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Data supplements

  • Supplemental materials for: Jiao et al

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Article Figures & Data

Figures

  • Figure 1

    Dietary iron modulates ferritin L, ferritin H, and TfR1 in the liver. Liver homogenates were analyzed for expression of ferritin H, ferritin L, and TfR1 by Western blotting as described in “Western blots.” Shown are means and SD; n = 5. Protein levels showed a statistically significant change in response to iron dose (P < .001 for ferritin H and L and < .05 for TfR1).

  • Figure 2

    Curcumin affects hematologic parameters of iron in mice fed a low-iron diet. Serum iron, transferrin saturation, hemoglobin, and hematocrit were measured in mice fed different concentrations of dietary iron containing either 0% or 2% curcumin. Shown are means and SD; n = 5. Curcumin caused a statistically significant reduction in all blood parameters in mice fed 5 mg iron (P ≤ .008).

  • Figure 3

    Curcumin-mediated reduction in hematologic parameters of iron metabolism in mice fed a low-iron diet depends on curcumin dose. (A) Plasma iron, transferrin saturation, hemoblogin, and hematocrit were measured in mice receiving 5 mg/kg dietary iron supplemented with curcumin at 0%, 0.2%, 0.5%, or 2.0%. Shown are means and SD; n = 5. The dose-dependent reduction in all parameters was statistically significant (P ≤ .009). (B) Representative peripheral blood smears from mice receiving 5 mg/kg dietary iron and 0%, 0.2%, 0.5%, or 2.0% curcumin.

  • Figure 4

    Curcumin reduces spleen iron and stainable iron in the bone marrow. (A) Spleen nonheme iron was measured in mice receiving 5 mg/kg dietary iron supplemented with curcumin at 0%, 0.2%, 0.5%, or 2.0%. Shown are means and SD; n = 5. Spleen iron showed a statistically significant change in response to curcumin dose (P = .005 for trend). (B) Bone marrow was obtained from decalcified femurs of representative mice and stained with hematoxylin and eosin and Perl stain. Mice had received either 5 mg/kg iron plus 0 curcumin, 1000 mg/kg iron plus 0 curcumin, and 1000 mg/kg iron plus 0.2% curcumin.

  • Figure 5

    Curcumin modulates iron-regulatory proteins in the liver. (A) TfR1 was analyzed in mice fed low-iron diets (containing either 5 mg/kg or 12 mg/kg dietary iron) in the presence or absence of 2% curcumin. Liver homogenates were prepared and TfR1 levels were assessed by Western blotting. Shown are means and SD; n = 5. (B) Ferritin was analyzed in mice fed normal or high-iron diets (containing either 50 mg/kg or 1000 mg/kg dietary iron) in the presence of absence of 2% curcumin. Total ferritin (ferritin H + ferritin L) was assessed by Western blotting in liver homogenates. Shown are means and SD; n = 5. (C) The ratio of active to total IRP was analyzed using bandshift assays as described in “RNA-binding protein gel-shift assay.” Liver homogenates were prepared from mice receiving 5 mg/kg dietary iron plus 0 curcumin, 1000 mg/kg dietary iron plus 0 curcumin, or 1000 mg/kg dietary iron plus 2% curcumin. Shown are means and SD; n = 3.

  • Figure 6

    Hepcidin is decreased by dietary iron deficiency or by curcumin. (A) Hepcidin mRNA was measured by real-time RT-PCR in livers of mice receiving dietary iron ranging from 1000 to 5 mg/kg diet (n = 5). Data were normalized to β-actin mRNA. The decline in hepcidin was statistically significant. (B) Hepcidin was measured in mice receiving 5 mg/kg dietary iron supplemented with different concentrations of dietary curcumin (n = 5). The difference between the 0% and 2.0% dietary curcumin group is of borderline significance compared using a 2-sample t test for specific comparisons (P = .08); other differences were not statistically significant. (C) HepG2 hepatocytes were treated with increasing concentrations of curcumin for 8 hours and hepcidin mRNA measured by real-time RT-PCR. Data were normalized to β-actin mRNA. The decline in hepcidin was statistically significant (P < .001 for trend); 12.5 and 25 μM curcumin were statistically different from control (P ≤ .001); 6.25 μM curcumin was not different from control). (D) Duplicate cultures of HepG2 cells were treated with the indicated concentrations of curcumin for 48 hours, and ferroportin levels assessed by Western blot. GAPDH was used as a loading control.

Tables

  • Table 1

    Effect of dietary iron on parameters of systemic iron

    Dietary iron, mg/kg dietPlasma iron, μg/dLTransferrin saturation, %Hb, g/dLHct, %Liver iron, μg/g wet weight*Spleen iron, μg/g wet weight*
    5165 ± 5332 ± 1313 ± 1.543 ± 538 ± 5120 ± 534
    12170 ± 1745 ± 414.9 ± 1.548 ± 1121 ± 29465 ± 118
    50129 ± 2635 ± 914.7 ± 0.446 ± 2229 ± 36727 ± 135
    1000167 ± 3141 ± 1314.7 ± 0.646 ± 2552 ± 1611216 ± 49
    • Data are mean and SD; n = 5. Modulation of dietary iron in C3H mice affects liver and spleen iron but does not affect plasma iron, transferrin saturation, hemoglobin, or hematocrit.

    • * Statistically significant response to dietary iron, P < .001. Plasma iron, transferrin saturation, hemoglobin, and hematocrit were not significantly different.