Effects and related mechanism of 5-aza-2'-deoxycytidine on endothelial function in rats with hyperhomocysteinemia

Abstract

To investigate the effects and related mechanism of 5-aza-2'-deoxycytidine(Aza) on endothelial function in Hyperhomocysteinemia rats. Adult male SD rats were divided into 3 groups (n=7 each): control group, hyperhomocysteinemia (HHcy) group and Aza group according to the random number table. Control group rats were fed with normal diet. HHcy group rats were fed with diet adding 3% L-methionine. Aza group rats were fed with diet adding 3% L-methionine and Aza (0.5 mg/kg) injection for consecutive three days per week for 8 weeks. After 8 weeks, content of rat plasma homocysteine (Hcy) was detected by enzyme linked immunosorbent assay (ELISA). The rat mesenteric artery endothelium-dependent diastolic function was detected. The nitric oxide synthase (eNOS) activity and asymmetric dimethyl fine ammonia acid (ADMA) content were detected by ELISA, and the content of nitric oxide was detected by nitrate reductase method in the mesenteric arteries. The mRNA expression of DNA methyl transferase 1 (DNMT1) and dimethyl arginine acid dimethylamine hydrolase 2 (DDAH2) in the mesenteric arteries were detected by real-time fluorescence quantitative PCR, and the protein expressions of DNMT1 and DDAH2 in the mesenteric arteries were detected by Western blot. The DDAH2 promoter methylation level in the mesenteric arteries was detected by nested methylation specific PCR. (1) The content of plasma Hcy was significantly higher in the HHcy group and Aza group compared to the control group ((29.00±0.94) μmol/L and (26.43±0.47) μmol/L vs.(10.34±0.63) μmol/L, both P<0.01), which was significantly reduced in the Aza group compared with the HHcy group (P<0.05). (2) Acetylcholine-mediated relaxation at various concentrations was significantly lower in the HHcy group and the Aza group compared with the control group (both P<0.05), which was significantly increased in Aza group compared with HHcy group (P<0.05). SNP-mediated relaxation at various concentrations was similar among the three groups(all P>0.05). (3) Compared with the control group, the content of nitric oxide in the HHcy group was significantly decreased ((0.52±0.01) μmol/g vs.(0.42±0.00) μmol/g, P<0.01), which could be increased by Aza((0.49±0.01) μmol/g, P<0.05); the eNOS activity in the HHcy group was significantly decreased ((0.74±0.01) U/mg vs. (0.57±0.00) U/mg, P<0.01), which could be significantly increased by Aza ((0.65±0.01) U/mg, P<0.01); the content of ADMA in the HHcy group was significantly increased ((0.34±0.01) μmol/g vs. (0.37±0.00) μmol/g, P<0.05), which could be significantly decreased by Aza ((0.32±0.01) μmol/g, P<0.05). (4) Compared with the control group, the relative expression of DDHA2 mRNA in the HHcy group was significantly decreased (0.15±0.01 vs.0.12±0.01, P<0.01), which could be significantly increased by Aza (0.13±0.01, P<0.05); the relative expression of DDHA2 protein in the HHcy group was significantly decreased (0.31±0.02 vs. 0.24±0.01, P<0.01), which could be significantly increased by Aza (0.28±0.01, P<0.01). Compared with the control group, the relative expression of DNMT1 mRNA in the HHcy group was significantly increased (0.23±0.01 vs.0.43±0.01, P<0.01), which could be significantly decreased by Aza (0.39±0.01, P<0.05); the relative expression of DNMT1 protein in the HHcy group was significantly increased (0.35±0.01 vs. 0.50±0.01, P<0.01), which could be significantly decreased by Aza (0.47±0.01, P<0.05). (5) Compared with the control group, the methylated/non methylated ratio of DDHA2 promoter in the HHcy group was significantly increased (1.04±0.03 vs. 1.26±0.03, P<0.01), which could be significantly decreased by Aza (0.80±0.03, P<0.01). Aza can inhibit the activity of DNMT1, reduce DDAH2 promoter methylation level, increase the expression of DDAH2, decrease the content of ADMA, increase eNOS activity and content of nitric oxide, thus lead to the improvement of endothelial dysfunction in mesenteric artery of Hyperhomocysteinemia rats.