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Astragalus polysaccharide upregulates hepcidin and reduces iron overload in mice via activation of p38 mitogen-activated protein kinase
Biochemical and Biophysical Research Communications 472 (2016) 163e168
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Astragalus polysaccharide upregulates hepcidin and reduces iron overload in mice via activation of p38 mitogen-activated protein kinase Feng Ren a, b, Xin-Hua Qian a, *, Xin-Lai Qian b, ** a b
Department of Neonatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pathology, Xinxiang Medical University, Xinxiang 453003, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Accepted 21 February 2016 Available online 23 February 2016
Thalassemia is a genetic disease characterized by iron overload which is a major detrimental factor contributing to mortality and organ damage. The hepcidin secreted by liver plays an essential role in orchestrating iron metabolism. Lowering iron load in thalassemia patients by means of increasing hepcidin might be a therapeutic strategy. In this study, we first found that astragalus polysaccharide (APS) significantly increased hepcidin expression in HepG2 and L-02 cell lines originating from hepatocytes and mice liver, respectively. Following treatment with APS, the iron concentrations in serum, liver, spleen, and heart were significantly reduced in comparison to saline treated control mice. In further experiments, upregulation of interleukin-6 (IL-6) and enhanced p38 MAPK phosphorylation were detected in APS treated cells and mice, and as documented in previous studies, IL-6 and P38 MAPK phosphorylation are involved in the regulation of hepcidin expression. We also found that the effects of APS on upregulating hepcidin and IL-6 expressions could be antagonized by pretreatment with SB203580, an inhibitor of p38 MAPK signaling. These findings suggest that activation of p38 MAPK and release of IL-6 might mediate induction of hepcidin by APS. It is concluded that APS might have therapeutic implications in patients with iron overload, especially for thalassemia patients. © 2016 Elsevier Inc. All rights reserved.
Keywords: Astragalus polysaccharide Thalassemia Iron load Hepcidin p38MAPK signaling pathway Interleukin-6
1. Introduction Patients with thalassemia are more likely to suffer from the fatal complication of iron overload, which causes damages to the liver, spleen, heart, and endocrine system [1]. Secondary iron overload can also result from long-term transfusion therapy in transfusiondependent thalassemia patients [2e4]. Hepcidin is secreted mainly by the liver and function as a key regulator of body iron homeostasis [5,6]. Hepcidin is homeostatically regulated by iron and erythropoietic activity, as well as inflammatory cytokines [7e9]. Many diseases associated with iron overload arise upon deregulation of hepcidin production. Hepcidin controls systemic iron homeostasis through rapid degradation of ferroportin, which
* Corresponding author. Department of Neonatology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China. ** Corresponding author. E-mail addresses: [email protected] (X.-H. Qian), [email protected] (X.-L. Qian). http://dx.doi.org/10.1016/j.bbrc.2016.02.088 0006-291X/© 2016 Elsevier Inc. All rights reserved.
decreases intestinal iron absorption and the mobilization of iron stores [10]. Elevated ferroportin expression secondary to hepcidin down-regulation is commonly associated with high level of iron in serum and organs [11,12]. Recent studies revealed that the expression of hepcidin was down-regulated among the thalassemia patients compared to the normal controls [13e15]. The iron overload disorder develops through a mechanism of excessive erythropoiesis, which suppresses hepcidin and increases iron absorption in thalassemia patients. Accumulated evidence suggests that therapeutic regimen targeting the hepcidin-ferroportin axis could have important clinical implications for thalassemia patients associated with iron overload. Astragalus polysaccharide (APS) is the major bioactive component of the extracts from roots of Astragalus membranaceus, a common Chinese medicinal herb. The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses [16]. Our previous study showed that APS could stimulate g-globin expression and hemoglobin F production and was safe in children with thalassemia [17e19]. However, it is still unknown whether APS has the ability to
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regulate hepcidin and reduce iron overload in mice via activation of p38MAPK. In this study, we investigated the role of APS in regulating hepcidin production and its efficacy on lowering iron overload. 2. Materials and methods 2.1. Animals C57BL/6 mice were purchased from Experimental Animal Center, Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). All mice were randomly divided into four groups (n ¼ 8): normal saline (NS) group, APS group, model plus NS group, and model plus APS group. Mice in the last two groups were intraperitoneally (i.p.) injected with iron dextran (Heshengtang Animal Pharmaceutical Company, Guangzhou, China) at 150 mg/kg every other day for 4 weeks. Meanwhile, the other two groups received isovolumic saline. After that, mice in the APS group and the model plus APS group were administered daily with APS 200 mg/kg of body weight for 7 days [20]. APS with a purity >98% was purchased from Cinorch Pharmaceutical Co., Ltd. (Tianjin, China). Simultaneously, the other two groups received isovolumic saline. At the end of the experiment, all animals were humanely euthanized, and the blood samples were harvested from the inferior vena cava and centrifuged at 4 C, 3000 revolutions per minute (rpm) for 10 min. Then the supernatant was collected and stored at 20 C. Liver, spleen, and heart were removed, rinsed and subjected to pathological examination and total protein extraction. All animal experiments were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. 2.2. Measurement of iron concentrations in mice Iron concentration in mice serum was assayed by automatic biochemistry analyzer (SIMENS Advia 2400). Iron depositions in liver, spleen, and heart were determined by atomic absorption spectrophotometer (AA240FS series by Varian Australia Pty Ltd.). 2.3. Histological analysis Iron depositions were identified by hematoxylin-eosin (H&E) and Prussian blue staining. Liver, spleen, and heart of mice in each group were fixed in 4% paraformaldehyde solution for 24 h and embedded in paraffin. 5 mm-thick sections were processed with H&E and Prussian blue staining for observation of iron depositions, respectively.
2.5. RT-PCR Total RNA was extracted from the cultured cells in accordance with the instructions for the Trizol total RNA extraction kit (Invitrogen, Carlsbad, USA) and the ratio of OD260 and OD280 was 1.8e2.0. The harvested RNA was diluted to a final concentration of 1 mg/mL, and stored at 70 C. The conditions for the first round of RT synthesis of cDNA were as follows: 42 C for 30 min, 99 C for 5 min and 5 C for 5 min. PCR reaction conditions were as follows: 94 C for 2 min, 94 C for 45sec, 50 C for 45sec, and 72 C for 45sec for a total of 35 cycles, then 72 C for 5 min; Primer sequences were as follows: 50 -CCTGACCAGTGGCTCTGTTT -30 , 50 -CACATCCCACACTTTGATCG-30 for hepcidin; 50 -GTGGGGCGCCCCAGGCACC A-30 , 50 -CTCCTTAATGTCACGCACGATTTC-30 for GAPDH. After 1.8% agarose gel electrophoresis with 1 mg/mL ethidium bromide dye, RTPCR products were observed with a GIS-2020 gel scanning image analytical system. By using 100 bp DNA Markers (TaKaRa Bio, Japan) as the standard molecular weight and GAPDH as an internal reference, the ratio of hepcidin to GAPDH was calculated. All PCR data in this paper were shown as the fold change of target gene normalized to GAPDH. 2.6. Western blot Cells and C57BL/6 mice liver tissues were washed twice with cold PBS and lysed on ice in cell lysis solution with protease inhibitors and quantified by Bradford method. 50 mg protein lysates were resolved on 10% SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and blocked in 5% nonfat dry milk in Tris-buffered saline (pH7.5). Membranes were immunoblotted overnight at 4 C with antihepcidin (Alpha Diagnostic International, San Antonio, USA), antiphosphorylated p38, anti-p38, anti-IL-1, anti-IL-6, anti-TNF-a (Cell Signaling Technology, Beverly, USA), and anti-b-actin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA), respectively, and followed by their respective secondary antibodies. Signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL). The intensity of the protein bands was analyzed by Quantity One (Bio-Rad Laboratories, Hercules, CA). All protein expression levels in this paper were shown as the fold change of target to b-actin. 2.7. Statistical analysis Data were presented as mean ± standard deviation (SD) for normal distribution. Groups were compared by one-way Analysis of variance (ANOVA) and multiple comparisons by LSD-t test by SPSS 21.0 (IBM SPSS for Windows, Version 21.0; IBM Corporation, Armonk, NY, USA). Outliers were excluded with larger or smaller than 2 SD. P < 0.05 was considered significant.
