorption spectrometry with a Solar M-6Atomic absorption spectrophotometer.7. Quantitative real-time RT-PCR analysisThe mRNA levels of Hepcidin,H-Ferritin,L-Ferritin,IL-6,MCP-1,TNF-a and MMP-2in the atherosclerotic plaques and the mRNA level of hepcidin in liver were quantified by RT-PCR.8. Statistical analysisAll analyses were performed using SPSS16.0(SPSS Inc., Chicago, IL). Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA with LSD post hoc test used for multiple comparisons. P<0.05was considered statistically significant.Results1. Detection of the restructuring adenovirus interference vectorsBoth the restructuring adenovirus expressing vector and interference vector were verified to be correct by sequencing validation. The virus tilter of Ad-hep is3.11×1012ifu/ml and the verus tilter of Ad-hep-shRNA is9.12×1012ifu/ml.2. The experssion of hepcidin in Atherosclerotic PlaqueRelative to the homolateral carotid arteries in the control group without atherosclerotic lesions, immunochemical staining and RT-PCR analysis revealed that both mRNA and protein expression levels of hepcidin were up-regulated in the carotid atherosclerotic plaques indicating the potential role of hepcidin in the pathogenesis of atherosclerosis. Hepcidin was located in macrophages ans SMCs.3. The result of transfection efficiency measurementElevated and comparable levels of fluorescent densities were observed in plaques of these infected carotid arteries, appearing at one week after infection and sustaining for additional two weeks. The ratio of the GFP positive staining area to the plaque area in the three treatment groups of mice two weeks after the infection was similar among these3groups.4. Effects of hepcidin on weightIn contrast, the local adenoviral gene delivery for three weeks hardly affected the weight.5. Effects of hepcidin on blood lipids and glucoseThe local adenoviral gene delivery for three weeks hardly affected the serum levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL),glucose.6. Effects of hepcidin on serum ironNeither the overexpression nor knockdown of hepcidin altered the serum iron by the collar placement in ApoE-/-mice relative to the control group.7. Effects of hepcidin on serum and liver hepcidinIn contrast, the local adenoviral gene delivery for three weeks hardly affected the serum hepcidin and the hepcidin mRNA expression in liver. 8. The expression of hepcidin in three groups after adenoviral deliveryImmunochemical staining and RT-PCR analysis confirmed the up-regulation of hepcidin in the carotid arteries of the Ad-hepcidine group and down-regulation of hepcidin in those of the Ad-hepcidin shRNA group.9. Effects of hepcidin overexpression or knockdown on lesion area in the three groupsNeither the overexpression nor knockdown of hepcidin altered the plaque size induced by the collar placement in ApoE-/-mice relative to the control group.10. Effects of hepcidin overexpression or knockdown on the plaque composition and stability of ApoE-/-mice in the three groupsThe plaque composition including macrophages, smooth muscle cells (SMCs), and collagen was significantly affected by hepcidin overexpression or knockdown. In the plaques of the Ad-hepcidin group, the relative contents of macrophages were increased whereas those of SMCs and collagen were decreased. In contrast, in the plaques of the Ad-hepcidin shRNA group, the relative contents of macrophages were reduced while those of SMCs and collagen were increased. The relative contents of lipids in plaques did not differ significantly among the Ad-EGFP, Ad-hepcidin and Ad-hepcidin shRNA groups. The plaque vulnerability index was elevated by the hepcidin overexpression but reduced by the hepcidin knockdown, respectively, suggesting that hepcidin plays a critical role in plaque destabilization rather than plaque formation.11. Effects of hepcidin overexpression or knockdown on the inflammatory cytokine expression in three groups of ApoE-/-miceThe expression levels of interleukin-6(IL-6), monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-alpha (TNF-α) and metalloproteinase-2(MMP-2) were substantially enhanced in both by hepcidin overexpression but were dramatically suppressed by hepcidin knockdown, suggesting that hepcidin destabilizes atherosclerotic plaques at least partly via exaggerating inflammatory responses in atherosclerotic lesions.12. Effects of hepcidin overexpression or knockdown on the levels of intracellular lipid in three groups of ApoE-/-miceThe expression level of ox-LDL in intraplaque macrophages was substantially enhanced by hepcidin over-expression but was dramatically suppressed by hepcidin knockdown.13. Effects of hepcidin overexpression or knockdown on the ferritin expression in three groups of