Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
ADAM17 Protein ; Alzheimer Disease/genetics* ; Amyloid beta-Peptides ; Drugs, Chinese Herbal/pharmacology* ; Hepcidins/genetics* ; Humans ; Pueraria

Hepcidin is a small cystein-rich cationic peptide produced mainly by the liver. It was initially isolated from human plasma and exhibited antimicrobial activity. Recently, several lines of evidence have suggested that hepcidin is a key regulator of iron metabolism at the whole body level and is relative to inflammation, infection, hypoxia and anemia. Hepcidin, is implicated in duodenal iron absorption and iron mobilization from reticuloendothelial macrophages. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. As both iron deficiency and iron excess are associated with cellular dysfunction, so hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease. To elucidate biological function of hepcidin further and use it for other research, it is necessary to produce enough hepcidin through DNA recombinant technique. As a highly successful system for the production of a variety of heterologous proteins, the methylotrophic Pichia pastoris system has the probability for a high level production of hepcidin. The subject of this paper is to summarize the regulation of hepcidin gene expression and the understanding of functions of hepcidin. At last, giving a prospect of production hepcidin by gene engineer.
Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; physiology ; Hepcidins ; Humans ; Iron ; metabolism ; Protein Engineering ; methods

OBJECTIVETo investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.

METHODSHCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.

RESULTSHCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.

CONCLUSIONSHCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

Antimicrobial Cationic Peptides ; genetics ; Cell Line ; Gene Expression Regulation, Viral ; Hepacivirus ; genetics ; Hepcidins ; Humans ; Plasmids ; Transfection ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo report the clinical data of a case of iron-refractory iron deficiency anemia (IRIDA), so as to improve the understanding of IRIDA.

METHODSThe IRIDA patient's hematological characteristics were summarized and analyzed. The hepcidin levels were tested by ELISA kit. The TMPRSS6 gene was amplified by PCR reaction and its mutation was analyzed by sequencing. The effect of TMPRSS6 gene mutation on TMPRSS6 protein tertiary structure was predicted by Swiss-Model.

RESULTSThe patient was characterized by typical microcytic hypochromic anemia, low transferrin saturation, more reduction of intracellular iron than exocellular iron. The plasma hepcidin level was 213.77 μg/L which was significantly higher than that of IDA patients [5.19(3.31-12.02) μg/L]. The patient also carried a homozygous missense mutation of K253E in exon 7 of TMPRSS6.

CONCLUSIONIn children and younger IDA patients with no reason for iron deficiency but unresponsiveness to routine iron treatment, the diagnosis of IRIDA needs to be considered. Serum hepcidin level and TMPRSS6 gene mutation should be detected.

Anemia, Iron-Deficiency ; blood ; genetics ; Female ; Hepcidins ; blood ; Humans ; Membrane Proteins ; genetics ; Mutation ; Protein Structure, Tertiary ; Serine Endopeptidases ; genetics ; Young Adult

Hepcidin are small cationic peptides with antibacterial activity expressed mainly in the liver of living organisms, and they play important roles in the host's immune response against microbial invasion and regulation of iron metabolism. Thus, they are considered to be good substitutes for traditional antibiotics. It is a good choice that the antimicrobial peptides are prepared by recombinant DNA expression. In the present study, two hepcidin mature peptide cDNAs from channel catfish (Ictalurus punctatus) (mCH) and tilapia (Oreochromis niloticus) (mTH) were connected by SOE-PCR in order to obtain more recombinant hepcidin with broad antimicrobial spectrum, and EcoR I and Not I sites were added to 5'- and 3'- ends of the fragment, respectively. The recombinant eukaryotic expression vector "pPIC9K-mCH-mTH" was successfully constructed, and transformed into Pichia pastoris GS115. The transformants containing multicopy gene insertion were selected by using different concentrations of G418 and other specific mediums, and identified by PCR for yeast genomic DNA. Expression was induced by adding 1% methanol at 30 degrees C for different times. Tricine-SDS-PAGE analysis demonstrated that the most appropriate expression time was 72 h, at which a high expression yield (77 mg/L) for the target protein was exhibited. The highly purified target protein was obtained from the fermentation supernatant by SP-Sepharose cation exchange chromatography. Bacteriostatic activity assay demonstrated that the fermentation supernatant containing the target protein and purified recombinant target protein had bacteriostatic activities against gram-positive and gram-negative bacterium. The present result provides the important initial value for industrial production of hepcidin antimicrobial peptide.
Animals ; Electrophoresis, Polyacrylamide Gel ; Fish Proteins ; biosynthesis ; genetics ; Fishes ; genetics ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hepcidins ; biosynthesis ; genetics ; Pichia ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