2.4. Cell lines and cell culture 3. Results Human normal liver cell line (L-02) and hepatocellular carcinoma cell line (HepG2) were purchased from the cell bank of Chinese Academy of Sciences. L-02 cells were maintained in DMEM (Hyclone, Logan city, USA) supplemented with 10% fetal bovine serum (FBS). HepG2 cells were maintained in RPMI 1640 (Hyclone, Logan city, USA) supplemented with 10% FBS. All the cell lines were incubated in a 5% CO2 humidified atmosphere at 37 C. APS with >98% purity was used in cell culture experiments as similarly described in previous publications [21]. For our experiments, cells were treated with 5 different concentrations of APS (0, 0.15, 0.30, 0.45 and 0.6 g/L) for various periods of time (0, 24, 48, 72, 96 or 120 h). The optimal induction concentration and time of APS were determined.
3.1. APS increases the expression of hepcidin in vitro and in vivo We first determined the effects of APS on hepcidin gene expression in HepG2 and L-02 cells. The cells were treated with 0, 0.15, 0.30, 0.45 and 0.60 g/L APS and the expression of hepcidin mRNA was analyzed at 48 h. RT-PCR results revealed that APS increased the expression of hepcidin in a concentration dependent manner. The cells treated with 0.15 g/L of APS displayed maximal increase of hepcidin expression (Fig. 1A and B). Next, we investigated the temporal dependence of hepcidin induction in response to 0.15 g/L APS. The HepG2 and L-02 cells were treated for up to 120 h, and the expression of the hepcidin
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Fig. 1. APS induces hepcidin expression in vitro and in vivo. The expression level of hepcidin gene was normalized to the corresponding level of GAPDH. (A) Representative electrophoresis images of hepcidin and GAPDH mRNA in HepG2 and L-02 cells cultured with various concentrations of APS for 48 h. (B) Expression levels of hepcidin mRNA in HepG2 and L-02 cells treated with various concentrations of APS for 48 h. (C) Representative electrophoresis images of hepcidin and GAPDH mRNA in HepG2 and L-02 cells cultured with 0.15 g/L of APS for various days. (D) Expression levels of hepcidin mRNA in HepG2 and L-02 cells treated with 0.15 g/L APS for various days. (E) Representative images of the protein level of hepcidin. Total proteins were isolated from HepG2 and L-02 cells which were cultured in 0.15 g/L APS for 48 or 72 h, respectively. The experiment was performed in the absence or presence of SB203580 (SB). (F) Protein expression levels of hepcidin in HepG2 and L-02 cells cultured in 0.15 g/L APS for 48 or 72 h, respectively. (G) Representative image of total protein from C57BL/6 mice liver tissues, harvested following 7 days of treatment with 200 mg/kg APS. (H) Protein expression levels of mice liver, harvested following 7 days of treatment with 200 mg/kg APS. *P < 0.05, vs. the negative control.
gene was analyzed by RT-PCR at different time points. The expression of hepcidin in HepG2 and L-02 cells reached peaks at 48 h and 72 h, respectively, and then gradually declined (Fig. 1C and D). Furthermore, the expression of hepcidin was also examined at the protein level in HepG2 and L-02 cells. HepG2 and L-02 cells untreated or treated with 0.15 g/L of APS for 48 h and 72 h respectively, were harvested and subjected to western blot analysis. The experiment was performed in the absence or presence of SB203580 (SB) (Merck KGaA, Darmstadt, Germany), a specific inhibitor of p38MAPK signaling pathway. The relative expression level of hepcidin was normalized to b-actin. The western blot results indicated that compared with HepG2 and L-02 cells untreated with APS, the expression levels of hepcidin in HepG2 and L-02 cells treated with APS increased 3.64-fold and 3.59-fold, respectively (P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h significantly reduced the effect of hepcidin induction by APS (P < 0.05) (Fig. 1 E and F). In addition, we determined the impact of APS on hepcidin protein level in liver tissues of C57BL/6 mice. The expression of hepcidin in APS group increased 2.24-fold compared to NS group (P < 0.05), while the hepcidin protein level induced in the model plus APS group increased 1.51-fold compared to the model plus NS group (P < 0.05) (Fig. 1G and H).
3.2. APS reduces the iron concentrations in the serum and organs in mice The iron concentration in iron overloaded C57BL/6 mice with or without APS treatment was determined. The results showed that the iron concentrations of serum, liver, spleen, and heart in iron overloaded mice increased 2.91, 10.51, 13.98 and 5.41- fold compared to normal mice, respectively (P < 0.05). Administration of APS reduced the iron level by 1.95, 4.56, 6.83 and 3.02 - fold compared to normal mice, respectively (P < 0.05) (S1).
3.3. APS administration reduces the deposition of hemosiderin effectively One of the consequences of iron overload is hemosiderosis, which is marked by the presence of hemosiderin in the liver, spleen, and heart. Hemosiderin is the complex formed of broken hemoglobin, ferric oxide (unused iron) and ferritin. The liver, spleen, and heart sections taken from the iron overloaded mice showed increased hemosiderin depositions. However, the liver, spleen, and heart sections taken from the APS-treated mice showed decrease in the depositions of hemosiderin (Fig. 2A and B). 3.4. APS activates p38 MAPK signaling pathways The level of phosphorylated p38 MAPK (p-p38) induced by APS was evaluated by western blot. In HepG2 cells, APS caused a 5.71fold increase in p-p38 in the absence of p38MAPK inhibitor SB (Fig. 3A and B, P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h, APS had no effect on p38 phosphorylation (P > 0.05). In L02 cells, in the absence of SB, APS caused a 21.33-fold increase in pp38 level (Fig. 3C and D) (P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h significantly reduced the effect of the APSmediated p-p38 induction (9.22-fold increase of p-p38, P < 0.05). These data provided evidences that APS activated the p38 MAPK signaling pathway in vitro. In order to confirm whether the p38 MAPK pathway was involved in APS induced hepcidin expression, we measured p38 phosphorylation and p38 total protein in liver tissues of C57BL/6 mice after APS treatment. The results in Fig. 3E and F indicated that the p-p38 MARK was significantly elevated in mice liver tissues, suggesting that the induction of hepcidin expression in C57BL/6 mice was closely associated with the activation of the p38 MARK pathway. 3.5. Effects of APS on the production of IL-1, IL-6, and TNF-a in vitro and in vivo In HepG2 and L-02 cells treated with APS, the expression of IL-6
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Fig. 2. APS reduces the iron concentrations in tissues. Representative histological analysis of iron deposits in liver, heart, and spleen following 7 days of APS therapy. Specimens were stained with (A). H&E and (B). Perl's Prussian blue iron stain. Brown hemosiderin (iron pigment) in H&E staining, and blue pigmentation in Perl's Prussian blue staining indicated the distribution of iron depositions. The magnification is 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. APS activates the p38 MAPK pathway in vitro and in vivo. Total proteins were isolated from HepG2 and L-02 cells treated with 0.15 g/L APS for 48 h or 72 h, respectively. The experiment was performed in the absence or presence of SB203580 (SB). Levels of phosphorylated p38 (p-p38) were normalized to the total p38 and untreated HepG2 or L-02 cell p-p38 levels were normalized to 1. (A) Representative images of the protein level of p-p38 in HepG2 cells. (B) Protein expression level of p-p38 in HepG2 cells treated with or without SB. (C) Representative images of the protein level of p-p38 in L-02 cells. (D) Protein expression levels of p-p38 in L-02 cells treated with or without SB. (E) Representative images of the protein level of p-p38 in mice liver tissues. (F) Protein expression level of p-p38 in mice liver tissues after administration of APS. *P < 0.05, vs. the negative control.
