Experimental designs

The 40 male newly weaned Specific Pathogen Free (SPF) SD rats, weighing 66.46 ± 4.22 g, were purchased from Beijing Changyang Xishan animal farm, animal Certificate No: scxk (Beijing): 2019-0010. After 1 week of adaptive feeding, they were randomly divided into control (C, n = 8) group and model (M, n = 32) group by simple randomization. C group was fed with normal diet and normal drinking water; M group was fed with iron deficiency diet (iron content was 21.33 mg/kg) and drinking deionized water to establish IDA model. After 6 weeks, they were randomly divided into four groups: iron deficiency group (DFe) [0 mg/(100 g·bw·d)], low iron group (LFe) [1.1 mg/ (100 g·bw·d)], medium iron group (MFe) [3.3 mg/(100 g·bw·d)], high iron group (HFe) [9.9 mg/(100 g·bw·d)] by block randomization, 2 ml for each animal for 4 weeks, C and DFe groups were perfused with 2 ml normal saline. In addition to the different iron content, the other nutritional components of the two diets were the same. Both of them added 1500 IU of VD3, and used shading cloth to prevent the rats from sunlight to synthesize VD3 through the skin. The temperature and relative humidity were 21 ± 1℃ and 46–60% respectively. In order to avoid iron pollution, SD rats were housed in stainless steel cages, with stainless steel food containers and plastic drinking bottles. After 4 weeks of intervention, the rats were anesthetized with 8% chloral hydrate. Blood samples were collected from the abdominal aorta for 8–10 ml. The blood was placed in a centrifugal tube for 30 min at room temperature and then centrifuged at 3000 r/min for 10 min to separate the serum. After washing the liver and kidney with normal saline, some of them were fixed by immersion in 4% paraformaldehyde, and the others were put into nuclease free tube and frozen at −80℃.

Main reagents and instruments

Iron Dextran (Yuanye Biology, S51662); VD3 (Beijing Solarbio, V8070); Tissue Iron Determination Kit (Nanjing Jiancheng, A039-2-1); hemoglobin (Hb) Kit (Nanjing Jiancheng, C021-1-1); red blood cell (RBC) diluent (Leagene Biotechnology, DA0150); 25-(OH)D3 kit (Jianglai Biological, JL20734); 1,25-(OH)D3 kit (Jianglai Biological, JL27246); transferrin (TF) (Jianglai Biotechnology, JL-31079); transferrin receptor (Trf) (Jianglai Biotechnology, JL-30566); serum iron (SI) (Nanjing Jiancheng, A039-1-1); iron-binding capacity (TIBC) (Nanjing Jiancheng, A040-1-1); CYP2R1 primary antibody (ABclonal, A10470); CYP27A1 primary antibody (ABclonal, A1982); CYP27B1 primary antibody (ABclonal, A1716); CYP24A1 primary antibody (ABclonal, A1805); Cy3-goat anti-rabbit IgG (Beyotime, A0516). Microplate reader (Bio-Rad, USA); DAPI (Aladdin, D106471); Paraffin embedding machine (Wuhan Junjie Electronics Co, Ltd, JT-12S); Microtome (Lycra Microsystems Co, Ltd, RM2016); Electron microscope (Nikon Instruments Co, Ltd, Eclipse Ci-L); PCR instrument (Hangzhou Bioet Company, Line Gene9640PCR).

Detection of hematological and serological indexes

ELISA kit was used to detect 25-(OH)D3, 1,25-(OH)2D3, TF, and Tfr; Biochemical kit was used to detect Hb, RBC,TIBC, SI, liver iron and kidney iron, and the operation was carried out strictly according to the operation procedures of the kit.

Western blotting was used to detect the expressions of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein

The expressions of CYP2R1 and CYP27A1 protein in liver, and the expressions of CYP27B1 and CYP24A1 protein in kidney were detected, each sample was taken 20 mg, and Radio Immunoprecipitation Assay (RIPA) lysate was added, The total protein was extracted, protein was prescribed by BCA Assay Kit, the total protein was sampled with 20 μg protein in 96-well plate, gel electrophoresis, transfer membrane, block, 4℃ primary antibody CYP2R1, CYP27A1, CYP27B1, CYP24A1 (1:500), β-actin (1:5000) were incubated overnight at room temperature, The secondary antibody (1:5000) was incubated for 2 h, CYP2R1, CYP27A1, CYP27B1, and CYP24A1 were detected by the luminescent solution, and the gray value was analyzed by Image J software.

Immunofluorescence staining of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein

The liver and kidney tissue specimens fixed with 4% paraformaldehyde, and then dehydrate after fixation, wax embedding, sectioning, dewaxing, hydration, gradient ethanol elution, antigen retrieval, CYP2R1, CYP27A1, CYP27B1, and CYP24A1 (diluted 1:100) primary antibody incubation, incubated with secondary antibody (diluted at 1:200) and counter-stain with diamidino-2-pheny1 indole (DAPI). The immunofluorescence intensity in the liver tissue was observed and photographed under a fluorescence microscope. The CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein staining were positive in red. The fluorescence intensity of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 protein was detected by Image Pro Plus image analysis software, and the integrated optical density (IOD) was calculated as the final average immunofluorescence intensity.

The mRNA expressions of CYP2R1, CYP27A1, CYP27B1 and CYP24A1 were detected by q-PCR

Total RNA was extracted from 20 mg liver and kidney tissues of rats. The cDNA was reverse transcribed and amplified by fluorescent quantitative PCR with β-actin as an internal reference. The upstream primer sequence of β-actin was 5´-TGTGATGGTGGGAATGGGTCAG-3´, downstream primer sequence 5´-TTTGATGTCACGCACGATTTCC -3´. CYP2R1 upstream primer sequence: 5´-TGCTACTACTCGTGCTGGTGGTC-3´, downstream primer sequence 5´-AGGGCCAGGGA GCAGATGTTG-3´. CYP27A1 upstream primer sequence: 5´-TCGCACCAATGTGAATCTGGC TAG-3´, downstream primer 5´-CTTCCACTGCTCCATGCTGTCTC-3´. CYP27B1 upstream primer sequence: 5´-GCACATAAACGGCAAGGCAAGTC-3´, downstream primer sequence 5´-AGCCAAGCCTCTCACCTCCTATG-3´. CYP24A1 upstream primer sequence 5´-GGAACTGT A CGCCGCTGTCAC-3´, downstream primer 5´-GCACGCTCTGAACTTCCTGAAGG-3´. PCR conditions: pre-denaturation 95℃, 30 s; denaturation: 95℃, 5 s, 60℃, 30 s (40 cycles); annealing: 95℃, 5 s, 60℃, 30 s, 95℃, 1 min; extension: 50℃, 30 s. The relative quantitative values were calculated according to the Ct values of the original q-PCR data.

Statistical analysis

Spss 25.0 software was used for statistical analysis of the data. The HB value and body weight of group C and group M were tested by t test; VD3 metabolism index, iron metabolism index, tissue iron and other indicators were compared by one-way ANOVA, LSD-t test or Dunnett's T3 test were used for pairwise comparison, P < 0.05 means the difference was statistically significant.