hSIH could be transferred by fecal microbiota transplantation, indicating the pivotal roles of intestinal flora in hSIH development. High-salt diet reduced the levels of B fragilis and arachidonic acid in the intestine, which increased intestinal-derived corticosterone production and corticosterone levels in serum and intestine, thereby promoting BP elevation. This study revealed a novel mechanism different from inflammation/immunity by which intestinal flora regulated BP, namely intestinal flora could modulate BP by affecting steroid hormone levels. These findings enriched the understanding of the function of intestinal flora and its effects on hypertension.

The abovementioned issues were investigated using various techniques including 16S rRNA gene sequencing, untargeted metabolomics, selective bacterial culture, and fecal microbiota transplantation. We found that high-salt diet induced hypertension in Wistar rats. The fecal microbiota of healthy rats could dramatically lower blood pressure (BP) of hypertensive rats, whereas the fecal microbiota of hSIH rats had opposite effects. The composition, metabolism, and interrelationship of intestinal flora in hSIH rats were considerably reshaped, including the increased corticosterone level and reduced Bacteroides and arachidonic acid levels, which tightly correlated with BP. The serum corticosterone level was also significantly increased in rats with hSIH. Furthermore, the above abnormalities were confirmed in patients with hypertension. The intestinal Bacteroides fragilis could inhibit the production of intestinal-derived corticosterone induced by high-salt diet through its metabolite arachidonic acid.

High-salt diet is one of the most important risk factors for hypertension. Intestinal flora has been reported to be associated with high salt–induced hypertension (hSIH). However, the detailed roles of intestinal flora in hSIH pathogenesis have not yet been fully elucidated.

Introduction

Editorial, see p 854

In This Issue, see p 807

Meet the First Author, see p 808

Hypertension is a common chronic disease and one of the most important risk factors of severe cardio-cerebrovascular events such as stroke and heart failure.1,2 Hypertension causes at least 45% and 51% of heart diseases and stroke deaths, respectively, worldwide each year. Currently, 45.6% of Americans experience hypertension according to the 2017 American College of Cardiology/American Heart Association Hypertension Guideline (systolic blood pressure [SBP] ≥130 mm Hg or diastolic blood pressure [DBP] ≥80 mm Hg).1 In China, nearly half of the adults (44.7%) experience hypertension according to the higher criteria (SBP ≥140 mm Hg or DBP ≥90 mm Hg).2 Therefore, hypertension has become one of the most serious public health problems in the world.3

Dietary habits are closely associated with the hypertension morbidity.4 Numerous studies have shown that high-salt diet (HSD) is an important independent risk factor for hypertension.5 The high salt–induced hypertension (hSIH) accounts for many patients with hypertension, and its pathogenesis currently is believed to be mainly associated with kidney sodium retention, elevated blood volume, and increased peripheral vascular resistance.6 However, the specific mechanisms for these pathological changes have not yet been fully elucidated. The reported mechanisms include aberrant activation of renin-angiotensin-aldosterone system (RAAS), sympathetic nervous and the kallikrein-kinin system,7 and vascular endothelial dysfunction.8 Among these mechanisms, the RAAS and MR (mineralocorticoid receptor) dysfunctions are critical for hypertension development, especially hSIH.9

The glucocorticoids (ie, cortisol in humans and corticosterone in rodents) play important roles in a variety of physiological and pathological processes. Adrenal cortex is the main site for corticosterone and cortisol synthesis and release. However, many key enzymes for sterol synthesis are also expressed in intestinal epithelium, thymus, brain, and other tissues able to synthesize bioactive glucocorticoids.10,11 The corticosterone produced by intestine is very low under physiological conditions but is significantly increased in an inflammatory environment.11 In intestinal bacteria-depleted mice that have undergone adrenalectomy, the intestinal-derived corticosterone can even restore plasma corticosterone level to normal.12 Corticosterone, cortisol, and aldosterone are the major regulators of sodium and potassium homeostasis, hemodynamics, and blood pressure (BP) in vivo. All these hormones can activate MR, resulting in sodium retention and BP elevation.13,14 Therefore, the abnormally elevated levels of these hormones are closely related to hypertension development.15,16