ApoE-/-miceThe the expression of ferritin mRNA and protein in the atherosclerotic plaque was up-regulated or down-regulated by the local adenoviral over-expression of hepcidin or by its shRNA, respectively. These results demonstrated that hepcidin controls iron trapping in e atherosclerotic lesions.14. Effects of hepcidin overexpression or knockdown on the non-heme iron in three groups of ApoE-/-miceThe non-heme iron level in the atherosclerotic plaque was up-regulated or down-regulated by the local adenoviral over-expression of hepcidin or by its shRNA, respectively. These results demonstrated that hepcidin controls iron trapping in e atherosclerotic lesions.Conclusions1. In a mouse model of atherosclerosis, the expression of hepcidin was upregulated in atherosclerotic plaque and located on macrophages and SMCs;2. Hepcidin plays a critical role in plaque destabilization.3. Hepcidin destabilizes atherosclerotic plaques via exaggerating inflammatory responses, promoting iron trapping and intracellular lipids accumaulation in atherosclerotic lesions. BackgroundThe stability of atherosclerotic plaque has been a hot topic in recent years. It has been recognized for years that leukocytes and platelets play an important role in the pathogenesis of atherosclerosis and erythrocytes have been traditionally deemed as an innocent bystander in the process of atherosclerosis. However, recent studies have suggested that erythrocytes are important factors contributing to atherosclerotic plaque growth and destabilization. More iron deposition and macrophages accumulation had been found in the atherosclerotic plaques which were characterized by accumulation of lipid contents and thin fribrous cap of plaque. Moreover, plaques within intraplaque hemorrhage are vulnerable to new plaque hemorrhage, which not only stimulates the progression of atherosclerosis but also promotes the transition from a stable to an unstable lesion. Our lab had established a rabbit model of intraplaque hemorrhage and demonstrated that erythrocytes induce plaque vulnerability in a dose-dependent way. However, the exact mechanisms underlying the erythrocyte-induced atherosclerosis remain obscure. Intraplaque hemorrhage can lead to accumulation of lipid contents, augmentation of inflammation and thin fribrous cap of plaque, which may be the main mechanisms of erythrocyte-induced plaque vunerability.Some substances released from erythrocytes should be related to the erythrocyte-induced vulnerability of plaque. Several lines of evidence have suggested that cellular iron signaling and iron-mediated oxidative damage are related to cardiovascular diseases. Iron plays important roles in the initiation and progression of atherosclerosis and a state of sustained iron depletion or mild iron deficiency protects against atherosclerosis. Ferritin are highly expressed in human and rabbit atherosclerotic plaques and iron chelation is benefitial to the endothelial function in patients with coronary artery disease. Accumulation of ferritin and redox-active iron may contribute to the oxidative stress in atherogenesis. In macrophage, with the presence of iron, the oxidants can be activated to form ROS via the iron-catalyzed Haber-Weiss reaction or Fenton reaction. ROS are cytotoxic because of its ability to initiate lipid peroxidation, damage membranes, oxidize sulfhydryl compounds, and inactivate enzymes and transporters. Oxidative reactions associated with the over-loaded iron and lipids facilitate macrophage apoptosis with the release of cellular contents into the lesion, which further enhances inflammatory responses such as recruitment of more monocytes to amply this process. We found in our earlier studies that postive erythrocyte staining paralleled with not only the iron deposition but also macrophage infiltration.Hepcidin is a recently discovered key hormone in the regulation of iron homeostasis and iron recycling. It has been proved that autocrine hepcidin leads to iron sequestration in human monocytes. The expression of hepcidin is stimulated by inflammation. Hepcidin binds to the iron transporter ferroportin1(Fpn1) on the cell surface, and induces Fpn internalization and degradation. As a result, the intracellular iron level is elevated.It has been proposed in2007that hepcidin promoted progression of atherosclerotic plaque by slowing or preventing the mobilization of iron from macrophages within atherosclerotic plaque. Hepcidin and metabolic syndrome are positively correlated. Recent research found that about15%of the metabolic syndrome patients were with iron overload, while about50%of the15%patients got NAFLD at the same time. Another report demonstrated recently that there was a positive correlation between hepcidin, macrophage