Molecular Mechanism Of Angelica Sinensis Polysaccharide Activating In Normal Iron Homeostasis

Hepcidin is a key negative regulator in body iron homeostasis. The abnormal hepcidin expression directly contributes to the pathogenesis of iron defecience or iron over-load, inducing deseases of disordered iron metabolism. Discovery and development of hepcidin modulator would be the new trend and ways to prevent and cure deseases of disordered iron metabolism. Angelica polysaccharide (ASP) is the active composition of Angelica sinensis (oliv.) Diels, and was demonstrated to participate in iron homeostasis. The purpose of this article is to define the effect of ASP inhibiting hepcidin expression involved in iron metabolism. Therefore, the activity of ASP suppressing hepcidin in normal rats as well as its mechanism were studied, and a stable transfected HepG2 cell line highly expressing human hepcidin was constructed which would be applied in the research on ASP affecting intestinal iron absorption in vitro.PartⅠStudy on Activity and Mechanism of ASP Suppressing Hepcidin in Normal RatsThe effects of Angelica sinensis polysaccharide (ASP) on the inhibition of hepcidin expression in normal rats, as well as the related mechanisms were studied. Total polysaccharide was extracted by boiling water and pureed after removing proteins by modulating pH. Carbohydrate content of ASP was always above 70% and stable in different samples.ASP solution was administrated to rats everyday for 14 d. Intraperitoneal injections of rhEPO were given to positive control group. Blood samples were taken before and after the administration. EPO and hepcidin levels in serum were determined by ELISA. The results showed that, ASP could stimulate the EPO secretion in body as that the EPO concentration reached the top merely 4 h after ASP administration; like EPO, ASP could significantly suppress hepcidin expression in normal rats, and the inhibition of hepcidin was positively correlated to the activation of EPO secretion. The effect of ASP on inhibiting hepcidin expression was demonstrated. It was speculated that the stimulation of erythropoiesis named EPO-like pathway was the main inhibition mechanism. This study lays the basis for further research on the related molecular mechanisms involved in hepcidin inhibition by ASP.To clarify the underlying mechanism in rhEPO and ASP suppressing hepcidin, we monitored the liver expressions of 6 proteins in 3 pathways which were previously demonstrated important for hepcidin regulation, including: STAT3/ phospho-STAT3 and STAT5/ phospho- STAT5 in JAK/STAT pathway, SMAD4 in HJV/BMP-SMAD pathway, and C/EBPα. Rats were grouped and administrated as above. Tissues were taken 8 h (rhEPO group) or 24 h (ASP and control group) after the last administration. Livers were perfused in order to remove the great amount of blood in liver. Signal proteins were determined by western blot. Molecular mechanisms involved in hepcidin suppression by ASP and EPO were primarily speculated as below: Fig.ⅠMolecular mechanism involved in hepcidin suppression by ASP and EPO. ASP down-regulats the transcription regulators STAT3/5 and SMAD4, and stimulats endogenous EPO secretion which further decreases the signal proteins in liver, as a result, hepcidin expression was suppressed. EPO inhibits the phosphorylation of STAT3/5 and the expression levels of C/EBPαand SMAD4, which causes the reduction of activating factors and hepcidin suppression.PartⅡModel for the Study on Regulation of Intestinal Iron Absorption—Establishment of Stable Transfected HepG2 Cell Line Expressing Human HepcidinTo construct eukaryotic expression vector containing full-length structural gene of human hepcidin and establish stable transfected HepG2 cell line with high expression of hepcidin. The synthesized DNA that encodes human hepcidin was inserted into vector pMD18-T after being identified by nucleotide sequencing. Digested by HindⅢand EcoRⅠ, the purified hepcidin gene fragment was subcloned into the eukaryotic expression vector pcDNA3.0. After analyzed by restrictive digestion reaction, the recombinant plasmid was transfected into HepG2 cells with liposome. Resistant cell clones were selected with G418, and expression of hepcidin in resistant clones were examined directly by ELISA. Recombinant plasmid pcDNA3-hepcidin was successfully constructed as indicated by restriction endonuclease analysis. Resistant HepG2 cell clones were obtained by selected with G418 for 3 weeks, the expression yield of hepcidin is 3 times as much as it is in common HepG2 cells. The stable transfected HepG2 cell line highly expressing human hepcidin was successfully constructed.