Therapeutic effect of folic acid combined with decitabine on diabetic mice

Abstract

Aim: To evaluate the therapeutic effect of folic acid combined with decitabine on diabetic mice.

Methods: The diabetic model of db/db mice were randomly divided into model group, folic acid group, decitabine group, folic acid combined with decitabine group, and C57 mice as normal control group. The density of retinal blood vessels and retinal thickness were detected by fundus photography and optical coherence tomography, respectively. Pathological changes of retina were observed by hematoxylin-eosin (HE) staining. The homocysteine (Hcy) in serum was detected by enzyme linked immunosorbent assay (ELISA). TdT-mediated dUTP nick-end labeling (TUNEL) was used to detect apoptosis in retinal tissue. Evans blue dye was used to detect the permeability of retinal blood vessels. The platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor receptor (VEGFR) protein were detected by Western blot. The 3-nitrotyrosine (3-NT) and 4-hydroxynonanine (4-HNE) were detected by immunohistochemistry.

Results: The density of retinal blood vessels, retinal thickness, retinal vascular permeability and the proportion of apoptotic cells of retinal tissue in the model group increased significantly than control group (P<0.05). The Hcy in serum and the levels of CD31, VEGFR, 3-NT, and 4-HNE in retinal tissue increased significantly in the model group (P<0.01). Folic acid and decitabine both reversed these changes significantly, and the combination of the folic acid and decitabine worked best.

Conclusion: The combination of folic acid and decitabine has a more significant protective effect on the retina in diabetic mice.

Keywords: apoptosis; decitabine; diabetic model folic acid; mouse.

Figure 1. The effect of folic acid combined with decitabine on the angiogenesis and thickness of retina in diabetic mice

The retinal vascularization was observed by fundus photography (FP), and the retinal thickness changes were observed by optical coherence tomography (OCT).

Figure 2. Effect of folic acid combined with decitabine on vascular permeability in retina of diabetic mice, and retinal permeability changes were detected by Evans blue staining

^aP<0.01 vs blank group, ^bP<0.01 vs model group, ^cP<0.01.

Figure 3. The effect of folic acid combined with decitabine on serum Hcy level in diabetic mice

ELISA assay was used to detect the level of Hcy in serum of mice. ^aP<0.01 vs blank group, ^bP<0.01 vs model group, ^cP<0.01.

Figure 4. The effect of folic acid combined with decitabine on the cell apoptosis of retina in diabetic mice

The cell apoptosis of retinal tissue was detected by Tunel staining (A, ×200), and the rate of apoptotic cells was analyzed using Image pro plus software (B). ^aP<0.01 vs blank group, ^bP<0.01 vs model group, ^cP<0.01.

Figure 5. The effect of folic acid combined with decitabine on the expression of 3-NT and 4-HNE in retina of diabetic mice

Immunohistochemistry was used to detect the changes of 3-NT and 4-HNE in the retina of mice (A, ×200), and the optical density of the region stained by 3,3′-diaminobenzidine dye was analyzed using Image pro plus software (B). 3-NT: 3-nitrotyrosine; 4-HNE: 4-hydroxynonenal. ^aP<0.01 vs blank group, ^bP<0.01 vs model group, ^cP<0.01.

Figure 6. The effect of folic acid combined with decitabine on the expression of CD31 and VEGFR in retina of diabetic mice

The expression of CD31 and VEGFR protein in the retina of mice was detected by Western blot, and the optical density of the bands was analyzed using Image pro plus software (B). CD31: Platelet endothelial cell adhesion molecule-1; VEGFR: Vascular endothelial growth factor receptor. ^aP<0.01 vs blank group, ^bP<0.01 vs model group, ^cP<0.01.