This study was aimed to investigate the effect of rhIL-6 and rhEPO on hepcidin mRNA expression in HepG2 cells and human primary hepatocytes, and mechanism of rhEPO in treatment of anemia of chronic disease (ACD). The HepG2 cells and human primary hepatocytes were cultured with medium containing different concentrations of rhIL-6 and rhEPO for a certain time, then mRNA was isolated and its RT-PCR was performed, the bands were photographed and analyzed by UVI band, the hepcidin and G3PDH mRNA ratio were semi-quantitatively analyzed. The expression levels of hepcidin in GepG2 cells and human primary hepatocytes at different conditions were compared. The results showed that the hepcidin mRNA expression in HepG2 cells and human primary hepatocytes could be enhanced by rhIL-6, the rhEPO could inhibit rhIL6-induced hepcidin mRAN expression. The rhEPO alone basically did not influence hepcidin mRNA expression in HepG2 cells. It is concluded that Hepcidin mRNA expression in HepG2 cells and human primary hepatocytes can be elevated by rhIL-6 with concentration- and time-dependent manner in certain range. rhEPO can inhibit this effect of rhIL-6.
Antimicrobial Cationic Peptides ; genetics ; metabolism ; Erythropoietin ; pharmacology ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Hepcidins ; Humans ; Interleukin-6 ; pharmacology ; RNA, Messenger ; genetics ; Recombinant Proteins ; pharmacology

OBJECTIVETo report novel mutations SEC23B gene in congenital dyserythropoietic anemia (CDA).

METHODSBy direct sequencing method, we sequenced CDAN1 and SEC23B genes in a Chinese CDA II patient, presented with chronic fatigue and dark urine, as well as his family members. Serum hepcidin was assayed by mass spectrometry.

RESULTSWe found a c.71G>A mutation and a c.74C> A mutation in the patient. In addition, a heterozygous c.55A>G mutation of HFE2 gene was found in some family members. The level of serum hepcidin of the patient was below the detection limit (<1 nmol/L).

CONCLUSIONContrary with what have been reported previously in the Europe, especially in the Italy, the gene mutations identified in this case was different and novel. The two novel mutations contribute to the diagnosis of CDAII and are the first report in East Asian CDAII patients.

Adolescent ; Adult ; Anemia, Dyserythropoietic, Congenital ; genetics ; Asian Continental Ancestry Group ; genetics ; GPI-Linked Proteins ; genetics ; Glycoproteins ; genetics ; Hepcidins ; blood ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Vesicular Transport Proteins ; genetics

OBJECTIVETo investigate the involvement of NF-kappaB (NF-kB) regulation of hepcidin gene transcription in acute phase response and its molecular mechanism.

METHODSFirst, a mouse model of acute phase response was established by intraperitoneal injection of LPS. The relationship between hepcidin expression and dose or time of LPS injection was assessed. Then, electrophoretic mobility shift assay (EMSA) was performed to explore the possibility of the involvement of NF-kB in regulation of hepcidin gene transcription. Next, pAVU6+27-NF-kB, NF-kB p65-specific siRNA expression vector was constructed and transfected into mouse primary hepatocytes using DOTAP liposomal transfection reagents. Hepcidin expression changes after silencing of NF-kB p65 and hepcidin expression after LPS induction were tested.

RESULTSHepcidin expression showed a time and dose-dependent manner with regard to LPS injection. At 10 h after 50 microg LPS injection, hepcidin expression reached its peak. The result of EMSA exhibited an evident lag band at -53 - -64 bp, indicating regulation of hepcidin gene expression by NF-kB. After mouse primary hepatocytes were transiently transfected with NF-kB p65-specific siRNAs, Western blot showed that inhibition rate of NF-kB expression was 50%-67%. Hepcidin expression of transfected hepatocytes dropped down obviously in comparison with that of untransfected hepatocytes, and could not be induced by LPS.

CONCLUSIONTranscription factor NF-kB is likely to be an important molecule in transcription regulation of hepcidin gene. As a key component, p65 subunit binds to hepcidin gene at -53 - -64 bp, and upregulates hepcidin expression.

