The following pure natural compounds extracted from Chinese medicinal plants were purchased from the National Institutes for Food and Drug Control of China (Beijing, China) with >99.5% purity: propyl gallate, C₁₀H₁₂O₅; resveratrol, C₁₄H₁₂O₃; astragaloside, C₄₁H₆₈O₁₄; curcumin, C₂₁H₂₀O₆; paeoniflorin, C₂₃H₂₈O₁₁; ligustrazine, C₈H₁₂N₂·HCl·2H₂O; ferulic acid, C₁₀H₁₀O₄; ginsenoside Rb1, C₅₄H₉₂O₂₃; wogonin, C₁₆H₁₂O₅; liquiritin, C₂₁H₂₂O₉; berberine, C₂₀H₁₈NO₄; icariin, C₃₃H₄₀O₁₅; epimedin A, C₃₉H₅₀O₁₉; epimedin B, C₃₈H₄₈O₁₉; and epimedin C, C₃₉H₅₀O₁₉. Dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) was used to dissolve these compounds, and the final concentration of DMSO in the culture medium was <0.5%.

The human hepatocellular carcinoma cell line, HepG2, and the mouse hepatocellular carcinoma cell line, Hepa 1-6, obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences were cultured in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS) (all from Gibco, Grand Island, NY, USA) at 37°C under a humidified atmosphere with 5% CO₂, as previously described (22,23). The cells were collected for RNA or protein extraction 24 h after being subjected to the various treatments (the cells were treated with the various compounds at concentrations of 5 µM for 24 h). Untreated (control) cells were cultured with the corresponding DMSO-containing medium.

The cytotoxicity of the natural compounds was evaluated using an MTT assay kit according to the manufacturer's instructions (Roche Life Science, Basel, Switzerland). Briefly, the HepG2 and the Hepa 1-6 cells were first serum-starved overnight, then inoculated into 96-well plates with 5,000 cells/well. The cells were then subjected to the different treatments. The cells were cultured for an additional 24 h, and subsequently, 20 µl MTT solution (5 mg/ml) was added to each well, followed by incubation for a further 4 h. At the end of the incubation period, 200 µl DMSO were added to each well, and the absorbance at 490 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

The HepG2 and the Hep 1-6 cells were seeded at 2×10⁵ cells/well in a 24-well plate 12 h prior to transfection. The cells were then co-transfected with 0.8 µg of a hepcidin-promoter-luciferase plasmid and 80 ng of Renilla luciferase plasmid with Lipofectamine 2000 in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The medium was replaced with RPMI-1640 or DMEM containing 10% FBS 6 h later, and the cells were then treated with each compound (at 5 µM) for 24 h. Afterwards, the cells were collected and washed with PBS, and finally lysed in lysis buffer (Promega, Madison, WI, USA). The cell lysates were then assessed for luciferase activity using the Dual-Luciferase Reporter assay system (Promega). The relative firefly luciferase activity for each sample was normalized to that of Renilla luciferase.

Total RNA was purified from the cells using TRIzol reagent according to the manufacturer's instructions (Invitrogen Grand, NY, Island, USA). Total RNA from liver specimens (obtained from mice, as described below) was also extracted using TRIzol reagent according to the manufacturer's instructions after the specimens were pulverized in liquid nitrogen. The mRNA expression levels were assessed by performing qPCR using SYBR-Green qPCR Master Mix (Qiagen, Valencia, CA, USA). Glyceraldehyde 3- phosphate dehydro genase (GAPDH) was used as a housekeeping gene for the normalization of relative gene expression. The primer sequences used were as follows: human hepcidin forward, 5′-CCTGACCAGTGGCTCTGTTT-3′ and reverse, 5′-CACATCCCACACTTTGATCG-3′; human GAPDH forward, 5′-GAAGGTGAAGGTCGGAGT-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; mouse hepcidin forward, 5′-CTGAGCAGCACCACCTATCTC-3′ and reverse, 5′-TGGCTCTAGGCTATGTTTTGC-3′; and mouse GAPDH forward, 5′-AAGGTCATCCCAGAGCTG′ and reverse, 5′-GCCATGAGGTCCACCACCCT-3′.

Following treatment with the compounds, HepG2 cells were harvested in cold PBS, and total proteins were then extracted using RIPA lysis buffer (Solarbio) supplemented with protease inhibitor cocktail (Roche Life Science). Interleukin (IL)-6 was used as positive control for p-Stat3 detection, which was purchased from Sigma Aldrich. Proteins were extracted following 24 h of administration at 100 ng/ml. Equal amounts of protein lysates for each sample were subjected to SDS-PAGE and western blot analysis following a standard procedure, as previously described (24,25). The primary antibodies used were as follows: anti-p-Smad1/5/8 (1:1,000; sc-12353) and anti-Smad1 (1:1,000; sc-7965) antibodies (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-p-Stat3 (1:1,000; 9145) and anti-Stat3 (1:1,000; 9139) antibodies (both from Cell Signaling Technology, Danvers, MA, USA); and anti-GAPDH (1:2,000; G8795; Sigma-Aldrich, St. Louis, MO, USA). Protein quantification was performed using ImageJ software (NIH, http://rsb.info.nih.gov/ij/).