increased 6.29-fold (P < 0.05) and 35.57-fold (P < 0.05) compared with the untreated control, respectively (Fig. 4AeD). It was notable that pre-treatment with SB reduced the effects of APS on IL-6 expression in HepG2 and L-02 cells with 42.47% and 51.71%, respectively. However, the protein expressions of IL-1 and TNF-a were not affected by APS treatment (Fig. 4AeD). To determine the effects of APS on cytokines in vivo, we examined IL-1, IL-6, and TNF-a protein expressions in C57BL/6 mice liver
tissues by western blot. As shown in Fig. 4E and F, compared to the NS group, the expression of IL-6 protein in APS group increased 1.72-fold (P < 0.05). When compared to the model plus NS group, the level of IL-6 of the model plus APS group increased 1.72-fold (P < 0.05). However, the expressions of IL-1 and TNF-a at protein level were not affected by APS treatment (Fig. 4 E and F). Taken together, our results demonstrated that APS significantly increased the expression of IL-6 in vitro and in vivo.
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Fig. 4. IL-6 is elevated after APS treatment in vitro and in vivo. The levels of interleukin-1 (IL-6), interleukin-6 (IL-1), and tumor necrosis factor-a (TNF-a) were determined by western blot. (A) Representative images of the protein levels of IL-6, IL-1, and TNF-a in HepG2 cells treated with APS, alone or in combination with SB pre-treatment. (B) Protein expression levels of IL-6, IL-1 and, TNF-a in HepG2 cells treated with APS, alone or in combination with SB pre-treatment. (C) Representative images of the protein level of IL-6, IL-1, and TNF-a in L-02 cells treated with APS, alone or in combination with SB pre-treatment. (D) Protein expression levels of IL-6, IL-1, and TNF-a in L-02 cells treated with APS, alone or in combination with SB pre-treatment. (E) Representative images of the protein level of IL-6, IL-1, and TNF-a in mice liver tissues after administration of 200 mg/kg APS for 7 days. (F) Protein expression levels of IL-6, IL-1, and TNF-a in mice liver tissues after administration of 200 mg/kg APS for 7 days. *P < 0.05, vs. the negative control.
4. Discussion As an important detrimental factor contributing to organ damage and mortality, iron overload is a major complication in patients with thalassemia [1,22]. Recent studies revealed that expressions of hepcidin mRNA and hepcidin protein were down-regulated in hepatic tissues obtained from thalassemic patients compared to healthy controls [23,24]. Decreased hepcidin expression led to inhibition of ferroportin degradation and iron increase from recycling macrophages and intestinal absorption [8]. However, in patients suffering thalassemia, the iron could not be effectively utilized by the erythrocytes, leading to iron overload in the presence of anemia. Selective modulation of hepcidin expression is a well-validated strategy to treat the secondary iron overload associated with thalassemia [3]. Identification of drugs that can upregulate hepcidin implicates clinical significance. Alkhateeb et al. found that ferristatin II increased hepcidin expression both in vivo and in vitro [25]. Kemna et al. reported that administration of lipopolysaccharide resulted in elevated hepcidin production in healthy humans [26]. However, to our knowledge, hepcidin-inducing natural drugs have not been identified yet. Astragalus as a traditional Chinese medicine has a long history for treatment of various human diseases. And it has been proven to be clinically safe without obvious adverse effects. Astragalus has a variety of pharmacological properties, including immunomodulatory, antioxidant, anti-stress, hepatoprotective, and anti-cancer functions [27]. APS as a major bioactive component of astragalus and exhibits potent hematopoietic activity. It was documented that APS was capable of stimulating erythroid colony-forming unit and erythroid burst-forming unit colony formation, culminating in increased production of erythrocytes and hemoglobin [28]. Our
previous study showed that APS had the ability to stimulate gglobin expression and hemoglobin F production and provided therapeutic effects on thalassemia [17,18]. In this study, we demonstrated that APS could stimulate the expression of hepcidin in hepatocytes, accompanied with reduced iron deposits. Activation of the p38MAPK pathway was required for APS-mediated upregulation of hepcidin. We showed that the administration of iron dextran significantly enhanced hepatic expression of hepcidin in mice. Pigeon et al. also reported that either high-iron diet or hepatic iron overload could lead to elevated expression of hepcidin in the liver of mice [29]. These findings indicated that hepcidin was an iron responsive gene. It was notable that hepcidin expression in iron overloaded mice injected with APS was increased, along with reduced serum iron and tissue iron load. Our data suggested that upregulation of hepcidin could be an important mechanism for APS-mediated prevention of iron depositions. Phosphorylation-dependent activation of p38MAPK is implicated in stress response. Various stimuli such as ultraviolet, radiation, and inflammatory cytokines are able to stimulate p38MAKP signaling [30]. It was reported that p38MAPK activation could promote the expression of inflammatory cytokines including IL-1, IL-4, IL-6, IL-8, and TNF-a. IL-6 was reported to have the ability to upregulate hepcidin via activation of the JAK/STAT pathways [31,32]. Our data showed that APS led to an enhancement of p38 MAPK phosphorylation and upregulation of IL-6 in hepatocytes. However, APS had little influence on the expression of IL-1 and TNF-a. Notably, these effects elicited by APS were significantly reversed by pharmacological inhibition of p38 MAPK with SB. These findings suggested that activation of p38 MAPK and release of IL-6 might mediate the induction of hepcidin by APS.
