| Iron is required to maintain multiple functions of the body,such as DNA synthesis and repair,oxygen delivery,and mitochondrial energy metabolism.Iron metabolism is regulated by hepcidin,which acts as a small molecule peptide hormone and plays a negative regulatory role in the regulation of iron metabolism.Hepcidin binds to FPN1 on the surface of hepatocytes,promotes internalization and degradation of FPN1 and regulates iron homeostasis.Under normal physiological conditions,the expression of hepcidin in the body was negatively correlated with serum iron levels.When serum iron is overloaded,the liver synthesizes and secretes increased hepcidin,which accelerates the degradation of FPN1,closes the export of iron to the blood,thereby reducing the transport of iron from intestinal epithelial cells and macrophages to the blood;Insufficient,the amount of hepcidin expressed in the liver is reduced,the opening of the FPN1 channel is increased,the iron stored in the liver is transported to the blood,and the small intestine is increased in iron absorption,thereby maintaining iron homeostasis.Chronic anemia is an important disease caused by the imbalance of hepcidin.The main cause of this disease is iron metabolism disorder caused by inflammatory stimulation or chronic infection and tumor.As an acute stress protein,hepcidin is involved in the regulation of iron balance in the body.Current research indicates that elevated hepcidin plays an important role in the pathogenesis of ACD.When the body is stimulated by inflammation or chronic infection such as sepsis,burns,inflammatory bowel disease,the inflammatory cytokine IL-6 stimulates the expression of hepcidin through the JAK-STAT signaling pathway,and the hepcidin synthesized by the liver increases.The serum hepcidin is significantly elevated in the patient,inhibiting the ingestion of iron from the intestinal epithelial cells and the release of iron from macrophages.At the same time,iron is blocked in the liver tissue and is difficult to be utilized by the body,eventually leading to hypocalcemia and induced iron deficiency.Anemia,which is characterized by decreased serum iron,difficulty in releasing iron in tissues,and increased expression of inflammatory mediators.However,existing treatments cannot treat chronic anemia,and treatment can only be achieved by treating inflammation.A variety of signaling pathways are involved in the expression,secretion,and maturation of the hepcidin gene into a biologically active hepcidin process,including HJV(hemojuvelin hepcidin regulatory protein)-BMP(bone morphogenetic protein).SMAD(drosophila mothers against decapentaplegic)signaling pathway,furin(Furin)-HJV signaling pathway.In addition,transmembrane protease serines 6,TMPRSS6,body iron levels,and inflammatory factors are factors that influence the expression of hepcidin.Studies have shown that heparin has good anti-hepcidin activity,however its strong anticoagulant activity makes its application against hepcidin activity affected by bleeding risk,so it has developed low anticoagulant activity and high anti-hepcidin activity.Heparin derivatives have become a research hotspot.Studies have found that heparin glucuronic acid o-diol cleavage derivatives have lower anticoagulant activity and better anti-hepcidin activity,while further N-acetylated heparin derivatives are more resistant to hepcidin.weak.Gs-Hp can quickly and effectively inhibit the expression of hepcidin in the liver,promote the transport of iron in the iron and other iron storage organs to the blood,thereby increasing the flow of iron in the blood.In mice that are chemically induced to produce inflammation,the heparin derivative inhibits the SMAD-BMP6 pathway and reduces the amount of hepcidin in the body.The mechanism of action is similar to the hepcidin binding protein HJV,which targets the BMP-HJV-SMAD pathway.Inhibition of IL-6 and iron on the induction of hepcidin.Compared with heparin,Gs-Hp has reduced anticoagulant activity while maintaining its high anti-hepcidin activity and anti-inflammatory and anti-tumor effects,so it is expected to be developed as a new drug for treating anemia caused by cancer and chronic diseases.Based on this,the subject is fitted into a heparin iron complex,which can reduce the anticoagulant activity of heparin and maintain the anti-hepcidin activity of heparin.At the same time,it can also supplement the lack of circulating iron in anemia patients,thus achieving treatment of chronic diseases.The purpose of anemia.In this study,heparin sodium was used as a control to investigate the effect of heparin iron on hepcidin expression in HepG2 cells.The effects of heparin iron on chronic inflammatory anemia model and acute inflammatory anemia model in mice were investigated,and its anti-hepcidin activity and resistance were investigated.The effect of inflammatory anemia.The main results of this research are as follows:1.Preparation and stability of heparin ironFour heparin iron complexes were prepared by ultrasonication of heparin sodium solution and DHOF solution,and their structures were determined to be spherical nanoparticles by particle size and potential,and transmission electron microscopy.The particle sizes of the four composites are all less than 100 nm,and the potential is relatively large,and the structure is relatively stable.The raw materials required for this method are easy to obtain,and the experimental steps are relatively simple and easy.After 8 weeks of stability experiments,the prepared heparin iron nanocomposites remained clear and transparent at room temperature,and no visible aggregation was observed by the naked eye.Through the investigation of particle size and potential measurement,it was found that the particle size of heparin iron nanocomposite increased with time,but remained within 200nm,which has good biological activity,indicating heparin prepared in this