All animal care and experimental procedures were approved by the Animal Ethics Committee at Xiyuan Hospital, China Academy of Chinese Medical Sciences (Beijing, China). Eight-week-old mice were used in this study. Wild-type (Wt) ICR mice were purchased from the Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed in a central specific pathogen-free (SPF) facility at the Beijing Xiyuan Hospital. Hepcidin-deficient [Hamp1^−/− or Hamp1-knockout (KO)] mice were originally provided by Dr Sophie Vaulont [Institut National de la Santé et de la Recherche Médicale (INSERM)] (26) and have been back-crossed into the C57BL/6 background (27). To deplete iron, the Hamp1^−/− mice were placed on a low-iron diet (4 ppm iron) 1 week prior to the administration of the compounds. This method was developed to reduce the serum iron content in the Hamp1^−/− mice in order to more clearly demonstrate changes in serum iron levels following the administration of the compounds. The mice were randomly grouped with 8 mice in each group (n=8). The compounds were administered intraperitoneally to the mice at a dose of 100 mg/kg body weight. The control mice received PBS containing DMSO at the relevant concentration. For the short-term treatment experiments, the mice were sacrificed 6 or 48 h after the injection of the compounds. With respect to the long-term treatments, the compounds were administered to the mice every 2 days, and the mice were sacrificed on days 4, 8 or 16. Lipopolysaccharide (LPS) was used as a positive control and was purchased from Sigma-Aldrich. Mice were administrated with LPS at 100 µg/mouse for 6 h. Blood was collected, and liver and spleen specimens from each mouse were isolated and individually separated. A small section of each liver specimen was quickly frozen in liquid nitrogen and then stored at −80°C for future RNA extraction.

The serum iron levels were determined using a serum iron detection kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hepatic and spleen iron contents were determined following the established protocols in our laboratory, as previously described (25). Briefly, liver and spleen specimens were first digested with an acid solution at 65°C for 72 h. Thereafter, chromagen working solution was added to each supernatant. Finally, the absorption at 535 nm was measured using a microplate reader (Thermo Fisher Scientific).

The liver specimens were fixed in 10% buffered formaldehyde, and embedded in paraffin. The deparaffinized sections were stained with hematoxylin and eosin (H&E), following a standard protocol, as previously described (25,28,29).

The SPSS 17.0 statistics package was used to analyze the experimental data. One-way analysis of variance (ANOVA) was used to differentiate the mean differences among groups compared to the control group. The differences between 2 groups were determined using an independent Student's t-test. All experimental data are presented as the means ± SD. A P-value <0.05 was considered to indicate a statistically significant difference.

The liver (where hepatocytes secrete hepcidin) is the predominant organ for the secretion of hepcidin into the circulation in order to regulate iron homeostasis (30). Thus, in this study, we used liver-derived cell lines. HepG2 and Hepa 1-6 are established hepatocyte cell lines representative of human cells and mouse cells, respectively. In an effort to investigate the potential role of natural compounds in the modulation of hepcidin expression, we screened 12 pure natural products using the dual hepcidin luciferase assay. The reporter construct was developed by our research group with the human hepcidin promoter fragment (1.6 kb) upstream of the firefly luciferase gene, as previousy described (31). The screening assay was performed in both the HepG2 and Hepa 1-6 cells. Prior to screening, cytotoxicity was evaluated by MTT assay. All these compounds exhibited no significant toxicity to both the HepG2 and Hepa 1-6 cells, with only a mild reduction in cell viability observed at higher concentrations of berberine, curcumin, icariin and wogonin (data not shown). Thus, we used a concentration of 5 µM in the initial screening assay, and cell viability was not impaired by any of the compounds (data not shown). Following treatment with the compounds for 24 h, hepcidin-luciferase activity was measured. As shown in Table I, propyl gallate, resveratrol, berberine, icariin and wogonin were observed to enhance hepcidin-luciferase activity by >2-fold in the HepG2 cells, compared to the untreated controls (P<0.05). Astragaloside, ligustrazine, ginsenoside Rb1 and liquiritin were found to enhance hepcidin-luciferase activity by approximately 50% in the HepG2 cells compared with the untreated controls (P<0.05). However, curcumin, paeoniflorin and ferulic acid failed to significantly alter hepcidin-luciferase activity in the HepG2 cells (P>0.05). Analogously, in the Hepa 1-6 cells, berberine, icariin and wogonin promoted hepcidin-luciferase activity by >2-fold compared with the untreated controls (P<0.05), which was consistent with the results observed in the HepG2 cells. Ferulic acid, ginsenoside Rb1 and liquiritin were able to stimulate hepcidin-luciferase activity by approximately 50% in the Hepa 1-6 cells in comparison with the untreated cells (P<0.05). The remaining compounds (propyl gallate, resveratrol, astragaloside, curcumin, paeoniflorin and ligustrazine) failed to significantly alter the hepcidin-luciferase activity in the Hepa 1-6 cells (P>0.05). Taken together, these findings indicated that berberine, icariin and wogonin exerted a robust stimulatory effect on hepcidin transcription in both the HepG2 and Hepa 1-6 cells. Thus, these 3 compounds were selected for use in our subsequent experiments.