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Conflicts of interest The authors have no conflict of interest. Acknowledgments This work was supported by Medical Science and Technology Project of Henan Province (201403132) and Scientific Research Fund of Xinxiang Medical University (2013QN109). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.02.088. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.02.088. References [1] R. Origa, R. Galanello, T. Ganz, N. Giagu, L. Maccioni, G. Faa, E. Nemeth, Liver iron concentrations and urinary hepcidin in beta-thalassemia, Haematologica 92 (2007) 583e588. [2] A. Ghavamzadeh, M. Mirzania, N. Kamalian, N. Sedighi, P. Azimi, Hepatic iron overload and fibrosis in patients with beta thalassemia major after hematopoietic stem cell transplantation: a pilot study, Int. J. Hematol. Oncol. Stem Cell Res. 9 (2015) 55e59. [3] S. Gardenghi, P. Ramos, M.F. Marongiu, L. Melchiori, L. Breda, E. Guy, K. Muirhead, N. Rao, C.N. Roy, N.C. Andrews, E. Nemeth, A. Follenzi, X. An, N. Mohandas, Y. Ginzburg, E.A. Rachmilewitz, P.J. Giardina, R.W. Grady, S. Rivella, Hepcidin as a therapeutic tool to limit iron overload and improve anemia in beta-thalassemic mice, J. Clin. Invest. 120 (2010) 4466e4477. [4] S. Gardenghi, M.F. Marongiu, P. Ramos, E. Guy, L. Breda, A. Chadburn, Y. Liu, N. Amariglio, G. Rechavi, E.A. Rachmilewitz, W. Breuer, Z.I. Cabantchik, D.M. Wrighting, N.C. Andrews, M. de Sousa, P.J. Giardina, R.W. Grady, S. Rivella, Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and upregulation of ferroportin, Blood 109 (2007) 5027e5035. [5] T. Ganz, E. Nemeth, Hepcidin and disorders of iron metabolism, Annu. Rev. Med. 62 (2011) 347e360. [6] T. Ganz, E. Nemeth, Hepcidin and iron homeostasis, Biochim. Biophys. Acta 1823 (2012) 1434e1443. [7] A.E. Armitage, L.A. Eddowes, U. Gileadi, S. Cole, N. Spottiswoode, T.A. Selvakumar, L.P. Ho, A.R. Townsend, H. Drakesmith, Hepcidin regulation by innate immune and infectious stimuli, Blood 118 (2011) 4129e4139. [8] H. Drakesmith, A.M. Prentice, Hepcidin and the iron-infection axis, Science 338 (2012) 768e772. [9] G. Nicolas, C. Chauvet, L. Viatte, J.L. Danan, X. Bigard, I. Devaux, C. Beaumont, A. Kahn, S. Vaulont, The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation, J. Clin. Invest. 110 (2002) 1037e1044. [10] B. Qiao, P. Sugianto, E. Fung, A. Del-Castillo-Rueda, M.J. Moran-Jimenez, T. Ganz, E. Nemeth, Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination, Cell Metab. 15 (2012) 918e924. [11] S.L. Ross, L. Tran, A. Winters, K.J. Lee, C. Plewa, I. Foltz, C. King, L.P. Miranda, J. Allen, H. Beckman, K.S. Cooke, G. Moody, B.J. Sasu, E. Nemeth, T. Ganz, G. Molineux, T.L. Arvedson, Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAKSTAT, Cell Metab. 15 (2012) 905e917. [12] I. De Domenico, D.M. Ward, J. Kaplan, Hepcidin regulation: ironing out the
details, J. Clin. Invest. 117 (2007) 1755e1758. [13] R. Origa, M. Cazzola, E. Mereu, F. Danjou, S. Barella, N. Giagu, R. Galanello, D.W. Swinkels, Differences in the erythropoiesis-hepcidin-iron store axis between hemoglobin H disease and beta-thalassemia intermedia, Haematologica 100 (2015) e169e171. [14] A. Aboul-Enein, A. El-Beshlawy, M. Hamdy, I. Shaheen, Z. El-Saadany, A. Samir, H.A. El-Samie, Peripheral expression of hepcidin gene in Egyptian betathalassemia major, Gene 564 (2015) 206e209. [15] G. Papanikolaou, M. Tzilianos, J.I. Christakis, D. Bogdanos, K. Tsimirika, J. MacFarlane, Y.P. Goldberg, N. Sakellaropoulos, T. Ganz, E. Nemeth, Hepcidin in iron overload disorders, Blood 105 (2005) 4103e4105. [16] M. Cargnello, P.P. Roux, Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases, Microbiol. Mol. Biol. Rev. 75 (2011) 50e83. [17] M. Yang, X.H. Qian, D.H. Zhao, S.Z. Fu, Effects of Astragalus polysaccharide on the erythroid lineage and microarray analysis in K562 cells, J. Ethnopharmacol. 127 (2010) 242e250. [18] Z.M. Lu, X.H. Qian, Z.W. Chen, C.H. Zhang, L.S. Guo, J. Chen, Prospective clinical study of radix astragali and its compound prescription for treatment of betathalassemia in children, Zhongguo Dang Dai Er Ke Za Zhi 14 (2012) 344e349. [19] B.S. Pace, X.H. Qian, J. Sangerman, S.F. Ofori-Acquah, B.S. Baliga, J. Han, S.D. Critz, p38 MAP kinase activation mediates g-globin gene induction in erythroid progenitors, Exp. Hematol. 31 (2003) 1089e1096. [20] A. Luan, F. Tang, Y. Yang, M. Lu, H. Wang, Y. Zhang, Astragalus polysaccharide attenuates isoproterenol-induced cardiac hypertrophy by regulating TNFalpha/PGC-1alpha signaling mediated energy biosynthesis, Environ. Toxicol. Pharmacol. 39 (2015) 1081e1090. [21] J. Zhang, J.Y. Gu, Z.S. Chen, K.C. Xing, B. Sun, Astragalus polysaccharide suppresses palmitate-induced apoptosis in human cardiac myocytes: the role of Nrf1 and antioxidant response, Int. J. Clin. Exp. Pathol. 8 (2015) 2515e2524. [22] B.A. El, T.M. El, M. Hamdy, G.M. El, K.A. Azim, D. Salem, F. Said, A. Samir, P.T. St, D.J. Pennell, Low prevalence of cardiac siderosis in heavily iron loaded Egyptian thalassemia major patients, Ann. Hematol. 93 (2014) 375e379. [23] G. Ravasi, S. Pelucchi, P. Trombini, R. Mariani, N. Tomosugi, G.L. Modignani, M. Pozzi, E. Nemeth, T. Ganz, H. Hayashi, D. Barisani, A. Piperno, Hepcidin expression in iron overload diseases is variably modulated by circulating factors, PLos One 7 (2012) e36425. [24] S.R. Pasricha, D.M. Frazer, D.K. Bowden, G.J. Anderson, Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with beta-thalassemia major: a longitudinal study, Blood 122 (2013) 124e133. [25] A.A. Alkhateeb, P.D. Buckett, A.M. Gardeck, J. Kim, S.L. Byrne, P.G. Fraenkel, M. Wessling-Resnick, The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro, Am. J. Physiol. Gastrointest. Liver Physiol. 308 (2015) G1019eG1026. [26] E. Kemna, P. Pickkers, E. Nemeth, H. van der Hoeven, D. Swinkels, Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS, Blood 106 (2005) 1864e1866. [27] B.M. Shao, W. Xu, H. Dai, P. Tu, Z. Li, X.M. Gao, A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb, Biochem. Biophys. Res. Commun. 320 (2004) 1103e1111. [28] X.L. Zhu, B.D. Zhu, Mechanisms by which Astragalus membranaceus injection regulates hematopoiesis in myelosuppressed mice, Phytother. Res. 21 (2007) 663e667. [29] C. Pigeon, G. Ilyin, B. Courselaud, P. Leroyer, B. Turlin, P. Brissot, O. Loreal, A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload, J. Biol. Chem. 276 (2001) 7811e7819. [30] G.L. Schieven, The p38alpha kinase plays a central role in inflammation, Curr. Top. Med. Chem. 9 (2009) 1038e1048. [31] X. Zhang, B.H. Rovin, Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines, Biochim. Biophys. Acta 1800 (2010) 1262e1267. [32] I. De Domenico, T.Y. Zhang, C.L. Koening, R.W. Branch, N. London, E. Lo, R.A. Daynes, J.P. Kushner, D. Li, D.M. Ward, J. Kaplan, Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice, J. Clin. Invest. 120 (2010) 2395e2405.
