Elucidation of Anti-Hypertensive Mechanism by a Novel Lactobacillus rhamnosus AC1 Fermented Soymilk in the Deoxycorticosterone Acetate-Salt Hypertensive Rats

Abstract

Dietary intake of fermented soymilk is associated with hypotensive effects, but the mechanisms involved have not been fully elucidated. We investigated the anti-hypertensive effects of soymilk fermented by L. rhamnosus AC1 on DOCA-salt hypertension from the point of view of oxidative stress, inflammatory response and alteration of the gut microbiome. The antioxidant assays in vitro indicated the ethanol extract (EE) of L. rhamnosus AC1 fermented soymilk showed better antioxidative effects than the water extract (WE). Those extracts displayed a hypotensive effect using a tail-cuff approach to measuring blood pressure and improved nitric oxide (NO), angiotensin II (Ang II), tumor necrosis factor-α (TNF-α) and interleukin factor-6 (IL-6) on DOCA-salt hypertensive rats. Furthermore, cardiac and renal fibrosis were attenuated by those extracts. The gut microbiota analysis revealed that they significantly reduced the abundance of phylum Proteobacteria, its family Enterobacteriaceae and genus Escherichia-Shigella. Moreover, metabolomic profiling revealed several potential gut microbiota-related metabolites which appeared to involve in the development and recovery of hypertension. In conclusion, fermented soymilk is a promising nutritional intervention strategy to improve hypertension via reducing inflammation and reverting dysbiotic microbiota.

Keywords: L. rhamnosus AC1; fermented soymilk; gut microbiota; hypertension.

Figure A1

Effects of administration of water extract (WE) and ethanol extract (EE) of L. rhamnosus AC1-fermented soymilk on the body weight (A), food intake (B), and water intake (C) in DOCA-salt hypertensive rats. Data are presented as means ± SEM (n = 5).

Figure A2

Effect of water extract and ethanol extract of L. rhamnosus-fermented soymilk on Alpha diversity of gut microbiota in DOCA-salt hypertensive rats at week 14.

Figure A3

Effect of water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on the microbial composition in the gut flora of DOCA-salt hypertensive rats at week 0. Percentage of community abundance on the phylum (A) and family levels (B).

Figure A4

Phylogenetic distribution of gut microbiota of rats in SHAM, DOCA, WE and EE at week 0. Indicator bacteria with LDA scores of 2 or greater in bacterial communities.

Figure A5

Effect of water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on predicted functions of gut microbiota in DOCA-salt hypertensive rats at week 14. Relative abundance of bacteria involved in tryptophan degradation (A), tryptophan biosynthesis (B), glutamate biosynthesis (C), arginine degradation (D), phenylethylamine degradation (E), polymyxin resistance (F) and allantoin degradation (G) are shown. Data are presented as means ± SEM (n = 4). Bar values bearing different letters were significantly different by Duncan’s multiple range test (p < 0.05).

Figure 1

Antioxidant abilities of water extract (WE) and ethanol extract (EE) of L. rhamnosus fermented soymilk in vitro. The scavenging effect of DPPH radicals (A); The ability for chelating Fe²⁺ (B); The reducing activity (C). Data presented as means ± SEM (n = 2). Bar values with different letters were significantly different by Duncan’s multiple range test (p < 0.05). ASA: Ascorbic acid; EDTA: Ethylenediaminetetraacetic acid.

Figure 2

Effects of administration of water extract (WE) and ethanol extract (EE) of L. rhamnosus AC1-fermented soymilk on DOCA-salt hypertensive rat. Timeline of animal experiments (A). Results of systolic blood pressure (B), the ratio of organ weight to body weight in the case of the kidney (C) and heart (D). Data are presented as means ± SEM (n = 5). Bar values bearing different letters were significantly different by Duncan’s multiple range test (p < 0.05). *, differences between the selected values were statistically different by Duncan’s multiple range test (p < 0.05).

Figure 3

Effects of the water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on tissue fibrosis in DOCA-salt hypertensive rats. Representative figure and quantification of renal perivascular fibrosis (A,B); Representative figure and quantification of heart perivascular fibrosis (C,D); Representative figure and quantification of aortic fibrosis (E,F). Data are presented as means ± SEM (n = 6). Bar values bearing different letters were significantly different by Duncan’s multiple range test (p < 0.05).

Figure 4

Effects of the water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on the NO content (A), levels of angiotensin II (B), TNF-α (C) and IL-6 (D) in the DOCA-salt hypertensive rats. Data are presented as means ± SEM (n = 6). Bar values bearing different letters were significantly different by Duncan’s multiple range test (p < 0.05).

Figure 5

Effect of water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on the microbial composition in the gut flora of DOCA-salt hypertensive rats at week 14. Percentage of community abundance on the phylum (A) and family levels (B).

Figure 6

Effect of water extract and ethanol extract of L. rhamnosus AC1-fermented soymilk on the microbial composition belonging to phylum Proteobacteria at week 14. Percentage of community abundance in SHAM (A), DOCA (B), WE (C) and EE (D).

Figure 7

Phylogenetic distribution of gut microbiota of rats in SHAM, DOCA, WE and EE at week 14. Indicator bacteria with LDA scores of 2 or greater in bacterial communities.

Figure 8

L. rhamnosus AC1-fermented soymilk extract alters potential gut microbiota-associated metabolites levels in DOCA-salt-induced hypertension. (A) Partial least squares-discrimination analysis (PLS-DA) score plots for discriminating the serum metabolome from Sham, DOCA, CAP, WE-treated and EE-treated groups (Upper: negative mode; Lower: positive mode). (B) Heatmap of the differential metabolites that were altered by DOCA and then regulated by WE. (C) Heatmap analysis of the spearman correlation of differential metabolites and gut microbiota after WE administration. Red represents positive correlation, and blue indicates negative correlation. (D) Heatmap of the differential metabolites that were altered by DOCA and then regulated by EE. (E) Heatmap analysis of the spearman correlation of differential metabolites and gut microbiota after EE administration. Red represents positive correlation, and blue indicates negative correlation. n = 4–6. * p < 0.05, ** p < 0.01. WE: water extract. EE: ethanol extract.

Figure 9

Proposed mechanism of L. rhamnosus AC1-fermented soymilk on DOCA-salt hypertensive rat. Four pathways are presented in this study, which are gut microbiota (yellow), oxidative stress (orange), RAAS (gray) and inflammation (blue). All the parameters are shown in the specific pathway. Black solid arrows represent a direct link between two parameters and black dotted arrows donate an indirect link. Red arrows mean the alteration of several biochemical parameters we evaluate. Green arrows indicate the active constituent in our fermented soymilk.