study.Iron nanocomposites have better stability.2.Detection of anticoagulant titer in vitro and in vivo of heparin ironThrough the detection of anticoagulant titer in vitro and in vivo of heparin iron,it was found that the obtained heparin iron can greatly reduce the anticoagulant activity of heparin.Through the anti-coagulation test of sheep plasma and the activity of FXa and FIIa,it was found that the anticoagulant titer of heparin iron was Half of heparin can effectively reduce the anticoagulant activity of heparin.After in vivo experiments in mice,heparin iron can effectively reduce the clotting time of heparin sodium,indicating that the heparin iron prepared by this subject has a good masking effect on heparin.The role of the test can be applied to mice in vivo to reduce the risk of heparin bleeding.3.MTT assay for the effect of heparin iron nanocomplex on the proliferation of tumor cell HepG2The effects of heparin sodium and heparin iron nanocomplex on the proliferation of HepG2 cells were detected by MTT colorimetric assay.The study found that heparin and heparin iron nanoparticles did not significantly inhibit the proliferation of tumor cells after treatment with 0-50?g/mL heparin and heparin iron nanoparticles for 48h.Among them,after high concentration(50?g/mL)administration,heparin iron 4 has obvious cell proliferation effect,while at other concentrations,several drugs have no obvious effect on the proliferation of tumor cells,indicating that the synthesized heparin Iron nanoparticles have no obvious cytotoxicity and can be safely used in animal experiments.4.Determination of heparin iron on hepcidin mRNA by qRT-PCRThe anti-hepcidin activity of four heparin irons was verified by qRT-PCR.The results showed that 4 heparin irons showed better concentration when the concentration was less than 0.5 ?g/mL in the absence of IL-6 stimulation.To reduce the expression of hepcidin mRNA,in the in vitro simulated inflammatory response experiment,the expression of hepcidin mRNA was minimized after administration of 0.4 ?g/mL heparin iron 2 under the stimulation of IL-6.The result is similar to that of heparin sodium,indicating that the heparin iron synthesized in this subject can maintain the anti-hepcidin activity of heparin sodium and can be studied in animals.5.Effect of heparin iron on the expression of hepcidin by ELISAThe effect of heparin iron on the expression of hepcidin was determined by ELISA.The results showed that hepcidin was highly expressed in the cell supernatant in the simulated inflammatory response system.After administration of 1.6 ?glmL heparin iron 2,hepcidin The expression level decreased to the level of IL-6,indicating that the heparin iron synthesized in this study has the effect of reducing the expression of hepcidin in the inflammatory response,and can be applied to the model of inflammatory anemia to verify its effect.6.Effect of heparin iron on hemoglobin content in chronic inflammatory miceHeparin iron has a significant effect on increasing hemoglobin content in an inflammatory anemia model.The hemoglobin level of the inflammatory anemia model group was significantly lower than that of the model group,and the heparin sodium group and the heparin iron group significantly increased the hemoglobin level;although the heparin group had significant differences in hemoglobin levels compared with the normal group.,but the average of the two is very close.It shows that heparin iron has similar physiological activity as heparin sodium,and can increase hemoglobin content by treating inflammation,and iron ions in heparin may also promote hemoglobin.7.Effect of heparin iron on the content of hepcidin in serum of chronic inflammatory miceHeparin iron has a significant effect on reducing the content of hepcidin.In the model of chronic inflammatory anemia,hepcidin is highly expressed,the heparin sodium group and the low dose of heparin iron have a better effect on reducing the content of hepcidin in inflammatory anemia,and basically return to normal values.EPO and diclofenac sodium do not have Reduce the role of hepcidin expression in an inflammatory anemia model.It is indicated that heparin iron has anti-inflammatory and anti-hepcidin activities similar to those of heparin sodium,and can fundamentally treat the symptoms of inflammatory anemia.8.Effect of heparin iron on hepcidin expression pathway in acute inflammatory model miceThe acute inflammatory anemia model of mice was replicated by intraperitoneal injection of LPS.The results showed that the levels of Idl mRNA,Socs3 mRNA,Crp mRNA and Hamp mRNA were significantly increased in the acute inflammatory anemia model group compared with the normal group(P<0.01).Acute hepatic anemia mice were treated with heparin sodium and heparin iron,intraperitoneal injection of 0.06mg/mL heparin iron,heparin sodium solution 0.2mL,heparin iron and heparin sodium group Idl mRNA,Socs3 mRNA,Crp mRNA,Hamp mRNA content Compared with the model group,the mice were significantly lower(P<0.01),and there was no significant difference between the Idl mRNA,Socs3 mRNA,Crp mRNA and Hamp mRNA levels in the normal control group(P>0.05).This result indicates that heparin iron and heparin sodium inhibit the BMP/SMAD pathway of Id1 mRNA,the STAT3 pathway of Socs3 mRNA,and the NF-kB pathway of Crp mRNA.Heparin iron and heparin sodium alleviate the symptoms of inflammation by inhibiting these three pathways and reduce the expression of hepcidin in the inflammatory response.In conclusion,in this study,a safe hepcidin inhibitor,heparin iron,was developed to inhibit the expression of hepcidin by inhibiting BMP/SMAD and JAK/STAT3 pathways,thereby demonstrating the role of correcting chronic inflammatory anemia in mice.It has become a potential drug for the targeted inhibition of hepcidin in the treatment of inflammatory anemia. |
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