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J. Liu et al.
of the investigated compounds, we examined the changes of iron indices in a hepcidin-deficient mouse model, Hamp1-/- mice, as previously described.33 Hamp1-/- mice do not produce functional hepcidin, and develop severe iron overload after weaning.34 We iron-depleted these mice on a low-iron diet (4 ppm) for 4 weeks according to an estab- lished protocol,35 so as to increase the sensitivity of Hamp1- /- mice to any hepcidin-independent serum iron-lowering effects of the compounds (time line in Online Supplementary Figure S10A). As shown in Online Supplementary Figure S10B-D, serum iron, hepatic and splenic iron levels were not significantly altered in Hamp1-/- mice, 6 or 24h follow- ing the administration of compounds 93, 156 and 165 at a dose of 30 mg/kg body weight, relative to the levels in untreated Hamp1-/- mice (P>0.05). These data indicate that
A
compounds 93, 156 and 165 predominantly target liver hepcidin in their modulation of iron homeostasis.
Compounds 93, 156 and 165 prevented iron overload in HFE-deficient (Hfe-/-) mice
The potential therapeutic effect of compounds 93, 156 and 165 was tested in iron overloaded Hfe-/- mice on a nor- mal diet. Consistent with previous reports, serum hep- cidin concentration in Hfe-/- mice progressively increased from 6 to 8 weeks (P<0.05) (Figure 5A), as the mice became iron-overloaded,36,37 so that the hepcidin level in Hfe-/- mice at the age of 10 weeks was comparable to that of Wt mice (P<0.05) (Figure 5A). Other iron parameters in Hfe-/- mice indicated that by week 10 rising hepatic iron caused hepcidin upregulation and accelerated iron accu-
B
Figure 4. Compounds 93, 156 and 165 regu- lated Gdf15, Twsg1 and Erfe in bone marrow cells. (A) Changes of Gdf15, Twsg1 and Erfe mRNA expression in bone marrow cells from wildtype (Wt) Balb/C mice upon administra- tion of the various compounds at a dose of 30 mg/kg for 24 h. (B) The variations of Gdf15, Twsg1 and Erfe mRNA expression in bone marrow cells from Hbbth3/+ mice treated with the various compounds at a dose of 30 mg/kg body weight for 24 h. *P<0.05; #P<0.001, relative to untreated control (Ctrl).
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haematologica | 2019; 104(9)