The Research On Effect Of Norcantharidin On Iron Metabolism In Inflammatory Condition

Objective:To explore the effect of norcantharidin on iron metabolism in vivo and in vitro inflammatory environment,and to prove whether norcantharidin has an effect on iron metabolism and related proteins by affecting the expression of IL-6 and hepcidin,so as to provide important ideas for the anticancer mechanism of norcantharidin.Methods:1.The CCK-8 method was used to detect and analyze the effect of norcantharidin on the viability of RAW264.7 murine macrophage cell line;2.Real-time PCR was investigated the effect of norcantharidin on the expression of IL-6 and hepcidin mRNA in RAW264.7 murine macrophage cell line and liver,spleen and duodenum of balb/c mice;3.Western bolt was used to detect the effects of norcantharidin on the expression of iron metabolism related proteins(TfR1,DMT1,Fpn1,Ferritin-l,IRP1)and pathway proteins(P-JAK2 and P-STAT3)in RAW264.7 murine macrophage cell line and liver,spleen and duodenum of balb/c mice;4.Immunofluorescence staining was used to detect the effects of norcantharidin on the expression of iron metabolism-related proteins TfR1,DMT1,Fpn1,Ferritin-L and IRP1 in liver,spleen and duodenum of balb/c mice;5.Iron kit was used to detect the effects of norcantharidin on the expression of iron content in tissues and serum iron content(SI),unsaturated iron binding capacity(UIBC)and transferrin saturation(TF% saturation).Results:1.Cell activity experiments showed that the maximum concentration of norcantharidin was 10?M when cell activity was not affected;2.The results of real-time PCR showed that the expression of IL-6 and hepcidin mRNA in cells and mice liver,spleen and duodenum was significantly increased compared with the control group,when the RAW264.7 treated with LPS(1 ?g/ml)for6 h and intraperitoneal injection of LPS(1 mg/kg)for 6h.While the expression of IL-6 and hepcidin mRNA were decreased in cells or mice treated with LPS and norcantharidin in acute inflammatory condition;3.The results of western bolt revealed that the expression of iron metabolism related proteins TfR1,DMT1 and Fpn1 were decreased,while Ferritin-L and IRP1 were increased,the pathway proteins P-JAK2 and P-STAT3 were increased after the cells treated with LPS(1 ?g/ml)for 6 h,compared with the control group,while the effect of norcantharidin(10 ?M)could reverse the effect of LPS;After intraperitoneal injection of LPS(1 mg/kg)for 6 h,the expression of iron metabolism related proteins TfR1,DMT1,Fpn1,IRP1 in the liver,spleen and duodenum of mice were decreased,the expression of iron storage protein Ferritin-L and pathway proteins P-JAK2 and P-STAT3 were increased.While continuous intraperitoneal injection of norcantharidin(2 mg/kg)for 3 days,the effect of LPS could be significantly reversed.4.The results of immunofluorescence staining indicated that the expression of iron metabolism related proteins,such as iron transport protein TfR1,DMT1,Fpn1 and iron regulatory protein IRP1 in the liver,spleen and duodenum of mice were decreased,the expression of iron storage proteinFerritin-L and pathway proteins P-JAK2 and P-STAT3 were increased after intraperitoneal injection of LPS(1 mg/kg)for 6 h.While continuous intraperitoneal injection of norcantharidin(2 mg/kg)for 3days,the effect of LPS could be significantly inhibited,namely the expression of iron input protein TfR1,DMT1,iron output protein Fpn1 and iron regulatory protein IRP1 were increased,while the expression of iron storage protein Ferritin-L and pathway proteins P-JAK2 and P-STAT3 were increased.5.The results of the iron kit showed that after 6h of intraperitoneal injection of LPS(1mg/kg),compared with the control group,the iron content in the liver,spleen and duodenum of mice were significantly increased;serum iron(SI)and serum transferrinsaturation(TF% saturation)were decreased.After 3 days of continuous intraperitoneal injection of norcantharidin(2 mg/kg),the iron content in liver,spleen and duodenum of mice were decreased significantly,and serum iron(SI)and transferrin saturation(TF% saturation)were increased,but there was no significant difference in the unsaturated iron-binding force(UIBC).Conclusion:Norcantharidin can inhibit the expression of hepcidin mRNA through IL-6/JAK2/STAT3 pathway,thus affecting the iron metabolism of cells and systems.