Icariin And Berberine Are Identified To Regulate Iron Homeostasis Through Altering Hepatic Hepcidin Expression

Iron is indispensable for the human being, involving in DNA synthesis, protein formation, oxygen transport and energy metabolism. Iron homeostasis is concertedly governed under normal settings, where the hepcidin-ferroportin axis is fundamentally responsible for the regulation of iron supply, utilization, recycling and storage. Hepcidin deficiency gives rise to enhanced dietary iron absorption and iron release from macrophages, and results in excess iron present in serum and organs, associated with dysfunctionality and morbidity of these organs. Hepcidin reduction has been demonstrated in a few types of hereditary hemochromatosis (HH) and P-thalassemia. Therapeutics that increase hepcidin concentration have been proposed to treat these iron overload diseases. In traditional Chinese medicine, many plants have been used in the treatment of these diseases, and are widely known to functionally enrich blood or promote blood circulation. Whether these effects are the result of an alteration in hepcidin production is unclear, but is an intriguing possibility. To date, natural compounds have not been closely investigated for this purpose.In the current study, we aimed to screen natural compounds. In the current study, hepcidin promoter-luciferase assay, real-time PCR and animal models were employed to study 14 natural compounds that are pure extracts from Chinese medicinal plants. Icariin and Berberine were found to incur a potent stimulatory effect on hepcidin transcription. Mechanistic studies indicated that Icariin and Berberine enhanced hepcidin expression through activating Stat3 signaling and Smad1/5/8 signaling. The induction of hepcidin was confirmed in mice upon Icariin administration, coupled with changes of serum and tissue iron concentrations. However, these changes were not observed in hepcidin-deficient (Hamp1-/-) mice upon Icariin, verifying hepatic hepcidin as the target of Icariin. To summarize, Icariin and Berberine exhibit a robust efficacy in inducing hepatic hepcidin to restrain dietary iron absorption and iron egress into circulation. This study signifies the potential application of certain natural compounds in treating iron disorders through modulating hepatic hepcidin expression.Study Ⅰ:Screening of natural compounds for modulating hepcidin expressionObjective:In an effort to investigate a potential role of natural compounds in modulating hepcidin expression..Methods:We screened 14 pure natural products with the dual hepcidin luciferase assay, The reporter construct was developed in our research group with the human hepcidin promoter fragment (1.6 kb) upstream of the firefly luciferase gene. The screening assay was carried out in both HepG2 and Hepal-6 cells. Prior to screening, cytotoxicity was evaluated through the MTT assay. These compounds did not show any cytotoxicity at concentrations up to 100 μM, as the representative results for Icariin. We thus used the concentration of 5 μM in the initial screening assay, and this concentration did not impair cell viability for all these compounds.Results:After treatment for 24 h, hepcidin-luciferase activity was recorded. Propyl Gallate, Esveratrol, Kutkin Ⅱ, Berberine, Icariin, Chrysaron and Wogonin were observed to enforce hepcidin-luciferase activity by approximately 2 fold in HepG2 cells, relative to the untreated control (P<0.05). Astragaloside, Ligustrazine, Ginsenoside Rbl and Liquiritin were found to increase hepcidin-luciferase activity by approximately 50% in HepG2 cells, compared to the untreated control (P<0.05). However, Curcumin, Paeoniflorin and Ferulic acid failed to significantly alter hepcidin-luciferase activity (P>0.05). Analogously in Hepal-6 cells, Kutkin Ⅱ, Berberine, Icariin, Chrysaron and Wogonin promoted hepcidin-luciferase activity by approximately 2 fold, compared to the untreated control (P<0.05), consistent with the results in HepG2 cells. Ferulic acid, Ginsenoside Rbl and Liquiritin were able to stimulate hepcidin-luciferase activity by approximately 50% in Hepal-6 cells, in comparison to untreated cells (P<0.05). The rest of the compounds including Propyl Gallate, Esveratrol, Astragaloside, Curcumin. Paeoniflorin and Ligustrazine did not significantly change the hepcidin-luciferase activity in Hepal-6 cells, relative to untreated cells (P>0.05).Conclusions:Considered together, Kutkin Ⅱ, Berberine, Icariin, Chrysaron and Wogonin revealed robust stimulatory effect on hepcidin transcription in both HepG2 and Hepa 1-6 cells.Study Ⅱ:Validation of screening data through qRT-PCR analysisObjective:To further confirm the screening results described above.Methods:The qRT-PCR was employed to determine hepcidin mRNA levels in response to these compounds. We chose Icariin, Berberine, Kutkin II and Chrysaron for further investigation here. Total RNAs were purified from cells using Trizol