There are about 1013 to 1014 bacteria in human intestine (ie, intestinal flora) that play pivotal roles in the development of various diseases, such as atherosclerosis, diabetes mellitus, malignant tumors, and autoimmune diseases.17 Recent studies have shown significant alteration of intestinal flora in patients and animals with hypertension, including the decrease in short-chain fatty acid–producing bacteria and the increase in proinflammatory bacteria and opportunistic pathogens.18,19 The BP of healthy animals can be markedly elevated by transplanting gut microbiota of patients or animals with hypertension.20,21 Some probiotics or prebiotics can also lower BP of patients or animals with hypertension.19,22 However, there are still few studies on the mechanisms of the relationship between intestinal flora and hypertension. Recently, Wilck et al23 have found that HSD significantly reduces intestinal Lactobacillus murinus in hypertensive FVB/N mice, while L murinus can metabolize tryptophan in the diet to produce indole, thereby inhibiting T lymphocyte differentiation into Th17 cells, alleviating inflammation and lowering BP. Nevertheless, the pathogenesis of hypertension is very complicated. In addition to inflammation, there should be other mechanisms by which intestinal flora regulates BP, such as gluco/mineralocorticoid. However, these issues have not yet been elucidated so far.

In this study, the relationship and mechanism between intestinal flora and hSIH were studied in Wistar rats using 16S rRNA gene sequencing, metabolomics, and functional research methods. We found that hSIH could be transferred by fecal microbiota transplantation, indicating the pivotal roles of intestinal flora in hSIH development. Moreover, HSD reduced the levels of Bacteroides fragilis and its metabolite arachidonic acid (AA) in the intestine, which increased intestinal-derived corticosterone production and serum and intestine corticosterone levels, thereby promoting BP elevation. Furthermore, the above abnormalities were also confirmed in patients with hypertension. Bfragilis and its metabolite AA could prevent the harmful effects of HSD. Our results revealed a novel mechanism through which intestinal flora could regulate BP by affecting steroid hormone levels.

Methods

The detailed descriptions of the Materials and Methods including 16S rRNA gene sequencing, untargeted metabolomics, selective bacterial culture, and fecal microbiota transplantation are available in the Data Supplement. Please see the Major Resources Table in the Data Supplement. All data supporting the findings of this study are available in the Data Supplement and online data repository.

Results

Abnormal Intestinal Flora Contributed to hSIH Development

In this study, Wistar male rats were fed with HSD (HSD group) and normal diet (control group). During feeding period, the body weights of rats in the 2 groups were not significantly different (Figure IA in the Data Supplement). The BP of HSD group rats gradually increased upon feeding, and reached the standard of hypertension (SBP/DBP, 167.9±13/125.4±17 mm Hg) at the fourth week, which was significantly higher than those in control group (SBP/DBP, 113.6±11/85.2±8 mm Hg) at the same time (Figure 1A and 1B). To identify the causal relationship between intestinal flora and BP elevation in HSD group rats, some HSD and control group rats were treated with quadruple antibiotic regimen to remove their original intestinal flora and then transplanted with different intestinal flora. The results showed that the antibiotic cocktail dramatically reduced the amplicon sequence variant richness of rat intestinal flora (82% and 88% decrease in control and HSD groups, respectively; Figure IB in the Data Supplement). The elevated SBP and DBP of HSD rats significantly decreased after antibiotic treatment and further decreased to near-normal levels after transplantation with intestinal flora from normotensive rats. In contrast, SBP and DBP elevated after antibiotics treatment in control group rats, and both SBP and DBP further increased after transplantation with intestinal flora from hypertensive rats (tail cuff, Figure 1C and 1D; telemetry, Figure 1E and 1F). These results implied that the abnormal intestinal flora play a key role in hSIH development and are one of the causes of hSIH, rather than being the result and accompanying phenomena of hypertension.