To confirm the screening results described above, RT-qPCR was employed to determine the hepcidin mRNA levels following treatment with the selected compounds. As shown in Fig. 1, hepcidin expression in the HepG2 cells was increased by approximately 2-fold by icariin at a concentration of 5 µM, and was further increased at concentrations of 20 and 50 µM by approximately 3-fold, compared with the untreated cells (P<0.05). Similarly, hepcidin expression in the Hepa 1-6 cells was induced by icariin (at concentrations of 5, 20 and 50 µM) in a dose-dependent manner, with a maximum increase observed at the concentration of 50 µM of approximately 3-fold compared to the controls (Fig. 1; P<0.05). Analogously, berberine increased hepcidin expression in the HepG2 cells by >3-fold at both 5 and 50 µM compared with the untreated cells (Fig. 1; P<0.05). In parallel to the HepG2 cells, hepcidin expression was elevated by approximately 4-fold in the Hepa 1-6 cells by berberine at both 5 and 50 µM (Fig. 1; P<0.05). By contrast, wogonin failed to alter hepcidin expression, and this was revealed by RT-qPCR (data not shown). Based on these results, the stimulatory effects of icariin and berberine on hepcidin expression were further demonstrated by the results of RT-qPCR, and we therefore selected these two compounds for further detailed evaluation.

To elucidate the molecular mechanisms responsible for the promotion of hepcidin expression by icariin and berberine, we explored the signaling pathways involved in the regulation of hepcidin expression. Although the regulation of hepcidin expression is rather complex, two signaling pathways are known to predominantly control hepcidin expression under normal and pathological conditions: the Stat3 pathway and the Smad1/5/8 pathway (6,8). Upon activation of one or both of these pathways, hepcidin expression is expected to rise, which inhibits dietary iron absorption, as well as iron egress from macrophages (6,8). Thus, in this study, we examined the activation of the Stat3 pathway and the Smad1/5/8 pathway in HepG2 cells following treatment with icariin or berberine. As shown in Fig. 2A, both icariin and berberine were found to markedly increase the p-Stat3 and p-Smad1/5/8 levels compared to the untreated control cells. Since IL-6 has been shown to increase hepcidin expression by activating the Stat3 signaling pathway (32,33), it was used in this study as a positive control to increase the p-Stat3 level (Fig. 2A). In addition, a compound (namely wogonin) that did not alter hepcidin expression was used as a negative control (Fig. 2A).

Furthermore, the dose-dependent effects of icariin and berberine on Stat3 and Smad1/5/8 were examined. As illustrated in Fig. 2B, icariin was observed to increase the level of p-Stat3, particularly at 50 µM. Icariin was also found to increase the level of p-Smad1/5/8, relative to the untreated cells, particularly at 50 µM (Fig. 2B). The quantitative analysis of Stat3 phosphorylation and Smad1/5/8 phosphorylation is shown in Fig. 2C and D, respectively. These observations suggest that icariin upregulates hepcidin expression through both the Stat3 and Smad1/5/8 pathways. Moreover, icariin at a higher concentration (at 50 µM) exhibited a greater ability to activate the Stat3 and Smad1/5/8 pathways than at the lower concentration (at 5 µM), in parallel to the greater induction of hepcidin expression observed with 50 µM icariin (Fig. 1). Similar to icariin, berberine was also demonstrated to activate both the Stat3 and Smad1/5/8 pathways, as indicated by a significant increase in the levels of p-Stat3 and p-Smad1/5/8 (Fig. 2A–D). It should be noted that neither icariin nor berberine altered the basal levels of total Stat3 and Smad1, as shown by the results of western blot analysis (Fig. 2A and B). Taken together, these results demonstrated that the promotion of hepcidin expression by icariin and berberine was due to the activation of both the Stat3 and Smad1/5/8 pathways; namely, these two signaling pathways jointly promoted hepcidin expression in hepatocytes in response to treatment with icariin and berberine. It should also be noted that it is not possible to exclude the involvement of other pathways which may also have been affected by icariin and berberine.

