Ascorbic Acid Protects against Hypertension through Downregulation of ACE1 Gene Expression Mediated by Histone Deacetylation in Prenatal Inflammation-Induced Offspring

Abstract

Hypertension is a major risk factor for cardiovascular and cerebrovascular disease. Prenatal exposure to lipopolysaccharide (LPS) leads to hypertension in a rat offspring. However, the mechanism is still unclear. This study unraveled epigenetic mechanism for this and explored the protective effects of ascorbic acid against hypertension on prenatal inflammation-induced offspring. Prenatal LPS exposure resulted in an increase of intrarenal oxidative stress and enhanced angiotensin-converting enzyme 1 (ACE1) gene expression at the mRNA and protein levels in 6- and 12-week-old offspring, correlating with the augmentation of histone H3 acetylation (H3AC) on the ACE1 promoter. However, the prenatal ascorbic acid treatment decreased the LPS-induced expression of ACE1, protected against intrarenal oxidative stress, and reversed the altered histone modification on the ACE1 promoter, showing the protective effect in offspring of prenatal LPS stimulation. Our study demonstrates that ascorbic acid is able to prevent hypertension in offspring from prenatal inflammation exposure. Thus, ascorbic acid can be a new approach towards the prevention of fetal programming hypertension.

Figure 1. Effects of a prenatal LPS exposure and the PDTC or the AA treatment on body weight, blood pressure, and urinary protein in offspring.

The body weight (a) of offspring was measured once a week from 4 to 12 weeks old. (n = 8 in each group). Systolic blood pressure (b, SBP) was measured by a tail-cuff method in 6, 8, 10 and 12 weeks old offspring. (n = 8 in each group). Urinary protein (c) was measured in 6 and 12 weeks old offspring. (n = 5 in each group). Data are expressed as mean ± SD. Different letters means significant differences (P < 0.05).

Figure 2. Effects of a prenatal LPS exposure and the PDTC or the AA treatment on renal cortex RAS mRNA and protein expression in offspring.

Renal cortex ACE1 (a), AT1a (b) and AT1b (c) mRNA expression were determined by real-time PCR in 6 and 12 weeks old offspring, GAPDH was taken as internal control. Renal cortex ACE1 and AT1 protein expression were assessed by immunoblotting in 6 (d) and 12 (e) weeks old offspring, GAPDH was taken as internal control. Data are expressed as mean ± SD. (n = 6 in each group). Different letters means significant differences (P < 0.05).

Figure 3. Effects of a prenatal LPS exposure and the PDTC or the AA treatment on oxidative stress in offspring.

Serum MDA (a) Serum SOD (b) Serum GSH (c) renal MDA (d) renal SOD (e) and renal GSH (f) were determined in 6 and 12 weeks old offspring. Data are expressed as mean ± SD. (n = 6 in each group). Different letters means significant differences (P < 0.05).

Figure 4. Effects of a prenatal LPS exposure and the PDTC or the AA treatment on renal cortex ACE1 DNA methylation in offspring.

(a) The positions of CpG island analysis, and ChIP analysis are shown schematically in which the numbers indicate the nucleotide location starting from transcription start site (TSS; +1). (b) is the selected amplicon of methylation analysis and the 42 CpG units. Unit 5*, 7*, 8*, 9*, 14* and 42* cannot be determined because of sequence problem. (c,d) Renal cortex ACE1 CpG island DNA methylation were determined by a MassARRAY Compact MALDI-TOF method in 6 and 12 weeks old offspring. Data are expressed as mean ± SD. (n = 6 in each group). ChIP; Chromatin immunoprecipitation. Different letters means significant differences (P < 0.05).

Figure 5. Effects of a prenatal LPS exposure and the PDTC or the AA treatment on renal cortex ACE1 histone acetylation in offspring.

(a,b) Renal cortex ACE1 histone H3 acetylation was assessed by chromatin immunoprecipitation in 6 and 12 weeks old offspring. (c,d) Renal cortex ACE1 HDAC1 was assessed by chromatin immunoprecipitation in 6 and 12 weeks old offspring. Data are expressed as mean ± SD. (n = 6 in each group). Different letters means significant differences (P < 0.05).