Figure 1. High-salt diet (HSD) induced hypertension in rats, which was transferable by fecal transplantation.A and B, The changes of systolic blood pressure (SBP; A) and diastolic blood pressure (DBP; B) of Wistar rats in HSD (n=20) and normal diet (control; n=23) groups measured by tail cuff. C–F, Changes of SBP and DBP measured by tail cuff (C and D; n=16) or telemetry (E and F; n=7) in rats with different treatments as described in the Materials and Methods. HSDFMToControl: intestinal flora of HSD group rats transplanted to control group rats. ControlFMToHSD: intestinal flora of control group rats transplanted to HSD group rats. Data presented as mean±SEM (A and B) and median (minimum to maximum; C–F). t test adjusted by Benjamini-Hochberg procedure to control false discovery rate (FDR) <5% was used for statistical tests in A and B. At FDR <0.05, *P<0.05, #P<0.001. One-way ANOVA with Tukey post hoc test was used in C–F. ^Adjusted P<0.05; ^^Adjusted P<0.01; ^^^Adjusted P<0.001. FMT indicates fecal microbiota transplantation.

HSD Induced Gut Dysbiosis Including the Reduction of Beneficial Bacteroides

To reveal HSD influences on intestinal flora, 16S rRNA gene sequencing was performed using feces from control and HSD group rats. The average sequencing depth was about 30 000 reads/sample without significant difference between control and HSD groups (Figure IC in the Data Supplement). This sequencing depth was saturated to detect intestinal bacteria (Figure 2A). Shannon index, Simpson index, and Pielou evenness were not significantly different between the 2 groups (Figure ID through IF in the Data Supplement), which is consistent with previously reported results.23 However, the principal component analysis and principal coordinate analysis based on amplicon sequence variants showed significant separation of the intestinal flora of rats in the 2 groups (Figure 2B). Compared with control group rats, the Firmicutes/Bacteroidetes ratio in HSD group rats was markedly increased, indicating a gut dysbiosis (Figure 2C). At phylum level, the proportion of Spirochaetes significantly increased, while Verrucomicrobia remarkably decreased in the intestine of high-salt hypertensive rats (Figure 2D). At a more detailed taxonomic level, HSD significantly changed the abundances of 31 intestinal genera or species, and 22 of them were remarkably reduced. Of the 22 bacteria, 12, 8, and 2 bacteria belonged to Bacteroidetes, Firmicutes, and Proteobacteria, respectively. The abundances of the remaining 9 bacteria, which belonged to Actinobacteria, Firmicutes, and Proteobacteria, were markedly increased (Figure 3A).

Figure 2. High-salt diet (HSD) significantly changed intestinal flora composition.A, Rarefaction curves of amplicon sequence variant (ASV) numbers detected by 200 random samplings in control (n=23) and HSD (n=20) groups. The slope of the curve for each sample is close to zero when the sequencing depth is sufficient to cover most of the intestinal flora. B, Analysis of β-diversity of intestinal flora in rats. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed to calculate the distances between fecal samples from the rats of control and HSD groups. Each point represents a sample. A clear separation is observed between the samples of control (n=23) and HSD (n=20) groups. C, The ratio of Firmicutes to Bacteroidetes (F/B) in control (n=23) and HSD (n=20) groups. Data were presented as median (minimum to maximum). Wilcoxon rank-sum test was used for statistical tests. $$$P<0.001. D, The pie chart of the 5 most abundant bacterial phyla in control (n=23) and HSD (n=20) groups. PC indicates principal component.