iron, MCP-1and vascular damage in patients with metabolic syndrome.In summary, it has been proven that erythrocytes and iron are associated with atherosclerosis and hepcidin is a key hormone in the regulation of iron balance and iron recycling, however, the role of hepcidin in the erythrocytosis-induced atherosclerotic progression is still unclear. In the present study, we studied the relationship between erythrocytes and the activation of macrophage in vitro and explored the potential role of hepcidin in this process and the underlying mechanisms. Objectives1. To observe the role of ox-LDL on the expression of hepcidin in macrophage.2. To explore the role of RBC in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.3. To explore the role of hepcidin in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.4. To explore the role of hepcidin in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in erythrophagocytosed macrophage.5. To explore the underlying mechanisms on hepcidin's role in the activation and apoptosis of macrophage after erythrophgocytosis.Methods1. Macrophage cultivation and erythrophagocytosisJ774macrophages were chosen for erythrophagocytosis and cultured in DMEM medium Erythrophagocytosis was performed as previously described with slight modifications, erythrocytes (2×109) were opsonized with goat anti-mouse IgG.The opsonized erythrocytes (2×107) were added to J774macrophage monolayers (2×106) and incubated for2hours. After uptake, noningested opsonized erythrocytes were removed using Red Blood Cell Lysis Buffer Control cells were subjected to the same lysis and washing steps as cells treated with erythrocytes.2. Transfection of siRNA targeting mouse hepcidin and the control siRNAHepcidin silence in cultured macrophages was achieved by hepcidin RNA interference approach.3. Quantification of transfection efficiencyMeasured the expression of hepcidin in both mRNA and protein levels and evaluted the transfection efficiency24hours after transfection.4. Cell grouping and treatmentPart Ⅰ. To explore the role of ox-LDL on the expression of hepcidin in macrophage, divide macrophages into the control group and ox-LDL stimulation group. Observe the expression of hepcidin in macrophage after different times of ox-LDL's stimulating with different concentrations.Part2. To investigate the role of RBC on intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.The cells were divided into groups of macrophages, macrophages+ox-LDL, erythrophgocytosed macrophges, erythrophgocytosed macrophges+ox-LDL. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part3. To investigate the role of hepcidin on ox-LDL-induced macrophage oxidative stress and apoptosis process. Macrophages will be divided into groups of control, oxLDL stimulation, hepcidin stimulation, and oxLDL+hepcidin stimulation. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part4. To investigate the role of hepcidin on ox-LDL-induced oxidative stress and apoptosis in erythrophgocytosed macrophge, the erythrophgocytosed macrophge were divided into groups of control, oxLDL stimulation, hepcidin stimulation, and oxLDL+hepcidin stimulation. The erythrophgocytosed macrophge were divided into into groups of hepcidin siRNA, control siRNA, hepcidin siRNA+ox-LDL, and control siRNA+ox-LDL. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part5. To investigate the mechanism of hepcidin's role on the activation and apoptosis of erythrophgocytosed macrophge. Cells were divided into groups of macrophages, erythrophgocytosed macrophge and erythrophgocytosed macrophge+hepcidin. After erythrophgocytosed macrophge were transfected the hepcidin siRNA or the control siRNA, treated them with or without ox-LDL stimulation, and accordingly divide erythrophgocytosed macrophge into groups of hepcidin siRNA, control siRNA, hepcidin siRNA+ox-LDL, and control siRNA+ox-LDL. Detect the expression level of Ferroportinl and Ferritin in each group.Part6. To further clarify that hepcidin is related to iron deposition in its role of erythrophgocytosed macrophges' activation and apoptosis, divide the cells into groups of macrophages, erythrophgocytosed macrophges, erythrophgocytosed macrophge+chelator (BPDL+DFO), and observe BPDL+DFO's effect upon erythrophgocytosed macrophges'oxidative stress, inflammation and apoptosis. Divide the cells into groups of macrophages, erythrophgocytosed macrophges, erythrophgocytosed macrophges+ox-LDL, and erythrophgocytosed macrophges+ox-LDL+chelator (BPDL+DFO), and observe the iron chelator's effect upon intracellular lipids accumulation in erythrophgocytosed macrophges.5. Quantitative Real-time-PCRThe J774macrophage with different treatments were extracted with TriZol Reagent and detected by RT-PCR.Hepcidin,Il-6, MCP-1, TNF-α, MMP-2, FPN1, L-ferritin