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Astragalus polysaccharide upregulates hepcidin and reduces iron overload in mice via activation of p38 mitogen-activated protein kinase Feng Ren a, b, Xin-Hua Qian a, *, Xin-Lai Qian b, ** a b
Department of Neonatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pathology, Xinxiang Medical University, Xinxiang 453003, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2016 Accepted 21 February 2016 Available online 23 February 2016
Thalassemia is a genetic disease characterized by iron overload which is a major detrimental factor contributing to mortality and organ damage. The hepcidin secreted by liver plays an essential role in orchestrating iron metabolism. Lowering iron load in thalassemia patients by means of increasing hepcidin might be a therapeutic strategy. In this study, we first found that astragalus polysaccharide (APS) significantly increased hepcidin expression in HepG2 and L-02 cell lines originating from hepatocytes and mice liver, respectively. Following treatment with APS, the iron concentrations in serum, liver, spleen, and heart were significantly reduced in comparison to saline treated control mice. In further experiments, upregulation of interleukin-6 (IL-6) and enhanced p38 MAPK phosphorylation were detected in APS treated cells and mice, and as documented in previous studies, IL-6 and P38 MAPK phosphorylation are involved in the regulation of hepcidin expression. We also found that the effects of APS on upregulating hepcidin and IL-6 expressions could be antagonized by pretreatment with SB203580, an inhibitor of p38 MAPK signaling. These findings suggest that activation of p38 MAPK and release of IL-6 might mediate induction of hepcidin by APS. It is concluded that APS might have therapeutic implications in patients with iron overload, especially for thalassemia patients. © 2016 Elsevier Inc. All rights reserved.
Keywords: Astragalus polysaccharide Thalassemia Iron load Hepcidin p38MAPK signaling pathway Interleukin-6
1. Introduction Patients with thalassemia are more likely to suffer from the fatal complication of iron overload, which causes damages to the liver, spleen, heart, and endocrine system [1]. Secondary iron overload can also result from long-term transfusion therapy in transfusiondependent thalassemia patients [2e4]. Hepcidin is secreted mainly by the liver and function as a key regulator of body iron homeostasis [5,6]. Hepcidin is homeostatically regulated by iron and erythropoietic activity, as well as inflammatory cytokines [7e9]. Many diseases associated with iron overload arise upon deregulation of hepcidin production. Hepcidin controls systemic iron homeostasis through rapid degradation of ferroportin, which
* Corresponding author. Department of Neonatology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China. ** Corresponding author. E-mail addresses: [email protected] (X.-H. Qian), [email protected] (X.-L. Qian). http://dx.doi.org/10.1016/j.bbrc.2016.02.088 0006-291X/© 2016 Elsevier Inc. All rights reserved.
decreases intestinal iron absorption and the mobilization of iron stores [10]. Elevated ferroportin expression secondary to hepcidin down-regulation is commonly associated with high level of iron in serum and organs [11,12]. Recent studies revealed that the expression of hepcidin was down-regulated among the thalassemia patients compared to the normal controls [13e15]. The iron overload disorder develops through a mechanism of excessive erythropoiesis, which suppresses hepcidin and increases iron absorption in thalassemia patients. Accumulated evidence suggests that therapeutic regimen targeting the hepcidin-ferroportin axis could have important clinical implications for thalassemia patients associated with iron overload. Astragalus polysaccharide (APS) is the major bioactive component of the extracts from roots of Astragalus membranaceus, a common Chinese medicinal herb. The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses [16]. Our previous study showed that APS could stimulate g-globin expression and hemoglobin F production and was safe in children with thalassemia [17e19]. However, it is still unknown whether APS has the ability to
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regulate hepcidin and reduce iron overload in mice via activation of p38MAPK. In this study, we investigated the role of APS in regulating hepcidin production and its efficacy on lowering iron overload. 2. Materials and methods 2.1. Animals C57BL/6 mice were purchased from Experimental Animal Center, Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). All mice were randomly divided into four groups (n ¼ 8): normal saline (NS) group, APS group, model plus NS group, and model plus APS group. Mice in the last two groups were intraperitoneally (i.p.) injected with iron dextran (Heshengtang Animal Pharmaceutical Company, Guangzhou, China) at 150 mg/kg every other day for 4 weeks. Meanwhile, the other two groups received isovolumic saline. After that, mice in the APS group and the model plus APS group were administered daily with APS 200 mg/kg of body weight for 7 days [20]. APS with a purity >98% was purchased from Cinorch Pharmaceutical Co., Ltd. (Tianjin, China). Simultaneously, the other two groups received isovolumic saline. At the end of the experiment, all animals were humanely euthanized, and the blood samples were harvested from the inferior vena cava and centrifuged at 4 C, 3000 revolutions per minute (rpm) for 10 min. Then the supernatant was collected and stored at 20 C. Liver, spleen, and heart were removed, rinsed and subjected to pathological examination and total protein extraction. All animal experiments were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. 2.2. Measurement of iron concentrations in mice Iron concentration in mice serum was assayed by automatic biochemistry analyzer (SIMENS Advia 2400). Iron depositions in liver, spleen, and heart were determined by atomic absorption spectrophotometer (AA240FS series by Varian Australia Pty Ltd.). 2.3. Histological analysis Iron depositions were identified by hematoxylin-eosin (H&E) and Prussian blue staining. Liver, spleen, and heart of mice in each group were fixed in 4% paraformaldehyde solution for 24 h and embedded in paraffin. 5 mm-thick sections were processed with H&E and Prussian blue staining for observation of iron depositions, respectively.
2.5. RT-PCR Total RNA was extracted from the cultured cells in accordance with the instructions for the Trizol total RNA extraction kit (Invitrogen, Carlsbad, USA) and the ratio of OD260 and OD280 was 1.8e2.0. The harvested RNA was diluted to a final concentration of 1 mg/mL, and stored at 70 C. The conditions for the first round of RT synthesis of cDNA were as follows: 42 C for 30 min, 99 C for 5 min and 5 C for 5 min. PCR reaction conditions were as follows: 94 C for 2 min, 94 C for 45sec, 50 C for 45sec, and 72 C for 45sec for a total of 35 cycles, then 72 C for 5 min; Primer sequences were as follows: 50 -CCTGACCAGTGGCTCTGTTT -30 , 50 -CACATCCCACACTTTGATCG-30 for hepcidin; 50 -GTGGGGCGCCCCAGGCACC A-30 , 50 -CTCCTTAATGTCACGCACGATTTC-30 for GAPDH. After 1.8% agarose gel electrophoresis with 1 mg/mL ethidium bromide dye, RTPCR products were observed with a GIS-2020 gel scanning image analytical system. By using 100 bp DNA Markers (TaKaRa Bio, Japan) as the standard molecular weight and GAPDH as an internal reference, the ratio of hepcidin to GAPDH was calculated. All PCR data in this paper were shown as the fold change of target gene normalized to GAPDH. 2.6. Western blot Cells and C57BL/6 mice liver tissues were washed twice with cold PBS and lysed on ice in cell lysis solution with protease inhibitors and quantified by Bradford method. 50 mg protein lysates were resolved on 10% SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and blocked in 5% nonfat dry milk in Tris-buffered saline (pH7.5). Membranes were immunoblotted overnight at 4 C with antihepcidin (Alpha Diagnostic International, San Antonio, USA), antiphosphorylated p38, anti-p38, anti-IL-1, anti-IL-6, anti-TNF-a (Cell Signaling Technology, Beverly, USA), and anti-b-actin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA), respectively, and followed by their respective secondary antibodies. Signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL). The intensity of the protein bands was analyzed by Quantity One (Bio-Rad Laboratories, Hercules, CA). All protein expression levels in this paper were shown as the fold change of target to b-actin. 2.7. Statistical analysis Data were presented as mean ± standard deviation (SD) for normal distribution. Groups were compared by one-way Analysis of variance (ANOVA) and multiple comparisons by LSD-t test by SPSS 21.0 (IBM SPSS for Windows, Version 21.0; IBM Corporation, Armonk, NY, USA). Outliers were excluded with larger or smaller than 2 SD. P < 0.05 was considered significant.