Figure 3. The composition, correlation network, and function of intestinal flora and their correlations with blood pressure (BP).A, Heat map of the relative abundances of the 31 most abundant intestinal bacteria, which significantly changed in high-salt diet (HSD; n=20) compared with those in control (n=23) group rats (false discovery rate [FDR]-adjusted P<0.05, FDR<5%). The color bar indicates Z score that represents the relative abundance. Z score <0 and >0 means the relative abundance is lower and higher than the mean. Row names thickened represent bacteria genus, otherwise represent bacteria species. B, Correlation network of gut microbiota in control (n=23) and HSD (n=20) groups. The significant strong Spearman correlations (|r|>0.6 and FDR-adjusted P<0.05, FDR<5%) among the intestinal bacterial genera are presented in the networks. The red and blue edges show positive and negative correlations, respectively. The spot colors represent different bacterial phyla. The thickness of the lines denotes correlation strength. The dotted rectangles indicate that the bacteria are emphasized in the Results section. C, Heat map of Spearman correlations between BP and the 31 most abundant bacterial genera, whose abundances changed significantly in HSD group rats. The color bar with numbers indicates the correlation coefficients. Row names thickened represent bacteria genus, otherwise represent bacteria species. n=23 in control and n=20 in HSD group. D, The relative abundances of the 6 most abundant bacterial genera that significantly correlated with BP in control (n=23) and HSD (n=20) groups. E, Dynamic changes of the relative abundances of Bacteroides in the HSD group rat intestine (n=6) during the feeding period. F, The significant differences in metagenomic functions of HSD (n=20) group rats compared with those of control (n=23) group rats. The legends on the right represent the predicted Kyoto Encyclopedia of Genes and Genomes pathways. This analysis is performed by PICRUSt software. Data were presented as mean±SEM (E) and median (minimum to maximum; D and F). t test (B and C) and Wilcoxon rank-sum test (A, D, and F) adjusted by Benjamini-Hochberg procedure to control FDR <5% were used for statistical tests. At FDR <5%, *FDR-adjusted P<0.05; +FDR-adjusted P<0.01; #FDR-adjusted P<0.001. One-way ANOVA test with Dunnett post hoc test was used in E. ^^Adjusted P<0.01 (zero week vs other groups). DBP indicates diastolic blood pressure; and SBP, systolic blood pressure.

Spearman correlation analysis showed that HSD significantly increased the positive correlations among many intestinal bacteria, especially Proteobacteria and Firmicutes, indicating that HSD might strengthen the symbiotic relationship between them. In high-salt hypertensive rats, the significant positive correlation between Bacteroides and Candidatus Arthromitus was lost, whereas it was seen between Bacteroides and Butyricmonas, Parabacteroides, Odoribacter, Sutterella, and Alistipes. Moreover, Clostridium and Butyricicoccus had no significant correlations with any other bacteria in the control group, but they significantly correlated with 02D06, Dehalobacterium, Oscillospira, and Butyricmonas in the HSD group (Figure 3B). The changes in correlations between these intestinal bacteria suggested that HSD could severely interfere with the symbiotic relationships among gut flora, which might be closely related to hSIH development.

To reveal the pathological significance of the markedly changed intestinal bacteria induced by HSD, the correlations between their abundances and BP were analyzed. It was observed that most of these intestinal bacteria strongly negatively correlated with BP, and only 7 bacteria significantly, positively correlated with SBP or DBP (Figure 3C). Among them, Lactobacillus and Bacteroides were the 2 most abundant bacteria, suggesting that they may play more important roles in hSIH pathogenesis (Figure 3D). Moreover, the intestinal Bacteroides abundance decreased dramatically in the first week of high-salt feeding and remained at a low level, while the BP increased distinctly in the second week of high-salt feeding and then increased rapidly afterward (Figures 1A, 1B, and 3E). This indicated that the significant change of Bacteroides occurs before that of BP. Thus, change in Bacteroides abundance was not a result of hSIH but a cause of hypertension, which was consistent with the results of gut flora transplantation. Additionally, phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis showed that many metabolic pathways and functions of intestinal flora including bacterial invasion of epithelial cells, steroid hormone biosynthesis, and glycerolipid metabolism were significantly enriched in the HSD group. The steroid hormone biosynthesis enrichment implied that the intestinal synthesis of steroids was increased in high-salt hypertensive rats (Figure 3F).

