The Role And Mechanism Of Hepatic Hepicdin In Sepsis

Part1The establishment of hepatic hepcidin knockdown mouse modelObjective:Hepcidin, mainly produced by hepatocytes, is a beta-defensin like cationic antimicrobial peptide. In addition to owning a weak bactericidal effect, hepatic hepcidin is also acting as an important iron regulation hormone. Inflammation or infection could up-regulate the expression of hepcidin in liver. Clinical studies show that, in patients with sepsis, the levels of hepcidin in serum and urine are significantly increased compared with the control groups. Thus, we hypothesize that hepatic hepcidin may play an important role in the regulation of infection and inflammation in sepsis. Therefore, this part of the study is designed that using the treatment with Adenoviral Vector-mediated Hepcidin-specific Short Hairpin RNA (Ad-shHepcl) by hydrodynamic injection via the tail vein, constructs the model of hepatic hepcidin knockdown(KD) mouse, for further analysis of the relationship between hepatic hepcidin and the development of sepsis. Methods:Mouse hepatic hepcidin expression was silenced using adenovirus-mediated hepcidin-specific short hairpin RNA (Ad-shHepc1) injected by hydrodynamic injection via the tail vein. The control group was injected the control adeno virus-mediated non-specific shRNA (Ad-shNeg) using the same method.13days after injection, samples such as peripheral blood, liver, spleen, lung and kidney were collected. The mRNA expression of hepcidin in tissues (liver, spleen, lung, kidney and peripheral leukocytes) was detected by real-time polymerase chain reaction (RT-PCR). And the protein levels of hepcidin in tissues (liver, spleen, lung and kidney) were also measured by method of immunohistochemistry (HIC). The iron content of spleen was assayed using the method of Prussian blue staining. The concentration of iron in serum was determined by atomic absorption spectroscopy. Nonheme iron concentrations in the liver and spleen tissues were determined using acid digestion method as previously described.Results:To compared with the control mice treated with Ad-shNeg, on the13th day after administration of adeno virus, hepcidin levels in the livers of the Ad-shHepc1challenged mice were significantly lower. The mRNA level and protein level of hepcidin in other tissues (spleen, lung, kidney and peripheral leukocytes) were comparable in the two groups. In spleen tissue, the iron content was significantly lower in the hepcidin knockdown mice than that in the control mice. Serum iron levels in the hepcidin knockdown mice were also significantly higher. In liver tissues, the iron content had a trend to be higher in the hepcidin knockdown mice, although there was no significant difference in the two groups.Conclusions:Administration of adenovirus-mediated hepcidin-specific short hairpin RNA (Ad-shHepc1) by hydrodynamic injection via the tail vein, can effectively inhibit the expression of hepcidin in liver, decrease iron content of spleen, increase serum iron concentration, and successfully establish the model of hepatic hepcidin knockdown mouse. Part2The impact of hepatic hepcidin on sepsisObjective:Hepcidin, mainly synthesised and secreted by liver, is a beta-defensin like cationic antimicrobial peptide, with extensive biological activity. Numerous studies confirm that inflammation can induce the up-regulation expression of hepatic hepcidin. Previous research has shown that the levels of hepcidin in critically ill patients are closely related to the plasma IL-6concentrations. Clinical study found that in patients with sepsis, hepcidin levels in serum and urine were significantly increased. In view of the establishment of hepatic hepcidin knockdown mouse model, this part of the study was designed to investigate the impact of hepatic hepcidin on the development of sepsis.Methods:Sepsis model was induced by cecal ligation and puncture (CLP) surgery in Ad-shHepcl or Ad-shNeg challenged mice. The mice were observed for7days to assess survival rate.24hours after the CLP surgery, samples such as peripheral blood, liver, spleen, lung and kidney were collected in both groups. Hepcidin expression in liver tissue of these septic mice was detected using real-time polymerase chain reaction and immunohistochemistry. Histological examination of liver and lung were conducted by the staining with hematoxylin and eosin (HE). Liver function was assessed by the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) tests. Apoptotic cell death in spleen was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Serum tumor necrosis factor alpha (TNF-a) and IL-6concentrations were measured using enzyme-linked immunosorbent assay. Blood and tissue bacterial infection in mice was detected using agar plate. Results:After CLP challenge, significantly lower hepcidin levels were also observed in the liver of the Ad-shHepcl-treated mice, but not in other tested tissues. Hepatic hepcidin knockdown mice showed significantly increased7-day mortality.24h after polymicrobial sepsis, hepatic hepcidin knockdown mice had exacerbated organ damage compared with Ad-shNeg challenged mice. The more severe oxidative stress was also observed in hepatic hepcidin knockdown mice than the control group. In addition, hepatic hepcidin knockdown mice showed compromised host inflammatory responses and bacterial clearance.Conclusions:Hepatic hepcidin knockdown mice after challenged with sepsis showed significantly increased mortality, exacerbated organ damage, oxidative stress and bacterial infection, as well as compromised host inflammatory responses. Hepatic hepcidin may play an important role in immune defense against infection and prognosis during sepsis. Part3The mechanism of hepatic hepcidin in sepsisObjective:Hepatic hepcidin is acting as a key hormone, which is involved the regulation of iron homeostasis. The up-regulated expression of hepatic hepcidin induced by stimulus such as infection or inflammation, may suggest that hepatic hepcidin plays an important role during these conditions. In view of the fact that hepatic hepcidin knockdown mice with sepsis show significantly decreased survival rate, therefore, this part of the study mainly focus on the mechanism of hepatic hepcidin in sepsis.Methods:Sepsis model was induced by cecal ligation and puncture (CLP) surgery in Ad-shHepcl or Ad-shNeg challenged mice. The changes of iron content between the two groups were observed in mice24hours after polymicrobial sepsis. The serum iron concentration was determined by atomic absorption spectroscopy. The iron content of spleen was assayed using the method of Prussian blue staining and determined by acid digestion method. In vitro, mouse macrophage RAW264.7cell lines were incubated with iron chelator deferoxamine (DFO) in various concentrations. The phagocytosis of macrophage were observed under a fluorescence microscope and detected by flow cytometer after incubated with fluorescent polystyrene microspheres. And the macrophage inflammatory response were also detected by quantitative real-time PCR after challenged with lipopolysaccharides (LPS). In some experiments, Ad-shHepcl administrated mice received the treatment with or without a low-iron diet in combination with deferoxamine (DFO). The changes of iron content and survival of mice subjected to CLP was monitored for7days. Results:After CLP challenge, the iron content in spleen tissue was significantly lower in the hepcidin knockdown mice than that in the control mice. In RAW264.7cells after treatment with various concentrations of desferoxamine, the phagocytosis were significantly compromised than those in the untreated macrophages. Furthermore, under lipopolysaccharides challenge, the mRNA levels of the inflammatory cytokines IL-6and TNF-a in desferoxamine-treated macrophages was significantly decreased. After treatment with a low-iron diet plus deferoxamine, serum iron concentration in the hepcidin knockdown mice were significantly decreased. When these mice were challenged with CLP surgery,7-day survival of these mice was significantly improved.Conclusions:Hepatic hepcidin knockdown mice with sepsis show disorders of iron metabolism, manifested as the significantly increased serum iron levels as well as the significantly decreased iron content in spleen. The lower intracellular iron content in macrophages significantly inhibits the function of macrophages in phagocytosis and inflammatory response ability. The treatment with a low-iron diet plus deferoxamine in hepatic hepcidin knockdown mice significantly improve iron over-load status, and obviously decrease the mortality of sepsis, the mechanism of which was closely related to the function of hepcidin in regulation of iron metabolism.