2.4. Cell lines and cell culture 3. Results Human normal liver cell line (L-02) and hepatocellular carcinoma cell line (HepG2) were purchased from the cell bank of Chinese Academy of Sciences. L-02 cells were maintained in DMEM (Hyclone, Logan city, USA) supplemented with 10% fetal bovine serum (FBS). HepG2 cells were maintained in RPMI 1640 (Hyclone, Logan city, USA) supplemented with 10% FBS. All the cell lines were incubated in a 5% CO2 humidified atmosphere at 37 C. APS with >98% purity was used in cell culture experiments as similarly described in previous publications [21]. For our experiments, cells were treated with 5 different concentrations of APS (0, 0.15, 0.30, 0.45 and 0.6 g/L) for various periods of time (0, 24, 48, 72, 96 or 120 h). The optimal induction concentration and time of APS were determined.
3.1. APS increases the expression of hepcidin in vitro and in vivo We first determined the effects of APS on hepcidin gene expression in HepG2 and L-02 cells. The cells were treated with 0, 0.15, 0.30, 0.45 and 0.60 g/L APS and the expression of hepcidin mRNA was analyzed at 48 h. RT-PCR results revealed that APS increased the expression of hepcidin in a concentration dependent manner. The cells treated with 0.15 g/L of APS displayed maximal increase of hepcidin expression (Fig. 1A and B). Next, we investigated the temporal dependence of hepcidin induction in response to 0.15 g/L APS. The HepG2 and L-02 cells were treated for up to 120 h, and the expression of the hepcidin
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Fig. 1. APS induces hepcidin expression in vitro and in vivo. The expression level of hepcidin gene was normalized to the corresponding level of GAPDH. (A) Representative electrophoresis images of hepcidin and GAPDH mRNA in HepG2 and L-02 cells cultured with various concentrations of APS for 48 h. (B) Expression levels of hepcidin mRNA in HepG2 and L-02 cells treated with various concentrations of APS for 48 h. (C) Representative electrophoresis images of hepcidin and GAPDH mRNA in HepG2 and L-02 cells cultured with 0.15 g/L of APS for various days. (D) Expression levels of hepcidin mRNA in HepG2 and L-02 cells treated with 0.15 g/L APS for various days. (E) Representative images of the protein level of hepcidin. Total proteins were isolated from HepG2 and L-02 cells which were cultured in 0.15 g/L APS for 48 or 72 h, respectively. The experiment was performed in the absence or presence of SB203580 (SB). (F) Protein expression levels of hepcidin in HepG2 and L-02 cells cultured in 0.15 g/L APS for 48 or 72 h, respectively. (G) Representative image of total protein from C57BL/6 mice liver tissues, harvested following 7 days of treatment with 200 mg/kg APS. (H) Protein expression levels of mice liver, harvested following 7 days of treatment with 200 mg/kg APS. *P < 0.05, vs. the negative control.
gene was analyzed by RT-PCR at different time points. The expression of hepcidin in HepG2 and L-02 cells reached peaks at 48 h and 72 h, respectively, and then gradually declined (Fig. 1C and D). Furthermore, the expression of hepcidin was also examined at the protein level in HepG2 and L-02 cells. HepG2 and L-02 cells untreated or treated with 0.15 g/L of APS for 48 h and 72 h respectively, were harvested and subjected to western blot analysis. The experiment was performed in the absence or presence of SB203580 (SB) (Merck KGaA, Darmstadt, Germany), a specific inhibitor of p38MAPK signaling pathway. The relative expression level of hepcidin was normalized to b-actin. The western blot results indicated that compared with HepG2 and L-02 cells untreated with APS, the expression levels of hepcidin in HepG2 and L-02 cells treated with APS increased 3.64-fold and 3.59-fold, respectively (P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h significantly reduced the effect of hepcidin induction by APS (P < 0.05) (Fig. 1 E and F). In addition, we determined the impact of APS on hepcidin protein level in liver tissues of C57BL/6 mice. The expression of hepcidin in APS group increased 2.24-fold compared to NS group (P < 0.05), while the hepcidin protein level induced in the model plus APS group increased 1.51-fold compared to the model plus NS group (P < 0.05) (Fig. 1G and H).
3.2. APS reduces the iron concentrations in the serum and organs in mice The iron concentration in iron overloaded C57BL/6 mice with or without APS treatment was determined. The results showed that the iron concentrations of serum, liver, spleen, and heart in iron overloaded mice increased 2.91, 10.51, 13.98 and 5.41- fold compared to normal mice, respectively (P < 0.05). Administration of APS reduced the iron level by 1.95, 4.56, 6.83 and 3.02 - fold compared to normal mice, respectively (P < 0.05) (S1).
3.3. APS administration reduces the deposition of hemosiderin effectively One of the consequences of iron overload is hemosiderosis, which is marked by the presence of hemosiderin in the liver, spleen, and heart. Hemosiderin is the complex formed of broken hemoglobin, ferric oxide (unused iron) and ferritin. The liver, spleen, and heart sections taken from the iron overloaded mice showed increased hemosiderin depositions. However, the liver, spleen, and heart sections taken from the APS-treated mice showed decrease in the depositions of hemosiderin (Fig. 2A and B). 3.4. APS activates p38 MAPK signaling pathways The level of phosphorylated p38 MAPK (p-p38) induced by APS was evaluated by western blot. In HepG2 cells, APS caused a 5.71fold increase in p-p38 in the absence of p38MAPK inhibitor SB (Fig. 3A and B, P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h, APS had no effect on p38 phosphorylation (P > 0.05). In L02 cells, in the absence of SB, APS caused a 21.33-fold increase in pp38 level (Fig. 3C and D) (P < 0.05). By contrast, pre-treatment with 10 mmol/L SB for 1 h significantly reduced the effect of the APSmediated p-p38 induction (9.22-fold increase of p-p38, P < 0.05). These data provided evidences that APS activated the p38 MAPK signaling pathway in vitro. In order to confirm whether the p38 MAPK pathway was involved in APS induced hepcidin expression, we measured p38 phosphorylation and p38 total protein in liver tissues of C57BL/6 mice after APS treatment. The results in Fig. 3E and F indicated that the p-p38 MARK was significantly elevated in mice liver tissues, suggesting that the induction of hepcidin expression in C57BL/6 mice was closely associated with the activation of the p38 MARK pathway. 3.5. Effects of APS on the production of IL-1, IL-6, and TNF-a in vitro and in vivo In HepG2 and L-02 cells treated with APS, the expression of IL-6
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Fig. 2. APS reduces the iron concentrations in tissues. Representative histological analysis of iron deposits in liver, heart, and spleen following 7 days of APS therapy. Specimens were stained with (A). H&E and (B). Perl's Prussian blue iron stain. Brown hemosiderin (iron pigment) in H&E staining, and blue pigmentation in Perl's Prussian blue staining indicated the distribution of iron depositions. The magnification is 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. APS activates the p38 MAPK pathway in vitro and in vivo. Total proteins were isolated from HepG2 and L-02 cells treated with 0.15 g/L APS for 48 h or 72 h, respectively. The experiment was performed in the absence or presence of SB203580 (SB). Levels of phosphorylated p38 (p-p38) were normalized to the total p38 and untreated HepG2 or L-02 cell p-p38 levels were normalized to 1. (A) Representative images of the protein level of p-p38 in HepG2 cells. (B) Protein expression level of p-p38 in HepG2 cells treated with or without SB. (C) Representative images of the protein level of p-p38 in L-02 cells. (D) Protein expression levels of p-p38 in L-02 cells treated with or without SB. (E) Representative images of the protein level of p-p38 in mice liver tissues. (F) Protein expression level of p-p38 in mice liver tissues after administration of APS. *P < 0.05, vs. the negative control.