HSD Significantly Changed the Metabolic Profiles of Intestinal Flora

As the microbe-host bridge, many metabolites of the intestinal flora could influence host physiology in the intestine or by entering the bloodstream. Therefore, the fecal samples from the control and HSD group rats were analyzed to explore the intestinal metabolic profiling using high-performance liquid chromatography–mass spectrometry. A total of 1708 and 1707 peak features were identified in positive (ES+) and negative (ES−) ion modes, respectively. These peaks were clustered using orthogonal partial least squares discriminant analysis, and their validation plots were obtained using 200 permutation tests. Results showed that the metabolic data clusters of control and HSD groups were separated from each other (Figure 4A and 4B). There were 955 and 641 peak features with significant changes in peak intensity in ES+ (505 and 450 upregulated and downregulated peaks, respectively) and ES− (333 and 308 upregulated and downregulated peaks, respectively) models, respectively (Figure 4C). These results indicated that HSD significantly altered the intestinal flora metabolic profiles. The significantly changed peak features were then analyzed using tandem mass spectrometry, and many metabolites were identified by the combination of precise molecular weight and structural information of compound structure database. These metabolites included amino acid derivatives, bile acids, lipids, fatty acids, glucocorticoids, and indole derivatives; many of them were produced or regulated directly by intestinal bacteria (Table I in the Data Supplement).

Figure 4. The significant changes in metabolic profiles of gut microbiota in high salt–induced hypertensive (hSIH) rats.A, The plots of OPLS-DA scores of all peak features in positive (ES+) and negative (ES−) ion modes from the untargeted metabolomics analysis of stool samples of the rats in high-salt diet (HSD; n=10) and control (n=10) groups. B, Validation of the OPLS-DA model via permutation test (times, 200). R2 measures the goodness of fit and Q2 measures the predictive ability of the model. The criterion for model validity is that the regression line of the Q2 points (blue dotted line) intersects the vertical solid line (left) below zero. C, Cloud plots of the peak features of intestinal metabolites that significantly changed in HSD group in positive ion mode (ES+; top) and negative ion mode (ES−; bottom). Yellow and blue circles indicate the significantly increased and decreased metabolites, respectively (fold change, >1.5; P<0.05), in HSD group compared with those of control group. The color tone indicates P: a dark color indicates a small P. The circle radius indicates the fold change of corresponding peak features. n=10. D, Heat map of Spearman correlations between the 31 most abundant bacteria whose abundances significantly changed in HSD rats and the 50 most abundant metabolites with important functions and significant differences. The color bar with numbers indicates the correlation coefficients. Row names thickened represent bacteria genus, otherwise represent bacteria species. n=10. E, Relationship between intestinal corticosterone level and the 4 most abundant bacterial genera whose abundances significantly changed in HSD rats, as well as the blood pressure. n=10. Wilcoxon rank-sum test (C) and t test adjusted by Benjamini-Hochberg procedure to control false discovery rate (FDR) <5% (D and E) were used for statistical tests. At FDR <5%, *FDR-adjusted P<0.05; +FDR-adjusted P<0.01; #FDR-adjusted P<0.001. r: the spearman correlation coefficient; P: statistical significance. Adj indicates adjusted; DBP, diastolic blood pressure; and SBP, systolic blood pressure.

