| Objective:To study the molecular mechanism that magnesium cantharidate improves Anemia of chronic disease by regulating the expression of Hepcidin,to verify whether magnesium cantharidate inhibits the expression of Hepcidin through the BMP6/SMAD pathway,thereby regulating the expression of iron metabolism-related proteins Tf R1,DMT1,and Fpn1,and has a positive effect on the body’s iron stability.The effect of state to achieve the effect of improving Anemia of chronic disease.Methods:1.The effect of magnesium cantharidate on viability of hep G2 cells was detected by CCK-8 method;2.Quantitative real time PCR(q RT-PCR)was used to detected Hepcidin m RNA expression of magnesium cantharidin in hep G2 cells and liver,spleen,and duodenum of Sprague Dawley(SD)rats;3.Western bolt(WB)was used to detect the effects of magnesium cantharidate on the expression of pathway proteins SMAD4 and P-Smad1/5/8 and iron metabolization-related proteins Tf R1,DMT1 and Fpn1 in hep G2 cells,liver,spleen and duodenum of SD rats;4.Establishment of anemia of chronic disease(ACD)model in rats for in vivo research;5.Changes of blood routine in rats after the action of magnesium cantharidate detected by automatic blood analyzer;6.The effects of magnesium cantharidate on iron content in liver,spleen and duodenum,iron content(SI),total iron binding force(TIBC)and transferrin saturation(Tf% saturation)in serum were examined by iron kits.Results:1.The results of the CCK-8 experiment showed that the maximum administration concentration of magnesium cantharidate was 0.75 μg/m L under the condition of minimal effect on cell viability;2.The results of q RT-PCR showed that Hepcidin expression was significantly increased in hep G2 cells treated with BMP6 cytokine(100ng/m L)for 8h compared with the control group.After magnesium cantharidate treatment,the expression of Hepcidin decreased compared with BMP6 cytokine induced group.In vivo experiment,compared with the control group,the expression of Hepcidin in ACD model group was significantly increased,while the expression of Hepcidin in liver,spleen and duodenum in magnesium cantharidate treatment group was down-regulated compared with the ACD model group;3.WB experiment results showed that compared with the control group,the expression of pathway proteins SMAD4 and p-SMAD1/5/8 was increased after BMP6 cytokine(100ng/m L)was applied to hep G2 cells,while the expression of iron metabolism-related proteins Tf R1,DMT1 and Fpn1 was decreased.After magnesium cantharidate treatment,the expression levels of SMAD4 and p-SMAD 1/5/8 were down-regulated and the expression levels of Tf R1,Fpn1 and DMT1 were up-regulated compared with the BMP6 cytokine induced group.In the in vivo experiment,compared with the control group,the expression levels of pathway proteins SMAD4 and p-SMAD 1/5/8 in liver,spleen and duodenum of ACD group were increased,and the expression levels of iron metabolism-related proteins Tf R1,Fpn1 and DMT1 were decreased.Compared with ACD model group,the expression levels of pathway proteins SMAD4 and p-SMAD 1/5/8 in liver,spleen and duodenum were down-regulated in magnesium cantharidate treatment group,and the expression levels of iron metabolism-related proteins Tf R1,Fpn1 and DMT1 were up-regulated;4.The observation of joint inflammation in rats showed that the feet were obviously red and swollen,and the blood routine examination of ACD rats showed that the red blood cell count and hemoglobin level were reduced;5.The results of blood routine tests by automatic hematology analyzer showed that compared with the control group,the red blood cell count and hemoglobin content of ACD group rats decreased,and the white blood cell count increased.Compared with ACD group,the content of white blood cell count in magnesium cantharidate treatment group was significantly decreased,while the content of red blood cell count and hemoglobin was significantly increased;6.Compared with the control group,the iron content in liver,spleen and duodenum,serum iron content,total iron binding capacity(TIBC)and transferrin saturation(Tf% saturation)of rats in ACD group were detected by iron kit.The results showed that the iron content in liver,spleen and duodenum of rats in ACD group was increased,the iron content in serum iron(SI)was decreased,and the total iron binding capacity was decreased,serum transferrin saturation(Tf% saturation)decreased.Compared with the ACD group,the iron content in liver,spleen and duodenum was decreased,the iron content in serum,the total iron binding capacity and the serum transferrin saturation(Tf% saturation)were increased in the magnesium cantharidate treatment group.Conclusion:Magnesium cantharidate can down-regulate the expression of Hepcidin by inhibiting the BMP/SMAD pathway,thereby regulating the changes in iron content and improving anemia of inflammation. |
PDF Full Text Request