increased 6.29-fold (P < 0.05) and 35.57-fold (P < 0.05) compared with the untreated control, respectively (Fig. 4AeD). It was notable that pre-treatment with SB reduced the effects of APS on IL-6 expression in HepG2 and L-02 cells with 42.47% and 51.71%, respectively. However, the protein expressions of IL-1 and TNF-a were not affected by APS treatment (Fig. 4AeD). To determine the effects of APS on cytokines in vivo, we examined IL-1, IL-6, and TNF-a protein expressions in C57BL/6 mice liver
tissues by western blot. As shown in Fig. 4E and F, compared to the NS group, the expression of IL-6 protein in APS group increased 1.72-fold (P < 0.05). When compared to the model plus NS group, the level of IL-6 of the model plus APS group increased 1.72-fold (P < 0.05). However, the expressions of IL-1 and TNF-a at protein level were not affected by APS treatment (Fig. 4 E and F). Taken together, our results demonstrated that APS significantly increased the expression of IL-6 in vitro and in vivo.
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Fig. 4. IL-6 is elevated after APS treatment in vitro and in vivo. The levels of interleukin-1 (IL-6), interleukin-6 (IL-1), and tumor necrosis factor-a (TNF-a) were determined by western blot. (A) Representative images of the protein levels of IL-6, IL-1, and TNF-a in HepG2 cells treated with APS, alone or in combination with SB pre-treatment. (B) Protein expression levels of IL-6, IL-1 and, TNF-a in HepG2 cells treated with APS, alone or in combination with SB pre-treatment. (C) Representative images of the protein level of IL-6, IL-1, and TNF-a in L-02 cells treated with APS, alone or in combination with SB pre-treatment. (D) Protein expression levels of IL-6, IL-1, and TNF-a in L-02 cells treated with APS, alone or in combination with SB pre-treatment. (E) Representative images of the protein level of IL-6, IL-1, and TNF-a in mice liver tissues after administration of 200 mg/kg APS for 7 days. (F) Protein expression levels of IL-6, IL-1, and TNF-a in mice liver tissues after administration of 200 mg/kg APS for 7 days. *P < 0.05, vs. the negative control.
4. Discussion As an important detrimental factor contributing to organ damage and mortality, iron overload is a major complication in patients with thalassemia [1,22]. Recent studies revealed that expressions of hepcidin mRNA and hepcidin protein were down-regulated in hepatic tissues obtained from thalassemic patients compared to healthy controls [23,24]. Decreased hepcidin expression led to inhibition of ferroportin degradation and iron increase from recycling macrophages and intestinal absorption [8]. However, in patients suffering thalassemia, the iron could not be effectively utilized by the erythrocytes, leading to iron overload in the presence of anemia. Selective modulation of hepcidin expression is a well-validated strategy to treat the secondary iron overload associated with thalassemia [3]. Identification of drugs that can upregulate hepcidin implicates clinical significance. Alkhateeb et al. found that ferristatin II increased hepcidin expression both in vivo and in vitro [25]. Kemna et al. reported that administration of lipopolysaccharide resulted in elevated hepcidin production in healthy humans [26]. However, to our knowledge, hepcidin-inducing natural drugs have not been identified yet. Astragalus as a traditional Chinese medicine has a long history for treatment of various human diseases. And it has been proven to be clinically safe without obvious adverse effects. Astragalus has a variety of pharmacological properties, including immunomodulatory, antioxidant, anti-stress, hepatoprotective, and anti-cancer functions [27]. APS as a major bioactive component of astragalus and exhibits potent hematopoietic activity. It was documented that APS was capable of stimulating erythroid colony-forming unit and erythroid burst-forming unit colony formation, culminating in increased production of erythrocytes and hemoglobin [28]. Our
previous study showed that APS had the ability to stimulate gglobin expression and hemoglobin F production and provided therapeutic effects on thalassemia [17,18]. In this study, we demonstrated that APS could stimulate the expression of hepcidin in hepatocytes, accompanied with reduced iron deposits. Activation of the p38MAPK pathway was required for APS-mediated upregulation of hepcidin. We showed that the administration of iron dextran significantly enhanced hepatic expression of hepcidin in mice. Pigeon et al. also reported that either high-iron diet or hepatic iron overload could lead to elevated expression of hepcidin in the liver of mice [29]. These findings indicated that hepcidin was an iron responsive gene. It was notable that hepcidin expression in iron overloaded mice injected with APS was increased, along with reduced serum iron and tissue iron load. Our data suggested that upregulation of hepcidin could be an important mechanism for APS-mediated prevention of iron depositions. Phosphorylation-dependent activation of p38MAPK is implicated in stress response. Various stimuli such as ultraviolet, radiation, and inflammatory cytokines are able to stimulate p38MAKP signaling [30]. It was reported that p38MAPK activation could promote the expression of inflammatory cytokines including IL-1, IL-4, IL-6, IL-8, and TNF-a. IL-6 was reported to have the ability to upregulate hepcidin via activation of the JAK/STAT pathways [31,32]. Our data showed that APS led to an enhancement of p38 MAPK phosphorylation and upregulation of IL-6 in hepatocytes. However, APS had little influence on the expression of IL-1 and TNF-a. Notably, these effects elicited by APS were significantly reversed by pharmacological inhibition of p38 MAPK with SB. These findings suggested that activation of p38 MAPK and release of IL-6 might mediate the induction of hepcidin by APS.
