Hepcidin: Expression And Regulation Of Iron-Related Proteins In Mice Brain

Iron is an essential element that is required for growth and survival. Nowadays, people have realized that many human diseases are associated with iron metabolism disorders. With the increase of old population, neurodegenerative diseases are paid attention gradually among these disorders related with iron. Recently, the brain iron metabolism research becomes very hot.Hepcidin was found to be a small cysteine-rich peptide with intrinsic antimicrobial activity. The first link between hepcidin and iron metabolism come from the study of Pigeon et al. The direct proof that hepcidin can modulate iron balance of the body was resulted from the observation of the mice whose hepcidin gene were knocked out and overexpressed. Therefore, hepcidin appears a key soluble signal among gut, liver, bone marrow and reticuloendothelial system, which was the long-postulated iron regulator. Obviously, plasma hepcidin inhibits iron uptake in the duodenum and iron release from macrophages through modulating the expression of DMTl and FP1. However, there is not a report on the expression of hepcidin in the brain up to now. Likewise, whether it can regulate brain iron metabolism has not to be answered. Therefor, in the dissertation, the expression of hepcidin in mice brain was identified and its role in the brain iron metabolism was investigated in vitro and in vivo. The results showed:1. Hepcidin mRNA was expressed in the mice brain.Total RNA was extracted from choroid plexus, cerebral cortex, diencephalon and mesencephalon, cerebellum, medulla oblongata and pons in mice. RT-PCR analysis demonstrated that hepcidin mRNA was expressed in all areas of mouse brain, in choroid plexus the mRNA expression was highest, although its level is generally lower than that of liver. The PCR products were purified and verified the sequence. Sequence analysis of the 137 bp fragment revealed the presence of 100% identity, compared with mouse muscles hepcidin antimicrobial peptide mRNA (cDNA clone MGC: 35592 IMAGE: 5097807). The result exhibited that the amplified products is hepcidin cDNA.2. Effect of hepcidin microinjection into the lateral cerebral ventricle on DMTl, FP1protein synthesis in different brain regions.After hepcidin was injected into the lateral cerebral ventricle for 12 h, the expression of DMT1 (+IRE) increased significantly (P<0.01); the expression of DMT 1 (-IRE) and FP1 decreased significantly (P<0.01) in cerebral cortex and in corpus striatum. But in substantia nigra, the DMT1 (+IRE) protein synthesis was significantly decreased (P<0.01); the DMT1 (-IRE) and FP1 protein synthesis was significantly increased (P<0.01) respectively. In choriod plexus of the lateral cerebral ventricle and the third ventricle, hepcidin induced the expression of DMT1 both (+IRE) and (-IRE) significantly higher (PO.01) than that of the control, but there was no significant change between two groups in the expression of FP1.3. In vitro, the cerebral cortex and hippocampus neurons were cultured successfully.Isolated neurons from the cerebral cortex and hippocampus of newborn SD rats were cultured for 7 days. The cell concentrition was approximate 2×l0⁶ each chamber. After identified by neuron-specific enolase, the purity of the cultural neurons was more than 95%. Then cells were used to experiment.4. Effect of hepcidin on iron uptake of cultured neurons.The iron absorption of neurons was elevated with the increase of the iron concentrition from 0 to 2 μM. When the iron concentrition was higher than 2μM, the level of cells uptake iron was unchangeable. Thus, we selected 2 μM to detect the ability of neurons iron uptake. Hepcidin (25μg/ml) was added in the media for 24 h, the cells were used to take up ⁵⁵Fe. The amount of ⁵⁵Fe in the hepcidin treated group cells was great higher than that of the control (P<0.05). Conclusions1. In the present study, hepcidin was identified in the mice brain by RT-PCR. The data showed that all 5 regions which were examined have the ability to express hepcidin mRNA, although the level is generally lower than that of liver. The futher investigation should be studied using in situ hybridization.2. After hepcidin injected into the lateral cerebral ventricle 12 h, the expression of DMT1, FP1 was significantly changed in cerebral cortex, corpus striatum, substantia nigra and choriod plexus. This result revealed hepcidin could modulate iron-transport molecules and regulate iron balance in the mouse. It is obvious that hepcidin appears specifically regional regulation. Because the mechanism of brain iron homeostasis is much more elaborate, the regulation of brain iron metabolism by hepcidin might be more complicated than other...