Effect of active component compound of Epimedii Folium,Astragali Radix,and Puerariae Lobatae Radix on expression of ADAM17 in HT22 cells by mediating hepcidin / 中国中药杂志

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.

Differences in Hepatic Expression of Iron, Inflammation and Stress-Related Genes in Patients with Nonalcoholic Steatohepatitis

Abstract: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. We have previously shown that hepatic reticuloendothelial system (RES) iron deposition is associated with an advanced degree of nonalcoholic steatohepatitis (NASH) in humans. In this study, we aimed to determine differentially expressed genes related to iron overload, inflammation and oxidative stress pathways, with the goal of identifying factors associated with NASH progression. Seventy five patients with NAFLD were evaluated for their biochemical parameters and their liver tissue analyzed for NASH histological characteristics. Gene expression analysis of pathways related to iron homeostasis, inflammation and oxidative stress was performed using real-time PCR. Gene expression was compared between subjects based on disease status and presence of hepatic iron staining. We observed increased gene expression of hepcidin (HAMP) (2.3 fold, p = 0.027), transmembrane serine proteinase 6 (TMPRSS6) (8.4 fold, p = 0.003), signal transducer and activator of transcription 3 (STAT3) (5.5 fold, p = 0.004), proinflammatory cytokines; IL-1β (2.7 fold, p = 0.046) and TNF-α (3.8 fold, p = 0.001) in patients with NASH. TMPRSS6, a negative regulator of HAMP, is overexpressed in patients with NASH and HIF1α (hypoxia inducible factor-1) is downregulated. NAFLD patients with hepatic iron deposition exhibited higher hepcidin expression (3.1 fold, p = 0.04) but lower expression of cytokines. In conclusion, we observed elevated hepatic HAMP expression in patients with NASH and in NAFLD patients who had hepatic iron deposition, while proinflammatory cytokines displayed elevated expression only in patients with NASH, suggesting a regulatory role for hepcidin in NAFL to NASH transition and in mitigating inflammatory responses.

Molecular cloning and antibacterial activity of hepcidin from Chinese rare minnow (Gobiocypris rarus)

Background Hepcidins, a kind of cysteine-rich antimicrobial peptides, play important roles in host immunological processes and iron regulation, which have been identified from several fish species. The rare minnow (Gobiocypris rarus), an endemic cyprinid fish in China, has been used extensively as model animal in laboratory. However, little is known about its hepcidin. Here, we report the cloning and characterization of a hepcidin gene from the liver of Chinese rare minnow. Results The full-length cDNA of rare minnow hepcidin is 662 bp, which contains an ORF of 273 bp encoding a prepropeptide of 90 amino acid residues. The predicted prepropeptide contains three domains: a signal peptide of 24 amino acids, a prodomain of 41 amino acids, and a mature peptide of 25 amino acids. Sequence alignment showed eight conserved cysteine residues in the mature peptide, which formed four disulfide bonds in spatial structure. The deduced structure of mature peptide showed a high degree of homology to the human hepcidin. Phylogenetic analysis showed that it had a close relationship with zebrafish hepcidin, and clustered in a clade with these from Cyprinidae. Synthetic peptide of rare minnow hepcidin could inhibit the growth of Gram positive bacterium Staphylococcus aureus and Gram negative bacteria Escherichia coli and Aeromonas hydrophila. Conclusion These results suggested that rare minnow hepcidin had typical structure of hepcidins and antibacterial activity. It could participate in innate immune response as an antibacterial agent and be used as antibiotic substance.

Sox2 function as a negative regulator to control HAMP expression

BACKGROUND: Hepcidin, encoding by HAMP gene, is the pivotal regulator of iron metabolism, controlling the systemic absorption and transportation of irons from intracellular stores. Abnormal levels of HAMP expression alter plasma iron parameters and lead to iron metabolism disorders. Therefore,itis animportant goal to understand the mechanisms controlling HAMP gene expression. RESULTS: Overexpression of Sox2 decrease basal expression of HAMP or induced by IL-6 or BMP-2, whereas, knockdown of Sox2 can increase HAMP expression, furthermore, two potential Sox2-binding sites were identified within the human HAMP promoter. Indeed, luciferase experiments demonstrated that deletion of any Sox2-binding site impaired the negative regulation of Sox2 on HAMP promoter transcriptional activity in basal conditions. ChIP experiments showed that Sox2 could directly bind to these sites. Finally, we verified the role of Sox2 to negatively regulate HAMP expression in human primary hepatocytes. CONCLUSION: We found that Sox2 as a novel factor to bind with HAMP promoter to negatively regulate HAMP expression, which may be further implicated as a therapeutic option for the amelioration of HAMP-overexpression-related diseases, including iron deficiency anemia.