based on the instructions from the manufacturer (Invitrogen). Total RNAs from tissue specimens were also extracted with Trizol reagent after the specimens were pulverized in liquid nitrogen following the manufacturer’s instructions. The mRNA expression level was probed through qRT-PCR analysis using SYBR Green qPCR master mix (Qiagen). Primer sequences are as follows. Human HAMP, forward: 5’-CCTGACCAGTGGCTCTGTTT-3’,reverse: 5’-CACATCCCACACTTTGATCG-3’; human GAPDH, forward: 5’-GAAGGTGAAGGTCGGAGT-3’,reverse:5’-GAAGATGGTGATGGGATTTC-3’; mouse HAMP, forward:5’-CTGAGCAGCACCACCTATCTC-3;,reverse: 5’-TGGCTCTAGGCTATGTTTTGC-3’; mouse GAPDH, forward: 5’-AAGGTCATCCCAGAGCTG, reverse:5’-GCCATGAGGTCCACCACCCT-3’; GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a housekeeping gene for the normalization of relative gene expression.Results:These compounds showed a substantial efficacy in increasing hepcidin-luciferase activity, as described above; however, only Icariin, Berberine, and Chrysaron were demonstrated to significantly enhance endogenous hepcidin expression in HepG2 cells by about 2-fold induction (P<0.05). Moreover, only Icariin and Berberine were verified to increase endogenous hepcidin expression by more than2 fold in Hepal-6 cells (P<0.05). These data together suggested that Icariin and Berberine have a great capability to induce hepatic hepcidin expression. Next, we further validated the activating effect of Icariin and Berberine on hepcidin expression through dose-dependent treatments. Hepcidin expression in HepG2 cells was increased by approximately 2 fold by Icariin at 5 μM, and further increased at 20 and 50 μM by approximately 3 fold, compared to untreated cells (P<0.05). Similarly, hepcidin expression of Hepal-6 cells was induced by Icariin in a dose-dependent manner from 5 to 50 μM, with a maximum increase at 50 μM by approximately 3 fold, relative to the control (P<0.05). In contrast to Icariin, Berberine promoted hepcidin expression in HepG2 cells by>3 fold at both 5 and 50 μM, compared to untreated cells (P<0.05). Analogous to HepG2 cells, hepcidin expression was elevated by about4 fold in Hepal-6 cells by Berberine at both 5 and 50 μM (P<0.05), suggesting that 5 μM has reached the greatest activity to stimulate hepcidin expression for Berberine. Conclusions:Since, the stimulating effect on hepcidin by Icariin and Berberine was further demonstrated by the qRT-PCR results, and we selected two compounds for further detailed evaluation.Study Ⅲ:Icariin and Berberine govern hepcidin expression through SMAD1/5/8 and STAT3 signalingObjective:To understand the molecular basis on which Icariin and Berberine promote hepcidin expression, we looked into the signaling pathways involved in hepcidin regulation.Methods:We used Western blotting analysis. After treatment with compounds, cells were harvested in cold PBS, and total proteins were then extracted with RIPA lysis buffer (Solarbio, China) supplemented with protease inhibitor cocktail (Roche). Equal amount of protein lysates for each sample were subjected to SDS-PAGE and Western blot analysis following a standard procedure, as described previously. The primary antibodies are listed below, anti-pSmad1/5/8 antibody (1:1000; Santa cruze Biotechnology), anti-Smad1 antibody (1:1000; Santa cruze Biotechnology), anti-pStat3 antibody (1:1000; Cell Signaling Technology), and anti-GAPDH (1:2000; Sigma-Aldrich).Results:Although the regulation of hepcidin is rather complicated, two signaling pathways are recognized to predominantly control hepcidin expression under the normal conditions, i.e. Stat3 pathway and Smadl/5/8 pathway. Upon activation of one or both of these pathways, hepcidin expression is expected to rise with resultant inhibition of dietary iron absorption and iron egress from macrophages. Thus, we examined the activation of the Stat3 pathway and Smad1/5/8 pathway in HepG2 cells upon treatment with Icariin or Berberine. Icariin was observed to increase the level of phosphorylated Stat3, especially at 50 μM. Meanwhile, Icariin was also observed to increase the level of phosphorylated Smadl/5/8, relative to untreated cells, especially at 50 μM. These observations suggested that Icariin upregulates hepcidin expression through both the Stat3 and Smad1/5/8 pathways. Moreover, Icariin at a higher concentration (at 50 μM) revealed a greater ability to activate the Stat3 and Smad1/5/8 pathways than the lower concentration (at 5 μM), in parallel to the greater induction of hepcidin expression for 50 μM Icariin. Analogous to Icariin, Berberine was also demonstrated to activate both the Stat3 pathway and Smad1/5/8 pathway, as evidenced by the significant increase in phosphorylated Stat3 and phosphorylated Smadl/5/8. The extent of the increase in phosphorylated Smadl/5/8 was similar in cells treated with 5 μM or 50 μM Berberine, consistent with the changes in hepcidin induction by Berberine at 5 μM or 50 μM. It should be noted that neither