and H-ferritin expression was normalized to that of β-actin.6. Western Blot AnalysisCollect the J774macrophage with different treatments, extract proteins, and detect protein expression levels of ferroportinl, H-Ferritin, L-Ferritin with Western Blot. IL-6, MCP-1and TNF-a. Sample loadings were normalized to β-actin expression.7. Immunofluorescence StainingExpression and localization of hepcidin and FPN1in J774macrophages or erythrophagocytosed macrophage were examined by immunofluorescent staining. The immunofluorescent staining of J774macrophages was then observed on a fluorescent microscopy.8.Detection of ApoptosisAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges,apoptosis was assessed by terminal deoxynucleotidyl transferase end-labelling (TUNEL staining. The number of TUNEL-positive cells was counted three times in randomly selected fields from each treatment.9.Quantification of ROS productionAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges, fluorescence measurement of ROS was performed with Flow Cytometerequipped with a488nm argon laser using conventional methods.10. Quantification of Intracellular LipidsAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges., the lipids of macrophages with different treatments were extracted with the Folch method and the intracellular TC, TG and LDL-C were measured by enzymatic assay.11. Statistical analysisAll analyses were performed using SPSS16.0(SPSS Inc., Chicago, IL). Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA with LSD post hoc test used for multiple comparisons. P<0.05was considered statistically significant. All experiments were repeated for at least3times.Results1. Macrophage swallowed the erythrocytesThe opsonized erythrocytes were added to J774macrophage monolayers (2×106) and incubated for2hours at37℃with a final phagocytosis ratio of red blood cells to J774cells at about9:1.2. Detection of hepcidin siRNA transfection efficiencyDesign the sequence of hepcidin siRNA and control siRNA.transfect macrophages and detect the hepcidin expression level before and after the macrophages transfection by real-time quantitative RT-PCR and immunofluorescence staining method. The hepcidin siRNA transfection efficiency was70%-80%, while the control siRNA had no effect on macrophage hepcidin expression.3. The expression of hepcidin was up-regulated in macrophages treated with ox-LDL stimulationDivide macrophages into the control group and ox-LDL stimulation group. Use50ug/ml ox-LDL to stimulate macrophages for0,1,2,4,8,16, and24hours. We found that the ox-LDL-induced up-regulation of hepcidin was transient, peaked at2hour after the stimulation and thereafter declined to the basal level within24hours, Using0ug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml of ox-LDL to stimulate macrophages for2hours and we found that the maximum effective dose was50μg/ml4. RBC enhanced the intracellular lipids accumulation, oxidative stress, inflammation and apoptosis of macrophage.Cells were divided into groups of control macrophages, macrophage+ox-LDL, erythrophagocytosed macrophages, and erythrophagocytosed macrophages+ox-LDL group. Detect the intracellular lipid levels, ROS production, apoptosis incidence and the expression of IL-6, MCP-1and TNF-a in mRNA and protein levels in each group. We found that erythrophagocytosed macrophages+ox-LDL group not only had a much higher level of intracellular lipid, ROS production, apoptosis and IL-6, MCP-1, and TNF-a expressions than that of the control group, but also was statistically different from the macrophages+ox-LDL group and the erythrophagocytosed macrophages group. ROS production, apoptosis rate and the level of cytokines in the macrophages+ox-LDL group and the erythrophagocytosed macrophages group became much higher than the control group, but there was no statistical difference between the two grouops. This confirmed that RBC itself has the effect on enhancing the oxidative stress, inflammation and apoptosis in macrophage, and has the effect of promoting the ox-LDL-induced oxidative stress, inflammation and apoptosis of macrophage. With the ox-LDL stimulating, intracellular lipid levels of macrophage were significantly elevated. Though RBC itself did not significantly improve the levels of intracellular lipids, it has a significant role in promoting ox-LDL-induced intracellular lipids accumulation.5. Hepcidin has no effect on ox-LDL-induced macrophage lipid accumulation, oxidative stress, inflammation and apoptosisMacrophages were divided into control group, ox-LDL treatment group, hepcidin treatment group, and ox-LD+hepcidin treatment group. Detect the levels of intracellular lipid, ROS production, apoptosis incidence