To explore the functional significance of these metabolic perturbations in the intestinal flora of high-salt hypertensive rats, the 50 annotated metabolites having the most significant statistical differences were selected, and the Spearman correlation coefficients between them and BP/differential bacteria were calculated. Significant correlations were observed between the changed intestinal flora, BP, and many altered metabolites (|r|>0.5, false discovery rate-adjusted P<0.05; Figure 4D). For example, dihydrotachysterol and acetylcholine significantly negatively correlated with SBP and DBP, whereas significantly positively and negatively correlated with Odoribacter and Flexispira, respectively. Prostaglandin E2 and leukotriene B4 strongly positively correlated with BP and had significant negative and positive correlations with Distasonis and Psychrobacter, respectively. It was worth noting that intestinal corticosterone concentration increased 4.4× in the HSD group. Corticosterone significantly negatively correlated with some highly abundant bacteria such as Bacteroides, Lactobacillus, and Alistipes. Moreover, it also significantly positively correlated with SBP and DBP (Figure 4E). Considering the pivotal roles of corticosterone in BP regulation, it is reasonable to assume that hSIH may not be only associated with the immunomodulatory effects of intestinal flora but also the increased corticosterone level caused by some reduced intestinal bacteria such as Bacteroides.

B fragilis Inhibited the Production of Intestinal-Derived Corticosterone Induced by HSD

The above results indicated that the highly abundant Bacteroides was the intestinal bacterial genus most associated with BP. To clarify the mechanism behind this association, a fecal Bacteroides strain was selectively cultured. The isolated bacterial strain was identified to be Bfragilis YCH46 by Sanger sequencing. The quantitative real-time polymerase chain reaction results further confirmed the significant decrease of Bfragilis YCH46 in the feces of HSD rats and patients with hypertension (Figure 5A). In addition to the significant increase in intestinal corticosterone level in HSD rats as revealed by metabolomics, ELISA results also demonstrated the significant increase in serum and intestinal corticosterone levels in patients and rats with hypertension (Figure 5B and 5C). Corticosterone can be transformed to aldosterone, which plays critical roles in BP regulation. However, compared with those in the rats with normal diet, the serum aldosterone level was significantly decreased in HSD rats (Figure S2A). The mRNA levels of Mr and the key enzymes in corticosterone synthesis, including Cyp11a1, Cyp11b1, and Hsd11b1, were markedly upregulated, whereas Hsd11b2 (hydroxysteroid 11-beta dehydrogenase 2), which could inactivate corticosterone, was significantly downregulated in the colon of hSIH rats (Figure 5D). These results suggested that HSD increases corticosterone synthesis and inhibits its degradation in rat intestine, leading to MR overactivation.

Figure 5. Bacteroides fragilis was significantly reduced in hSIH rats and could inhibit the intestinal corticosterone synthesis.A, The changes in abundance of B fragilis YCH46 in the intestinal flora of patients (n=8) and rats (n=8) with hypertension (HTN) and their corresponding healthy control groups (n=4) determined by quantitative real-time polymerase chain reaction. B, The serum corticosterone concentrations in patients (n=11) and rats (n=11) with HTN and their corresponding healthy control groups (n=11) determined by ELISA. C, The fecal corticosterone concentrations in patients with HTN (n=9) and healthy individuals (control; n=9) determined by ELISA. D, The mRNA expression levels of Mr and the key enzymes (Cyp11a1, Cyp11b1, Hsd11b1, and Hsd11b2) of corticosterone synthesis and activity in colon tissues of high-salt diet (HSD; n=6) and control (n=4) rats. E, Effects of B fragilis YCH46 on the level of corticosterone produced by the colon tissues of C57BL/6 mice cultured in vitro. SnCtrl+M2GSC and SnHSD+M2GSC: supernatants of normal and HSD mice colon fragments cultured in M2GSC medium. SnCtrl+B.f.S and SnHSD+B.f.S: supernatants of normal and HSD mice colon segments cultured in B fragilis YCH46-cultured M2GSC medium supernatant. n=5. F–J, The influences of B fragilis YCH46 on the mRNA expression levels of Mr (F), Cyp11a1 (G), Cyp11b1 (H), Hsd11b1 (I), and Hsd11b2 (J) in C57BL/6 mice colon tissues cultured in vitro. n=3. Ctrl+M2GSC and HSD+M2GSC: colon segments of mice fed normal and HSD cultured in M2GSC medium. Ctrl+B.f.S and HSD+B.f.S: colon segments of mice fed normal and HSD cultured in Bfragilis YCH46-cultured M2GSC medium supernatant. Data presented as median (minimum to maximum). Wilcoxon rank-sum test was used for statistical tests in A–C. $$P<0.01; $$$P<0.001. Wilcoxon rank-sum test adjusted by Benjamini-Hochberg procedure to control false discovery rate (FDR) <5% was used in D. At FDR <5%, +FDR-adjusted P<0.01. One-way ANOVA adjusted by Dunnett post hoc test was used for statistical tests in E–J. ^Adjusted P<0.05; ^^adjusted P<0.01; ^^^adjusted P<0.001 (SnHSD+M2GSC in E or HSD+M2GSC in F–J vs other groups). Ctrl indicates control.

