Maternal N-Acetylcysteine Therapy Prevents Hypertension in Spontaneously Hypertensive Rat Offspring: Implications of Hydrogen Sulfide-Generating Pathway and Gut Microbiota

Abstract

Hypertension can come from early life. N-acetylcysteine (NAC), a hydrogen sulfide (H₂S) precursor as well as an antioxidant, has antihypertensive effect. We investigated whether maternal NAC therapy can protect spontaneously hypertensive rats (SHR) male offspring against hypertension. The pregnant rats were assigned to four groups: SHRs without treatment; Wistar Kyoto (WKY) without treatment; SHR+NAC, SHRs received 1% NAC in drinking water throughout pregnancy and lactation; and, WKY+NAC, WKY rats received 1% NAC in drinking water during pregnancy and lactation. Male offspring (n = 8/group) were killed at 12 weeks of age. Maternal NAC therapy prevented the rise in systolic blood pressure (BP) in male SHR offspring at 12 weeks of age. Renal cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulphurtransferase (3MST) protein levels and H₂S-releasing activity were increased in the SHR+NAC offspring. Maternal NAC therapy increased fecal H₂S and thiosulfate levels in the SHR+NAC group. Additionally, maternal NAC therapy differentially shaped gut microbiota and caused a distinct enterotype in each group. The protective effect of maternal NAC therapy against hypertension in SHR offspring is related to increased phylum Actinobacteria and genera Bifidobacterium and Allobaculum, but decreased phylum Verrucomicrobia, genera Turicibacter, and Akkermansia. Several microbes were identified as microbial markers, including genera Bifidobacterium, Allobaculum, Holdemania, and Turicibacter. Our results indicated that antioxidant therapy by NAC in pregnant SHRs can prevent the developmental programming of hypertension in male adult offspring. Our findings highlight the interrelationships among H₂S-generating pathway in the kidneys and gut, gut microbiota, and hypertension. The implications of maternal NAC therapy elicited long-term protective effects on hypertension in later life that still await further clinical translation.

Keywords: N-acetylcysteine; developmental origins of health and disease (DOHaD); gut microbiota; hydrogen sulfide; hypertension; oxidative stress; renin-angiotensin system; thiosulfate.

Figure 1

Effects of maternal N-acetylcysteine (NAC) therapy on systolic blood pressure. The data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR. †In many instances the error bars of the figure are contained within the symbols.

Figure 2

(A) Effect of maternal N-acetylcysteine (NAC) therapy on mRNA expression of H₂S-generating enzymes in male offspring kidneys at 12 weeks of age. (B) Representative Western blots show cystathionine γ-lyase (CSE, ~45 kDa), cystathionine β-synthase (CBS, ~61 kDa), and 3-mercaptopyruvate sulfurtransferase (3MST, ~52 kDa) bands in male offspring kidneys at 12 weeks of age. Relative abundance of renal cortical (C) CSE, (D) CBS, and (E) 3MST were quantified. Data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR.

Figure 3

Effects of maternal N-acetylcysteine (NAC) therapy on (A) renal H₂S-releasing activity, (B). Plasma H₂S level, (C) fecal H₂S level, (D) plasma thiosulfate level, and (E) fecal thiosulfate level. Data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR; ^§ p < 0.05 vs. WKY+NAC.

Figure 4

Effects of maternal N-acetylcysteine (NAC) therapy on the renin-angiotensin system. The data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR.

Figure 5

Effects of maternal N-acetylcysteine (NAC) therapy on the gut microbiome in male offspring at 12 weeks of age. (A) Variation in fecal bacterial α-diversity analyzed by the Shannon’s diversity indexes. (B) β-diversity changes in gut microbiota across groups by the Principal Coordinate Analysis (PCoA). (C) Relative abundance of the five major phyla of the gut microbiota among the four groups. In descending order, they are: Firmicutes (orange), Bacteroidetes (blue), Actinobacteria (green), Verrucomicrobia (purple), and Proteobacteria (light blue). (D) The Firmicutes to Bacteroidetes (F/B) ratio. Relative abundance of the phyla (E) Actinobacteria and (F) Verrucomicrobia. Abundance = the number of bacterial operational taxonomic unit (OTU) sequences detected in each sample. Data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR; ^† p < 0.05 vs. WKY+NAC.

Figure 6

Effects of maternal N-acetylcysteine (NAC) therapy on the gut microbiome in male offspring at 12 weeks of age. Relative abundances of the genera (A) Bifidobacterium, (B) Lactobacillus, (C) Turicibacter, (D) Akkermansia, (E) Holdemania, and (F) Allobaculum. Abundance = the number of bacterial operational taxonomic unit (OTU) sequences detected in each sample. Data are shown as means ± SEM; N = 8/group. * p < 0.05 vs. WKY; ^# p < 0.05 vs. SHR; ^† p < 0.05 vs. WKY+NAC.

Figure 7

Effect of maternal N-acetylcysteine (NAC) therapy on the gut microbiome in male offspring at 12 weeks of age. Linear discriminant analysis effect size (LEfSe) was applied to identify enriched bacterial species. The threshold of the linear discriminant was set to 2. Different taxonomic levels of bacteria are given reaching from phylum (p) and class (c) via order (o) and family (f) down to genus (g) and species (s). Accordingly, the phylum Actinobacteria is presented as p_Actinobacteria (i.e., level_name). Most enriched and depleted bacterial taxa in the (A) SHR (red) versus SHR+NAC group (green) and (B) WKY (red) versus WKY+NAC group (green) are shown.