Acute-Phase Reaction ; chemically induced ; genetics ; Animals ; Anti-Bacterial Agents ; Antimicrobial Cationic Peptides ; genetics ; Gene Expression Regulation ; Hepcidins ; Lipopolysaccharides ; Mice ; NF-kappa B ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transcription, Genetic

The aim of this study was to investigate the changes of iron levels and hepatic regulatory molecules expression involved in iron metabolism in non-diabetic obese/type 2 diabetic rat models. Male Wistar rats were divided into 3 groups: control group, non-diabetic obese group and type 2 diabetic group (n=20 each). The rats were evaluated physiologically and biochemically. The hepatic histopathological changes were observed using haematoxylin and eosin (HE) staining. The mRNA expression patterns of hepcidin, interleukin-6 (IL-6), hypoxia-inducible factor (HIF) and ferroportin (Fpn) in the rat liver in control group, non-diabetic obese group and type 2 diabetic group were analyzed by real-time RT-PCR. The protein expression patterns of hepcidin in liver of each group were further analyzed by immunohistochemistry and Western blotting. As compared with control group, the ferritin in non-diabetic obese group and type 2 diabetic group was increased significantly (P<0.001). However, there was no significant difference in soluble transferring receptor (sTfR):ferritin ratio among the three groups (P>0.05). The real-time RT-PCR, immunohistochemistry and Western blotting results all revealed that the expression levels of hepcidin in non-diabetic obese group and type 2 diabetic group were elevated significantly as compared with those in control group (P<0.001). The expression levels of hepcidin mRNA between non-diabetic obese group and type 2 diabetic group showed no significant difference (P>0.05). However, the protein expression levels of hepcidin in type 2 diabetic group were significantly higher than those in non-diabetic obese group (P<0.05). Compared to control group, the expression levels of IL-6 mRNA in non-diabetic obese group and type 2 diabetic group were increased significantly and the expression levels of Fpn mRNA decreased (P<0.05). However, the expression levels of HIF mRNA had no significant difference among three groups. It is suggested that iron metabolism is substantially disturbed in non-diabetic obese and type 2 diabetic rats probably by the abnormal expression of hepcidin in chronic inflammatory status. The increased hepcidin may restrain the iron release from the cells by affecting the expression of Fpn, which probably associates with the development of diabetic complication.
Animals ; Diabetes Mellitus, Type 2 ; complications ; metabolism ; Hepcidins ; genetics ; metabolism ; Interleukin-6 ; genetics ; Iron ; metabolism ; Male ; Obesity ; complications ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Real-Time Polymerase Chain Reaction

This study was purposed to investigate the effect of multiple myeloma patients' sera on hepcidin mRNA expression of Hep-3b hepatoma cell line and effect of human interleukin-6 (IL-6) antibody or recombinant human erythropoietin (rhEPO) on hepcidin mRNA expression. The clinical information and serum of multiple myeloma patients were collected. Their sera of a final concentration of 10% were added into Hep-3b cell medium. The mRNA from Hep-3b cells was extracted, and hepcidin mRNA expression was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). A final concentration of 10 ng/ml human IL-6 antibody and 2 U/ml rhEPO were added into the medium respectively. The results showed that the sera of untreated multiple myeloma patients elevated hepcidin mRNA expression of Hep-3b cells, compared with healthy controls and iron deficiency anemia patients. This effect was fully neutralized by human IL-6 antibody or rhEPO. The hemoglobin (Hb) level was stable during the follow up of regularly treated multiple myeloma patients and the effect of MM patient serum on Hep-3b cell hepcidin mRNA expression was reduced. It is concluded that the hepcidin mRNA expression of Hep-3b cell can be increased by untreated multiple myeloma patient serum. This promotive effect can be antagonised by IL-6, which suggests that IL-6 may be possible to elevate expression level of hepcidin in Hep-3b cells and results in anemia of chronic disease (ACD). The above mentioned promotive effects also can be suppressed by rhEPO, which indicates that the rhEPO may possess curative effect for ACD disease. During short-term follow-up of treated patients with multiple myeloma the Hb level is stable, the influence of patients serum on hepcidin mRNA of Hep-3b cells decreases, which shows the stabilization of disease and amelioration of ACD patient status.
Adult ; Aged ; Antibodies, Monoclonal ; pharmacology ; Antimicrobial Cationic Peptides ; genetics ; Cell Line, Tumor ; Erythropoietin ; blood ; pharmacology ; Female ; Hepcidins ; Humans ; Interleukin-6 ; immunology ; Male ; Middle Aged ; Multiple Myeloma ; genetics ; metabolism ; RNA, Messenger ; genetics