Yan-Zhong Chang

Role of hepcidin in murine brain iron metabolism

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism.

Hepcidin Is Involved in Iron Regulation in the Ischemic Brain

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain.

Fasudil hydrochloride hydrate, a Rho‐kinase inhibitor, suppresses isoproterenol‐induced heart failure in rats via JNK and ERK1/2 pathways

The Rho‐kinase (ROCK) plays an important role in the pathogenesis of heart injury. Recent cellular and molecular biology studies indicated a pivotal role of the RhoA/ROCK cascade in many aspects of cardiovascular function such as heart failure, cardiac hypertrophy, and ventricular remodeling following myocardial infarction. However, the signal transduction of RhoA/ROCK and its down‐stream signaling pathways remains elusive, and the mechanism of ROCK‐mediated isoproterenol (ISO)‐induced heart failure is still not thoroughly understood. In the present study, we investigated the effect of the ROCK inhibitor, fasudil hydrochloride hydrate, on ISO‐induced heart failure and the potential relationship of RhoA/ROCK to the extracellular signal‐regulated kinases (ERK) and the c‐jun NH 2‐terminal kinase (JNK) pathways. Male Sprague‐Dawley (SD) rats, maintained on a normal diet, were randomly divided into four groups given control, ISO alone, ISO with low‐dose fasudil, or ISO with high‐dose fasudil treatments. Fasudil effectively inhibited ISO‐induced heart failure, as evaluated by biometric, hemodynamic, and histological examinations. Consistently, ISO‐induced ROCK‐1 mRNA expression and myosin phosphatase target subunit‐1 (MYPT‐1) phosphorylation were markedly suppressed by fasudil. In addition, fasudil significantly decreased ISO‐induced JNK activation, ERK translocation to the nucleus and subsequent c‐fos, c‐jun expression and upregulated c‐FLIPL expression. Taken together, these results indicate that the RhoA/ROCK pathway is essential for ISO induced heart failure, which can be effectively suppressed by fasudil. J. Cell. Biochem. 112: 1920–1929, 2011.

Effect of erythropoietin on hepcidin, DMT1 with IRE, and hephaestin gene expression in duodenum of rats

BackgroundErythropoietin (Epo) is the central regulator of red blood cell production and can stimulate proliferation and differentiation of erythroid progenitor cells. Now, recombinant human erythropoietin (rHuEpo) is widely used in patients with renal disease, chronic anemia, and iron deficiency of early childhood. It has been reported that the enhanced erythropoiesis associated with erythropoietin therapy increases intestinal iron absorption, but the molecular mechanisms underlying are unknown. Therefore, we have investigated the effect of rHuEpo on duodenal iron transport protein synthesis in rats.MethodsMale Sprague-Dawley rats weighing 250 g were randomly divided into two groups: (1) rHuEpo injection group (rHuEpo, 500 IU/day, s.c.), and (2) control group (injection of the same volume of saline). After 3 days injection, blood parameters, serum iron status, and non-heme iron concentrations in the liver and duodenum were examined at the fifth day. The mRNA levels and protein synthesis of duodenal divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin (Hp) were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. Hepatic hepcidin mRNA expression was analyzed by RT-PCR.ResultsrHuEpo injection significantly stimulated erythropoiesis and decreased serum iron status, non-heme iron concentrations in the liver and duodenum. DMT1 (+IRE) and Hp expression in duodenum were increased significantly. However, DMT1 (−IRE) and FPN1 expression had no apparent change. Hepatic hepcidin mRNA expression was decreased dramatically, reaching an almost undetectable level in rHuEpo-treated rats.ConclusionsrHuEpo administration improved the duodenal iron absorption by increasing the expression of DMT1 (+IRE) and Hp.

Effect of iron liposomes on anemia of inflammation

Supplementation with iron-fortified foods is an effective method for treating iron deficiency diseases. However, traditional iron agents used to treat anemia of inflammation (AI) have little effect. In this study, two types of iron liposomes, heme liposomes (HEME-LIP) and ferric citrate liposomes (FAC-LIP), were prepared by the rotary-evaporated film-ultrasonication method, and the encapsulation efficiencies, microstructures, size distributions and zeta potentials were assessed. Both types of iron liposomes showed stable physical characteristics. When used to treat rat models of AI, FAC-LIP and HEME-LIP could increase serum iron levels by 119% and 54% higher than did ferric citrate (FAC) and heme, respectively. Furthermore, the hepcidin, a key regulator of iron homeostasis was up-regulated by these iron liposomes, especially by HEME-LIP. These results indicate that the absorption of iron liposomes was improved over that of unencapsulated iron agents. Thus, iron liposomes may be used to fortify food in treating iron deficiency diseases, especially AI.