Icariin nor Berberine changed the basal level of total Stat3 and Smadl, as evidenced by the Western blotting analysis.Conclusions:These results together demonstrated that the promotion of hepcidin expression by Icariin and Berberine was due to the activation of both STAT3 and SMAD1/5/8 pathways, namely, these two signaling pathways jointly promoted hepcidin expression in hepatocytes in response to Icariin and Berberine.Study Ⅳ:Icariin regulates hepatic hepcidin expression in miceObjective:To validate the stimulatory effect of the compounds, animal studies were further performed.Methods:Wild-type (Wt) ICR mice were randomly grouped with 8 mice in each group (n= 8). Compounds were intraperitoneally administrated in mice with a dose at 100 mg/kg body weight. The control mice received PBS with a corresponding concentration of DMSO only. Regarding the acute treatment, mice were sacrificed 6 or 48 h after injection. With respect to the chronic treatment, mice were administrated every 2 days, and mice were sacrificed on day 4,8 and 16. Blood was collected, and liver and spleen specimens for each mouse were individually separated and weighed. A small piece of each liver specimen was quickly frozen in liquid nitrogen and then saved at -80℃ for future RNA extraction.Results:Since Icariin and Berberine promoted hepcidin expression and activated the Stat3 and Smadl/5/8 pathways similarly, we only selected Icariin for the following animal experiments. Consistent with the in vitro results, Icariin was observed to enhance hepatic hepcidin expression. The hepcidin mRNA level was induced by approximately 50%(P<0.05) immediately 6 h after Icariin administration, and a maximum induction was reached (more than 2.5 fold, P<0.05) on day 2 post Icariin administration, compared to untreated mice. The stimulatory effect on hepatic hepcidin expression was weakened from day 4 to the end of the administration on day 16. As a result of hepatic hepcidin alterations, serum iron was correspondingly changed. The serum iron concentration was greatly reduced at 6 h by approximately 40% in mice treated with Icariin, compared to untreated mice (P<0.05). The concentration of serum iron was consecutively reduced during the Icariin administration on day 2 (P<0.05). Serum iron rose from day 4 to day 8, still lower than that of untreated mice, and reached a similar level to that in untreated mice on day 16. The findings with serum iron changes were in agreement with the alterations of hepatic hepcidin expression in response to Icariin. Subject to hepatic hepcidin changes, splenic iron concentrations were also accordingly changed. Total splenic iron was continuously increased since 6 h to the end of the administration on day 16 (P<0.05). In contrast, total hepatic iron was not significantly altered upon Icariin administration (P>0.05), in agreement with the constant hepatic iron mass in response to exotic stimuli. Additionally, no significant hepatotoxicity was demonstrated in liver specimens from mice administrated with Icariin at the current dose after the short-term and long-term administration. No noticeable alterations were found in liver specimens from mice treated with Icariin for 48 h. This piece of evidence based on histological examination indicated the safety of Icariin for in vivo administration.Conclusions:These results collectively suggested that Icariin elicited a robust ability to modulate hepatic hepcidin concentration, associated with according changes of systemic iron levels.Study Ⅴ:Icariin fails to change body iron mass in hepcidin-deficient miceObjective: To confirm our finding that Icariin functions to regulate iron homeostasis through enforcing hepatic hepcidin expressionMethods: We used a novel model of hepcidin knockout mice, namely Hampl-/- mice.Hamp1-/- mice do not produce functional hepcidin in circulation, and these mice thus develop severe iron overload in serum and organs after weaning. To discriminate a clearer change of serum iron, we depleted body iron by putting these mice in low-iron diet. After iron-depletion, we were able to significantly reduce the serum iron level of Hampl-/- mice. Hampl-/- mice were randomly grouped with 8 mice in each group (n= 8). Compounds were intraperitoneally administrated in mice with a dose at 100 mg/kg body weight. The control mice received PBS with a corresponding concentration of DMSO only. Regarding the acute treatment, mice were sacrificed 6 or 48 h after injection.Results:The serum iron level was not significantly altered in Hampl-/- mice upon the administration of Icariin at the same dose as that used above for Wt mice, i.e. at 100 mg/kg body weight, for 48 h (P>0.05). Moreover, there are no significant changes in total hepatic and splenic iron in Icariin-treated Hampl-/- mice, compared to untreated control mice.Conclusions:This piece of data further demonstrated that Icariin predominantly targets hepatic expression to modulate iron homeostasis.