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Conflicts of interest The authors have no conflict of interest. Acknowledgments This work was supported by Medical Science and Technology Project of Henan Province (201403132) and Scientific Research Fund of Xinxiang Medical University (2013QN109). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.02.088. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.02.088. References [1] R. Origa, R. Galanello, T. Ganz, N. Giagu, L. Maccioni, G. Faa, E. Nemeth, Liver iron concentrations and urinary hepcidin in beta-thalassemia, Haematologica 92 (2007) 583e588. [2] A. Ghavamzadeh, M. Mirzania, N. Kamalian, N. Sedighi, P. Azimi, Hepatic iron overload and fibrosis in patients with beta thalassemia major after hematopoietic stem cell transplantation: a pilot study, Int. J. Hematol. Oncol. Stem Cell Res. 9 (2015) 55e59. [3] S. Gardenghi, P. Ramos, M.F. Marongiu, L. Melchiori, L. Breda, E. Guy, K. Muirhead, N. Rao, C.N. Roy, N.C. Andrews, E. Nemeth, A. Follenzi, X. An, N. Mohandas, Y. Ginzburg, E.A. Rachmilewitz, P.J. Giardina, R.W. Grady, S. Rivella, Hepcidin as a therapeutic tool to limit iron overload and improve anemia in beta-thalassemic mice, J. Clin. Invest. 120 (2010) 4466e4477. [4] S. Gardenghi, M.F. Marongiu, P. Ramos, E. Guy, L. Breda, A. Chadburn, Y. Liu, N. Amariglio, G. Rechavi, E.A. Rachmilewitz, W. Breuer, Z.I. Cabantchik, D.M. Wrighting, N.C. Andrews, M. de Sousa, P.J. Giardina, R.W. Grady, S. Rivella, Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and upregulation of ferroportin, Blood 109 (2007) 5027e5035. [5] T. Ganz, E. Nemeth, Hepcidin and disorders of iron metabolism, Annu. Rev. Med. 62 (2011) 347e360. [6] T. Ganz, E. Nemeth, Hepcidin and iron homeostasis, Biochim. Biophys. Acta 1823 (2012) 1434e1443. [7] A.E. Armitage, L.A. Eddowes, U. Gileadi, S. Cole, N. Spottiswoode, T.A. Selvakumar, L.P. Ho, A.R. Townsend, H. Drakesmith, Hepcidin regulation by innate immune and infectious stimuli, Blood 118 (2011) 4129e4139. [8] H. Drakesmith, A.M. Prentice, Hepcidin and the iron-infection axis, Science 338 (2012) 768e772. [9] G. Nicolas, C. Chauvet, L. Viatte, J.L. Danan, X. Bigard, I. Devaux, C. Beaumont, A. Kahn, S. Vaulont, The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation, J. Clin. Invest. 110 (2002) 1037e1044. [10] B. Qiao, P. Sugianto, E. Fung, A. Del-Castillo-Rueda, M.J. Moran-Jimenez, T. Ganz, E. Nemeth, Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination, Cell Metab. 15 (2012) 918e924. [11] S.L. Ross, L. Tran, A. Winters, K.J. Lee, C. Plewa, I. Foltz, C. King, L.P. Miranda, J. Allen, H. Beckman, K.S. Cooke, G. Moody, B.J. Sasu, E. Nemeth, T. Ganz, G. Molineux, T.L. Arvedson, Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAKSTAT, Cell Metab. 15 (2012) 905e917. [12] I. De Domenico, D.M. Ward, J. Kaplan, Hepcidin regulation: ironing out the
details, J. Clin. Invest. 117 (2007) 1755e1758. [13] R. Origa, M. Cazzola, E. Mereu, F. Danjou, S. Barella, N. Giagu, R. Galanello, D.W. Swinkels, Differences in the erythropoiesis-hepcidin-iron store axis between hemoglobin H disease and beta-thalassemia intermedia, Haematologica 100 (2015) e169e171. [14] A. Aboul-Enein, A. El-Beshlawy, M. Hamdy, I. Shaheen, Z. El-Saadany, A. Samir, H.A. El-Samie, Peripheral expression of hepcidin gene in Egyptian betathalassemia major, Gene 564 (2015) 206e209. [15] G. Papanikolaou, M. Tzilianos, J.I. Christakis, D. Bogdanos, K. Tsimirika, J. MacFarlane, Y.P. Goldberg, N. Sakellaropoulos, T. Ganz, E. Nemeth, Hepcidin in iron overload disorders, Blood 105 (2005) 4103e4105. [16] M. Cargnello, P.P. Roux, Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases, Microbiol. Mol. Biol. Rev. 75 (2011) 50e83. [17] M. Yang, X.H. Qian, D.H. Zhao, S.Z. Fu, Effects of Astragalus polysaccharide on the erythroid lineage and microarray analysis in K562 cells, J. Ethnopharmacol. 127 (2010) 242e250. [18] Z.M. Lu, X.H. Qian, Z.W. Chen, C.H. Zhang, L.S. Guo, J. Chen, Prospective clinical study of radix astragali and its compound prescription for treatment of betathalassemia in children, Zhongguo Dang Dai Er Ke Za Zhi 14 (2012) 344e349. [19] B.S. Pace, X.H. Qian, J. Sangerman, S.F. Ofori-Acquah, B.S. Baliga, J. Han, S.D. Critz, p38 MAP kinase activation mediates g-globin gene induction in erythroid progenitors, Exp. Hematol. 31 (2003) 1089e1096. [20] A. Luan, F. Tang, Y. Yang, M. Lu, H. Wang, Y. Zhang, Astragalus polysaccharide attenuates isoproterenol-induced cardiac hypertrophy by regulating TNFalpha/PGC-1alpha signaling mediated energy biosynthesis, Environ. Toxicol. Pharmacol. 39 (2015) 1081e1090. [21] J. Zhang, J.Y. Gu, Z.S. Chen, K.C. Xing, B. Sun, Astragalus polysaccharide suppresses palmitate-induced apoptosis in human cardiac myocytes: the role of Nrf1 and antioxidant response, Int. J. Clin. Exp. Pathol. 8 (2015) 2515e2524. [22] B.A. El, T.M. El, M. Hamdy, G.M. El, K.A. Azim, D. Salem, F. Said, A. Samir, P.T. St, D.J. Pennell, Low prevalence of cardiac siderosis in heavily iron loaded Egyptian thalassemia major patients, Ann. Hematol. 93 (2014) 375e379. [23] G. Ravasi, S. Pelucchi, P. Trombini, R. Mariani, N. Tomosugi, G.L. Modignani, M. Pozzi, E. Nemeth, T. Ganz, H. Hayashi, D. Barisani, A. Piperno, Hepcidin expression in iron overload diseases is variably modulated by circulating factors, PLos One 7 (2012) e36425. [24] S.R. Pasricha, D.M. Frazer, D.K. Bowden, G.J. Anderson, Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with beta-thalassemia major: a longitudinal study, Blood 122 (2013) 124e133. [25] A.A. Alkhateeb, P.D. Buckett, A.M. Gardeck, J. Kim, S.L. Byrne, P.G. Fraenkel, M. Wessling-Resnick, The small molecule ferristatin II induces hepatic hepcidin expression in vivo and in vitro, Am. J. Physiol. Gastrointest. Liver Physiol. 308 (2015) G1019eG1026. [26] E. Kemna, P. Pickkers, E. Nemeth, H. van der Hoeven, D. Swinkels, Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS, Blood 106 (2005) 1864e1866. [27] B.M. Shao, W. Xu, H. Dai, P. Tu, Z. Li, X.M. Gao, A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb, Biochem. Biophys. Res. Commun. 320 (2004) 1103e1111. [28] X.L. Zhu, B.D. Zhu, Mechanisms by which Astragalus membranaceus injection regulates hematopoiesis in myelosuppressed mice, Phytother. Res. 21 (2007) 663e667. [29] C. Pigeon, G. Ilyin, B. Courselaud, P. Leroyer, B. Turlin, P. Brissot, O. Loreal, A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload, J. Biol. Chem. 276 (2001) 7811e7819. [30] G.L. Schieven, The p38alpha kinase plays a central role in inflammation, Curr. Top. Med. Chem. 9 (2009) 1038e1048. [31] X. Zhang, B.H. Rovin, Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines, Biochim. Biophys. Acta 1800 (2010) 1262e1267. [32] I. De Domenico, T.Y. Zhang, C.L. Koening, R.W. Branch, N. London, E. Lo, R.A. Daynes, J.P. Kushner, D. Li, D.M. Ward, J. Kaplan, Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice, J. Clin. Invest. 120 (2010) 2395e2405.