Decreased DMT1 and Increased Ferroportin 1 Expression Is the Mechanisms of Reduced Iron Retention in Macrophages by Erythropoietin in Rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague–Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then 55Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT‐PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo‐treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo‐treated rats. J. Cell. Biochem. 104: 629–641, 2008.

Specific Hemosiderin Deposition in Spleen Induced by a Low Dose of Cisplatin: Altered Iron Metabolism and Its Implication as an Acute Hemosiderin Formation Model

Cisplatin is one of the commonly-used chemotherapeutic drugs to efficiently treat malignant tumors in clinic, however, the adverse effects of cisplatin such as nephrotoxicity, neurotoxicity, and hemolytic uremic syndrome are often observed at its clinical doses (approximately 60 mg/m(2)), which limit its broader application. In earlier studies, little attention was paid to the subtle changes in the architecture of lymphatic organs after low doses of cisplatin treatment. This paper reviews current understanding of cisplatin-induced erythrocyte injury, and presents our latest finding that a low dose of cisplatin (3.6 mg/m(2)/day, 14 days) could induce specific hemosiderin deposition in spleen of both normal and hepatoma-22 (H22) inoculated Balb/C mice. This dose of cisplatin significantly inhibited H22-induced acute ascites development. No significant toxicity was induced by this dose of cisplatin to tissues except for hemosiderin accumulation in the spleen of both normal and H22 tumor-bearing mice. Increased splenic iron content and erythrocyte injury were observed after treatment with the low dose of cisplatin. The mRNA levels of ferroportin (FPN1) and ferritin were upregulated by 25 and 5-fold in spleen, respectively. Overexpression of FPN1 and ferritin protein were also been observed at protein levels by Western blotting analysis. In addition, the mRNA expression of hepcidin was also increased, suggesting blockage of iron recycling through FPN1 in spleen with cisplatin treatment. In conclusion, cisplatin treatment damages the erythrocytes which accumulate in the red pulp of spleen with defective recycling of FPN1 and ferritin protein. Hepcidin inhibits the function of FPN1 as iron-exporter leading to iron overloaded inside ferritins of splenic cells, which are stained with abnormal hemosiderin accumulation. These results demonstrate that cisplatin-caused hemosiderin deposition in spleen provides a valuable clue for understanding the molecular basis of toxicity of cisplatin and hemosiderin accumulation and iron metabolism in vivo.

Hepcidin, an antimicrobial peptide is downregulated in ceruloplasmin-deficient mice

Hepcidin, a principle regulator of iron metabolism, is synthesized by the liver. Contradictory results have been reported on the regulation of hepcidin expression in response to serum transferrin saturation and liver iron content. In the present study, we explore the expression of murine hepcidin mRNA and further analyze the relationship between liver hepcidin mRNA expression, liver iron stores, and serum iron level utilizing ceruloplasmin gene knockout mice. We find that hepcidin expression correlates significantly with serum transferrin saturation, whereas there is a negative correlation of hepcidin expression with liver tissue iron level.

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron

Abstract Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinsons disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP+-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.

Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis

Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). In addition, inflammation is tightly linked to iron metabolism dysregulation. However, it is not clear whether the brain inflammation-induced iron metabolism dysregulation contributes to the NDs pathogenesis. Herein, we demonstrate that the expression of the systemic iron regulatory hormone, hepcidin, is induced by lipopolysaccharide (LPS) through the IL-6/STAT3 pathway in the cortex and hippocampus. In this paradigm, activated glial cells are the source of IL-6, which was essential in the iron overload-activated apoptosis of neurons. Disrupting astrocyte hepcidin expression prevented the apoptosis of neurons, which were able to maintain levels of FPN1 adequate to avoid iron accumulation. Together, our data are consistent with a model whereby inflammation initiates an intercellular signaling cascade in which activated microglia, through IL-6 signaling, stimulate astrocytes to release hepcidin which, in turn, signals to neurons, via hepcidin, to prevent their iron release. Such a pathway is relevant to NDs in that it links inflammation, microglia and astrocytes to neuronal damage.