Inhibition Of Hepcidin Expression By Silkworm Feces Extract In Iron Deficiency Rats And Acute Inflammation Mice

Silkworm Feces Extract(SFE),as active pharmaceutical ingredients of Shengxueningpian,is a water-soluble iron complex which consists of sodium iron chlorophyllin and chlorophyll derivative.Recently researches showed that sodium iron chlorophyllin,which is a heme analogue,has anti-anemia,anti-inflammatory,antioxidant and other biological activity.In this paper,the SFE was used as the research object.We established animal models of iron deficiency anemia in rats and acute inflammation in mice with or without SFE treatment.We also established IL-6,BMP6 or LPS induced Hep G2 cells hepcidin over-expression cell models in the presence or absence of SFE.The effects of SFE on iron deficiency anemia and acute inflammation were evaluated by a series of methods.The aim of this study was to clarify the inhibitory effects and mechanisms of SFE on hepcidin expression.This study may provide a theoretical basis and new directions for the clinical application of SFE.Part One:The inhibitory effects and mechanisms of SFE on hepcidin expression in IDA ratsIn this part,the IDA model was established by feeding an iron-deficient diet combined with repeating bleeding from the orbital vein plexus every other day.The rats were intragastrically administered with 1m L distilled water or SFE(27 mg/kg or 54 mg/kg)each day.We monitored serum iron and blood routine every week.After 4 weeks,the hematological parameters were detected by automatic hematology analyzer.In SFE treatment groups,the Hb,RBC,HCT,MCV,MCH and MCHC were significantly higher than those of model group,and RDW-CV were significantly lower than that of model group.Serum iron and tissue iron,which were detected by atomic absorption spectrometry,were significantly increased compared with the model group.The kit was used to detect TIBC,and the results showed that SFE significantly reduced serum TIBC and increased serum TSAT.These results indicatd that SFE could effectively improve iron deficiency anemia.To clarify whether SFE could inhibit hepcidin expression,serum hepcidin was detected by ELISA,and western blot was performed to detect the expression of hepcidin and ferroportin1.The results showed that SFE significantly down-regulated hepcidin expression and up-regulated ferroportin1 expression.Then,western blot was used to detect hepcidin upstream related proteins and related proteins in IRP/IRE pathway.RIP was used to detect the affinity of IRP to IRE.These results showed that SFE could down-regulate the expression of p-JAK2,p-STAT3,BMP6,p-SMAD1/5/8,SMAD4 and p38 MAPK,thereby inhibiting hepcidin expression.Besides,SFE could activate the IRP/IRE pathway,upregulate the expression of IRP1 and IRP2,increase the affinity of IRPs and IRE,thus up-regulated Tf R1 expression and down-regulated ferritin expression,then promoted tissue iron release.In conclusion,the present study demonstrated that SFE not only replenished iron supply as an iron source,but also increased IRPs expression and suppressed hepcidin expression,which promoted the release of stored iron.In this manner,SFE supplied sufficient iron to meet erythropoietic needs,resulting in a significant preventative effect on iron deficiency anemia.This part of the study provided new ideas for the treatment of iron deficiency anemia and theoretical basis for the clinical application of SFE.Part Two: The inhibitory effects and mechanisms of SFE on hepcidin expression in acute inflammatory miceIn this part of the experiment,the mice were intragastrically administered with SFE(50 mg/kg)for 6 weeks,and then the lipopolysaccharide(LPS,2 ?g/g)was intraperitoneal injected to induce acute inflammation.Observed H&E staining of liver tissue sections,the boundary between cells was unclear and the inflammatory cells infiltrated severely in model group.While the cell morphology and inflammatory cell infiltration of were significantly improved in SFE group.The serum SOD activity and MDA content were detected by the kit.The serum SOD activity of mice had no significant changes and the serum MDA content was rapidly increased in model group.However,the serum SOD activity of micen was significantly increased in SFE group,and serum MDA content was significantly lower than that of the model group.Western blot was performed to detect the expression of hepcidin and related proteins,the results showed that the expression of hepcidin,NF-?B and p-STAT3 in SFE group were significantly lower than those of model group.In summary,SFE could effectively alleviate acute inflammation and oxidative damage induced by LPS.And SFE could also effectively inhibit the over-expression of hepcidin and the activation of inflammatory pathway stimulated by LPS.This part of the study may provide a new direction for the clinical application of SFE.Part Three: The inhibitory effects and mechanisms of SFE on hepcidin expression in vitroTo further clarify the mechanism of SFE inhibiting the expression of hepcidin,we constructed hepcidin over-expression models induced by IL-6,BMP6 or LPS in vitro.Hep G2 cells were stimulated by IL-6(50 ng/m L),BMP6(25 ng/m L)or LPS(1 ?g/m L)with or without SFE(200 ?g/m L).Western blot and q RT-PCR analyses were conducted to explored the expression of related proteins and hepcidin m RNA.Results showed that the expression of hepcidin m RNA,p-STAT3,and p-JAK2 was significantly increased after IL-6 stimulation,while co-cultured with SFE significantly reversed the induction of IL-6.The expression of hepcidin m RNA,p-SMAD1/5/8 and SMAD4 was significantly increased after stimulation with BMP6,while the intervention of SFE significantly counteracted the induction of BMP6.LPS significantly up-regulated the protein expression levels of hepcidin,NF-?B,and p-STAT3,while the SFE co-culture group significantly inhibited the induction of LPS.All in all,in vitro experiments further proved that SFE could inhibit JAK2/STAT3 and BMP6/SMAD pathways,thereby down-regulating hepcidin expression.