Table 1.
Specific primers used for Q-PCR.
Fig 1.
Effect of 1,25(OH)2D3 deficiency on serum biochemistry.
(A) Deletion efficiency of the Cyp27b1 allele was analyzed using genomic DNA from the tail. (B) Quantitative PCR assay for Cyp27b1 mRNA in colons. (C) Serum calcium, (D) serum phosphorus and (E) serum 1,25(OH)2D3 from 10-month-old Cyp27b1-/- and WT mice (n = 5; mean±SEM). **: p< 0.01.
Fig 2.
1,25(OH)2D3 deficiency induced chronic colonic inflammation.
(A) Body weight from 3 month, 6 month and 10 month mice of Cyp27b1-/- and WT. The full length of colon (B and C) and the ratio of length vs body weight (D) from WT and Cyp27b1-/- mice with 10 month age. Values are mean ± SEM, n = 8. *P < 0.05, * * P < 0.01. (E) HE staining of colon section from WT and Cyp27b1-/- mice. Magnification, ×200. Bar, 50μm. Immunohistochemical analyses of CD3 (F) and F4/80 (G) expression in colon tissues from the WT and Cyp27b1-/- mice at 10 month. Magnification, ×400. Bar, 25μm. The histogram shows the mean percentage of the CD3 or F4/80 positive cells determined from five randomly selected fields. Magnification, ×400. Bar, 25μm. Values are mean ± SEM, n = 5. *P < 0.05.
Fig 3.
1,25(OH)2D3 deficiency induced DNA damage and cellular senescence.
Immunohistochemical analyses of 8-OHdG (A) γH2AX (B) expression and analysis of SA-β-gal positive cells (C) in the colon from the WT and Cyp27b1-/- mice. The positive cells in lamina proppria are marked by arrow. The histogram shows the mean percentage of the 8-OHdG or γH2AX, SA-β-gal positive cells determined from five randomly selected fields. Magnification, ×400. Bar, 25μm. Values are mean ± SEM, n = 5. *P < 0.05. (D): Western blot analyses of colonic γH2AX expression in the WT and Cyp27b1-/- mice, β-actin was used as a loading control.
Fig 4.
1,25(OH)2D3 deficiency induced the genes expression of the senescence-associated inflammatory cytokines.
Immunohistochemical analyses of IL-1ɑ (A) IL-6 (B) IL-8 (C) HGF1 (D) MMP3 (E) expressions in the colon from the WT and Cyp27b1-/- mice. Positive cells in lamina propria were labeled by arrow. The histogram shows the mean percentage of the positive area determined from five randomly selected fields. Magnification, ×400. Bar, 25μm. Values are mean ± SEM, n = 5. *P < 0.05, ** P < 0.01. (F) Real-time RT-PCR was performed on extracts of colon from WT and Cyp27b1-/- mice for the gene expression of IL-1α, IL-1β, IL-6, IL-8, HGF1, MMP-3 and MMP-13. (G) Western blot analyses of colonic NF-κB (p105 and p50) expression in the WT and Cyp27b1-/- mice, β-actin was used as a loading control.
Fig 5.
1,25(OH)2D3 deficiency induced the ROS production and decreased antioxidants.
(A) Flow cytometry analysis of ROS in colons from the three groups of 10-month-old mice. (B) H2O2 and (C) Total SOD level of colons measured by spectrophotometry. (D)Real-time RT-PCR was performed on extracts of colon from WT and Cyp27b1-/- mice for the gene expression of antioxidants. (E) Western blot analyses of colonic SOD2 and Prdx2 expression in the WT and Cyp27b1-/- mice, β-actin was used as a loading control.