Effects And Mechanisms Of Erythropoietin On Hepcidin And Tumor Growth In Mice Myeloma Models

【BACKGROUND】Anemia of chronic disease(ACD) is an anemic syndrome concomitant with acute and chronic inflammatory disorders,infections and malignancies.Iron metabolic dysfunction is considered to be the most important pathogenesis.Hepcidin,a newly found 25-amino acid peptide,is thought to be the central regulator in iron metabolism.It binds to the ferroportin(FPN),induces the internalization and degradation of the hepcidin-FPN1 complex to decrease iron export.On the other hand,it inhibits the expression of intestinal iron absorption through inhibiting the related proteins,such as divalent metal transporter 1(DMT1),and leads directly to iron metabolic dysfunction in ACD.There has been no study about hepcidin in tumoral ACD model yet.EPO is a useful drug to treat ACD.It has been proved that EPO can inhibit the expression of hepcidin in inflammatory conditions both in vitro and in vivo,so as to correct ACD,but little is known about the concrete mechanism.Besides,EPO also probably stimulates the proliferation of tumor when it corrects anemia,but there is no definite conclusion when it comes to multiple myeloma(MM).【OBJECTIVE】1.To find out whether ACD will form in the longstanding tumor-loading mouse MM model.2.To find out whether the hepatic hepcidin level will rise in the MM mouse model,and whether the effect targets,DMT1 and FPN,will change correspondingly.3.To understand whether EPO will inhibit hepcidin expression in the mouse model of MM and what is the possible mechanism.4.To understand whether EPO can stimulate the growth of MM.【METHODS】1.Myeloma cell is injected subcutaneously in mouse so as to establish the longstanding tumor-loading model.Indexes of anemia and iron metabolism in mouse are detected.2.The method of real time-PCR is used to detect the mRNA expressions of hepatic hepcidin,DMT1 and FPN in liver and duodenum.3.The mechanism of EPO is explored through the comparison of hepatic hepcidin, duodenal DMT1 and FPN mRNA expressions,and the expressions of hepcidin regulators such as IL-6,BMP4,TMPRSS6 and GDF-15 between normal tumor-loading mice and those treated with short period of EPO,long period of EPO,and anti IL-6 antibody.4.The expressions of EPOR in MM cell strain and MM patient's primary tumor cells are measured through flow cytometer.5.CCK-8 is used to detect the proliferative influence of EPO on the myltiple myeloma cell strain cultured in vitro.6.The tumor-loading model of MM is used to observe the effect of EPO on tumor proliferation.【RESULTS】1.The mice begin to be anemic and have decreased serum iorn and transferrin saturation 6 weeks after tumor implantation.Iron stain of bone marrow smears shows iron deficiency in red blood cells and iron overload in macrophages.2.The hepatic hepcidin mRNAs of MM tumor-loading mice are higher than those of normal even-aged mice,the intestinal DMT1,FPN and hepatic FPN mRNA are obviously decreased,and the hepatic DMT1 mRNA is unchanged.3.All of short period of EPO,long period of EPO and anti IL-6 antibody can inhibit hepatic hepcidin mRNA level obviously,but long period of EPO and anti IL-6 antibody can increase intestinal DMT1 and FPN mRNA levels,and only long period of EPO can increase hepatic FPN mRNA.4.Hepatic IL-6 mRNA level decreases after EPO treatment.Short period of EPO decreases hepatic BMP4 mRNA level,but long period of EPO does not have such effect.5.EPOR is expressed in both MM cell strain and MM patient's primary cells,but EPO can not stimulate the proliferation of MM cells in vitro and in vivo.【CONCLUSIONS】1.Longstanding tumor-loading model of MM is a tumor-ACD model.2.Hepatic hepcidin mRNA increases in tumor-loading mouse of MM.3.EPO inhibits obviously the expression of hepatic hepcidin mRNA4.Short period of EPO inhibits hepcidin through decreasing IL-6,serum iron and BMP4.5.MM cells express EPOR,but EPO does not stimulate the proliferation of MM.