Figure 3.

Microbiota was involved in VD-regulated intestinal immunity. (a) The proximal promoter of il22 (−2719 to + 506 bp) in zebrafish was amplified, and cloned into the luciferase reporter plasmid pGL3 as pGL3-il22. Meanwhile, pGL3 plasmid without il22 promoter was used as control. pGL3 or pGL3-il22 was microinjected into zebrafish embryos at one or two-cell stage, followed by the incubation with control buffer or 1,25(OH)₂D₃ (10 nM). After 24 h, the relative luciferase activity in zebrafish embryos was assessed (n = 6–9 replicates/group, 10–15 larvae/replicate). (b) The zebrafish at 3 mpf were treated with antibiotics mixture or control buffer for one week, and the gene expression of il22, zfbd1, zfbd2 and zfbd3 in zebrafish intestine was assessed (n = 8/group). (c) After the zebrafish were treated with antibiotics mixture for one week, they were conventionally raised for another week (CONVED group). The zebrafish in control group were conventionally raised for 2 weeks. The gene level of il22, zfbd1, zfbd2 and zfbd3 in zebrafish intestine was analyzed (n = 8/group). (d-e) WT and cyp2r1 mutant zebrafish were treated with or without antibiotics for 3 weeks, and the gene expression of il22, zfbd1, zfbd2, zfbd3 in the gut was analyzed (n = 8/group). (f-g) The gene expression of il22, zfbd1, zfbd2, zfbd3 in the gut of zebrafish fed with 0 or 800 IU/kg VD₃ diets for 4 weeks with or without antibiotic treatment (n = 8/group). **p < 0.01, ***p < 0.001, ns: non-significance. See also Figures S3.