To determine whether the in vitro effects of icariin and berberine can be reproduced in vivo, we administered both compounds to mice. As shown in Fig. 3A, consistent with the in vitro results, icariin enhanced hepatic hepcidin mRNA expression by approximately 50% (P<0.05) 6 h following the administration of icariin, and a maximum induction (>2.5-fold, P<0.05) was observed on day 2 post-icariin administration, compared with the untreated mice (Fig. 3A). The stimulatory effects on hepatic hepcidin expression declined at later time points (Fig. 3A). As a result of alterations in hepatic hepcidin expression, serum iron levels were correspondingly altered, as shown in Fig. 3B. The serum iron concentration was markedly reduced at 6 h by approximately 40% in the mice treated with icariin, compared with the untreated mice (Fig. 3B; P<0.05). The serum iron concentration was consecutively lower following treatment with icariin compared to the controls on days 2, 4 and 8 (P<0.05), reaching similar levels to those of the untreated mice on day 16 (Fig. 3B). Subject to the changes in hepatic hepcidin levels, the spleen iron concentrations were also altered accordingly. As shown in Fig. 3C, the total spleen iron levels were continuously higher following treatment with icariin than those of the untreated mice from 6 h to the end of the administration on day 16 (P<0.05). By contrast, the changes in total hepatic iron levels were less significantly altered upon icariin administration than the spleen iron levels, although they were also higher compared to the controls (Fig. 3D; P>0.05); these results are in agreement with those of previous studies showing the constant hepatic iron content in response to external stimuli (28,29). Notably, berberine did not promote hepcidin expression in mice (Fig. 4A), and no significant alterations in serum, liver and spleen iron levels were observed (Fig. 4B–D); the in vivo results obtained for hepcidin expression were inconsistent with the in vitro results described above.

To further investigate our in vivo findings, ligustrazine (which showed a limited ability to alter hepcidin expression in vitro, as shown in Table I) was administered to the mice as a control. Ligustrazine did not alter the hepcidin expression levels or the body iron status (data not shown). Furthermore, LPS was used as a positive control to induce hepcidin expression in the mice, as previously described (29). It has been previously established that LPS increases hepcidin expression through an inflammatory mechanism (34–36). Exposure to LPS significantly increased hepatic hepcidin expression and there were corresponding alterations in body iron status (data not shown). These data further supported our findings regarding the icariin-mediated regulation of hepcidin expression.

No significant hepatotoxicity was observed in the liver specimens from the mice administered icariin. As shown in Fig. 5, no noticeable alterations (no disordered hepatic cords or enlarged central veins) were observed in the liver specimens obtained from the mice treated with icariin for 48 h. This piece of evidence based on histological examination indicated the safety of icariin for in vivo administration.

To further examine the regulatory effects of icariin on hepcidin expression, 3 analogues of icariin, epimedin A, B and C (which harbor structural similarities to icariin, as depicted in Fig. 6) were also administered to the mice. As shown in Fig. 7A, epimedin A, B and C all significantly increased hepatic hepcidin expression, particularly epimedin B and C (P<0.05). As a result, the serum, spleen and hepatic iron levels were altered accordingly, with more potent effects being observed following the administration of epimedin C (Fig. 7B–D). Collectively, these results suggested that icariin and its analogues exhibited a robust ability to modulate hepatic hepcidin concentrations in mice, which were associated with corresponding alterations in systemic iron levels.

To confirm the above-mentioned finding that icariin regulates iron homeostasis by altering hepatic hepcidin expression, we used Hamp1^−/− mice, as previously described (26). Hamp1^−/− mice do not produce functional hepcidin, and these mice thus develop severe iron overload in the serum and organs after weaning. To more clearly demonstrate changes in serum iron levels in the animals that were already iron-loaded, we depleted body iron by placing these mice on a low-iron diet. In other words, this regime would in fact increase the sensitivity of these Hamp1^−/−mice to the administration of the compounds. Following iron depletion, we observed a significant reduction in the serum iron levels in the Hamp1^−/− mice (data not shown). As shown in Fig. 8A, the serum iron levels were not significantly altered in the Hamp1^−/− mice following the administration of icariin at the same dose as that administered to the Wt ICR mice, as described above, i.e., at 100 mg/kg body weight, for 6, 24 or 48 h (P>0.05). Moreover, there were no significant changes observed in the total hepatic or spleen iron levels in the icariin-treated Hamp1^−/− mice, compared with the untreated control mice (Fig. 8B and C). These data further demonstrate that icariin predominantly targets hepatic hepcidin expression in order to modulate iron homeostasis.