Study Of The Effect And Mechanism Of Iron Dextran On Vitamin D₃ Metabolism In SD Rats With Iron Deficiency Anemia

Objective:In this study,SD rats with iron deficiency anemia（IDA）model were used to explore the relationship between iron metabolism and vitamin D（VD）metabolism,and to further explore whether iron affects the levels of 25-（OH）D₃and 1,25-（OH）₂D₃by regulating VD hydroxylase.Methods:After 40 male SD rats with a body weight of 66.46±4.22g,they were fed adaptively for 1 week,they were randomly divided into control group（Control,C,n=8 normal diet）and model group（Model,M,n=32 iron-deficiency diet）according to their body weight.IDA model was established in the latter group,both of them drank deionized water freely.During the modeling period,the mental status,activity status,skin and mucous membrane color of rats in each group were observed to determine the modeling situation.After 6 weeks,the M group was randomly divided into the Deficiency iron（DFe）[0mg/（100g·bw·d）],Low iron（LFe）[1.1mg/（100g·bw·d）],Medium iron（MFe）[3.3mg/（100g·bw·d）],High iron（HFe）[9.9mg/（100g·bw·d）]according to the level of Hemoglobin（Hb）,8 rats in each group,2ml/d iron dextran was given by gavage for 4 weeks.After 4 weeks,the rats were anesthetized with 8%chloral hydrate 5ml/kg.The blood was collected from the abdominal aorta,and liver and kidney tissues were collected.The separated serum and tissues were packed and frozen at-80°C for testing.ELISA method was used to detect the levels of 25-（OH）D₃and 1,25-（OH）₂D₃in rat serum,iron metabolism-related indicators;Western blotting,immunofluorescence staining and q-PCR to detect liver CYP2R1,CYP27A1,kidney CYP27B1,CYP24A1 expression at the translation level and transcription level.Results:1.The mental state of DFe group was poor,and the levels of hemoglobin（Hb）,red blood cell（RBC）,liver iron and kidney iron were significantly lower than the other four groups.2.The levels of total iron binding capacity（TIBC）,divalent metal transporter 1（DMT1）,transferrin（TF）,transferrin receptor（Tfr）and membrane transporter 1（FPN1）in DFe group were higher than those in other groups,the levels of Tfr and FPN1 were significantly increased（P<0.05）;Serum iron（SI）level was the lowest in DFe group.3.Compared with group C,the serum levels of 25-（OH）D₃and 1,25-（OH）₂D₃in DFe group were significantly lower（P<0.05）,and the levels of 25-（OH）D₃and1,25-（OH）₂D₃were also increased with the increase of iron concentration.4.By correlation analysis,the levels of 25-（OH）D₃and 1,25-（OH）₂D₃were negatively correlated with TIBC,DMT1,TF,Tfr and FPN1,but not with SI.5.The protein and mRNA expression levels of CYP2R1,CYP27A1 and CYP24A1 in DFe group were significantly lower than those in the other four groups,while the protein and mRNA expression levels of CYP27B1 in DFe group was significantly higher than those in the other four groups.6.Immunofluorescence results showed that the Integrated optical density（IOD）values of CYP2R1,CYP27A1 and CYP24A1 in DFe group were lower than those in C group,and the expression of CYP27B1 was the highest in DFe group,which was significantly higher than that in C group（P<0.05）.7.The agarose gel electrophoresis showed that the expression of amplification bands of hydroxylase in each group was consistent with the quantitative results of q-PCR.Conclusion:1.Iron deficiency lowers iron reserves in the liver and kidneys,which may affect the activity of hydroxylase.2.Iron may reduce the levels of 25-（OH）D₃and 1,25-（OH）₂D₃by regulating the translation and transcription levels of hydroxylase.3.In the case of sufficient iron,the VD hydroxylation of Cytochrome P450（CYP450）enzyme family can effectively use iron as a cofactor to promote the hydroxylation of VD;under the condition of iron deficiency,the expression of VD hydroxylase in liver is down-regulated and that in kidney is up-regulated.Or it may be due to iron deficiency caused by CYP2R1 and CYP27B1,resulting in the barrier of hydrogenase active center,the conversion to the active form of VD capacity is weakened.