and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels in each group. We found that all the detection targets in the hepcidin treatment group showed no significant difference from the control group. All detection targets of the ox-LDL+hepcidin treatment group were significantly higher than those of the control group, but showed no significant difference from those of the ox-LDL treatment group. This confirmed that hepcidin has no significant impact upon ox-LDL induced lipid accumulation, oxidative stress, inflammation and apoptosis in macrophage.6Hepcidin's role in enhancing the erythrophagocytosed macrophages's intracellular lipids accumulation, oxidative stress, inflammation and apoptosis with or without ox-LDL treatment The erythrophagocytosed macrophages were divided into groups of control, oxLDL treatment, hepcidin treatment, oxLDL+hepcidin treatment. Detect intracellular lipid levels, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels. We found that the ox-LDL+hepcidin treatment group's intracellular lipid levels, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression levels were not only significantly higher than those of the control group, but also also significantly different from those of the oxLDl treatment group and the hepcidin treatment group. The ROS production, apoptosis rate and IL-6, MCP-1and TNF-a expressions of the oxLDL treatment group and hepcidin treatment group were significantly higher than those of the control group, but no statistcal difference between the two groups was found. The intracellular lipid levels of the oxLDL treatment group were significantly lower than those of the oxLDL+hepcidin treatment group, but significantly higher than those of the Hepcidin treatment group, which confirmed that the hepcidin itself does not increase the intracellular lipid level of erythrophagocytosed macrophages, but significantly increase the accumulation of lipid in erythrophagocytosed macrophages induced by ox-LDL. erythrophagocytosed macrophages were treated with or without oxLDL-stimulated after they were transfected with hepcidin siRNA or control siRNA. And then erythrophagocytosed macrophages were divided into the hepcidin siRNA group, control siRNA group; hepcidin siRNA+ox-LDL group, and the control siRNA+ox-LDL-group. Detect respectively the intracellular lipid level, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels in each group.All the detected targets of the hepcidin siRNA group decreased significantly compared with the control siRNA group. With the ox-LDL stimulation, All the detected targets of the hepcidin siRNA group remained significantly lower than those of the control siRNA group, but all the detected targets of both the two groups were higher than those of the ox-LDL(-) group.7. The effect of erythrophagocytosis and hepcidin on the expression of Ferroportin1and ferritinAfter erythrophagocytosis, we observed a time-dependent up-regulation of L-ferritin and H-ferritin which are iron-storage proteins, and ferroportinl, an iron-export protein. The expression of Fpnl reached a peak at4hour, declined thereafter, and returned to the basal level by24hour after erythrophagocytosis, whereas the expression of H-ferritin and L-ferritin reached a peak at4hour and6hour, respectively, and sustained at least by24hour after erythrophagocytosis.Hepcidin further up-regulated the H-ferritin and L-ferritin expression whereas down-regulated the Fpn1expression in the erythrophagocytosed macrophages. After the transfection of hepcidin siRNA, erythrophagocytosed macrophages's the H-Ferritin and L-Ferritin expression, with or without ox-LDL stimulation, significantly decreased compared with the control siRNA group.8. Iron chelator's impact on the activation and apoptosis of erythrophagocytosed macrophagesIntracellular iron of macrophage was scavenged by iron chelators including desferrioxamine (DFO) and ferrous chelator2,2'-bipyridyl (BPDL). The increased pro-inflammatory cytokine production, ROS formation and apoptosis in macrophages after erythrophagocytosis were inhibited by adding of DFO and BPDL Meanwhile, the H-Ferritin and L-Ferritin expression levels in the iron chelators groups were significantly reduced in the erythrophagocytosed macrophages.Conclusions1. The expression of hepcidin was time-and dose-dependently up-regulated in macrophages treated with ox-LDL stimulation.2.The intracellular lipid levels, oxidative stress, inflammation and apoptosis could be promoted by RBC with or without ox-LDL treatment3. Hepcidin enhanced the ox-LDL-induced pro-atherogenic activation of macrophages only in the setting of erythrophagocytosis.4. Hepcidin plays an important enhancing role in the pro-atherogenic activation and apoptosis by promoting the intracellular iron depositin.