The correlation analysis illustrated that Bacteroides abundance significantly negatively correlated with intestinal corticosterone level. Therefore, Bacteroides effects on corticosterone production in HSD-fed C57BL/6 mice colon tissues were examined. The results indicated that the changes in corticosterone level and the expressions of Mr and the foregoing enzymes of corticosterone synthesis in HSD mice were consistent with those in the HSD rats. Moreover, the culture supernatant of Bfragilis YCH46 could significantly reduce the corticosterone level and reverse the abnormal expressions of Mr, Cyp11a1, Cyp11b1, Hsd11b1, and Hsd11b2 in high salt–fed mice colon tissues (Figure 5E through 5J). Namely, Bfragilis effectively counteracted HSD effects on intestinal corticosterone metabolism and dramatically reduced intestinal corticosterone production level.

AA Produced by Bfragilis Inhibited Corticosterone Production in the Intestine

The aforementioned correlation analysis indicated that 3 intestinal metabolites, namely 4,7,10,13,16,19-docosahexaenoic acid, eicosapentaenoic acid, and AA, significantly negatively correlated with corticosterone level and BP but positively correlated with Bacteroides (Figure 6A). These results suggested that the 3 metabolites might be the key bridges between Bacteroides and its inhibitory effect on corticosterone synthesis in the intestine. The results further showed that AA significantly reduced the increased expressions of Mr, Cyp11a1, Cyp11b1, and Hsd11b1 in the intestinal segments of HSD-induced mice and dramatically upregulated Hsd11b2 expression, which was suppressed by HSD (Figure 6B through 6F). Meanwhile, AA could also significantly reduce the HSD-induced increased intestinal corticosterone production (Figure 6G). However, eicosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid had no significant effects on the abovementioned gene expression and intestinal corticosterone production (Figure IIB through IIF in the Data Supplement; Figure 6G). AA concentration was much higher in B fragilis YCH46 group than in M2GSC and other groups, indicating that AA was produced by Bfragilis (Figure 6H). Furthermore, AA was also significantly decreased in feces of patients with hypertension, which was consistent with the metabolomics results of rat feces (Figure 6I). All these results demonstrated that the intestinal Bfragilis could produce AA to inhibit the intestinal corticosterone synthesis and attenuate the HSD-induced intestinal corticosterone increase, thereby exerting an antihypertensive effect.

Figure 6. Arachidonic acid (AA) produced by Bacteroides fragilis inhibited the corticosterone synthesis in the intestine.A, Heat map of Spearman correlations between intestinal metabolites 4,7,10,13,16,19-docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and AA levels and amounts of Bacteroides, corticosterone, and blood pressure. The color bar with numbers indicates the correlation coefficients. n=10. B–F, The influence of AA on the mRNA expression levels of Mr (B), Cyp11a1 (C), Cyp11b1 (D), Hsd11b1 (E), and Hsd11b2 (F) in C57BL/6 mice colon tissues cultured in vitro. Ctrl+AA (Ctrl+ethanol [EtOH]): colon segments of normal diet–fed mice treated with 0.25 mmol/L AA (ethanol). High-salt diet (HSD)+AA (HSD+EtOH): colon segments of HSD-fed mice treated with 0.25 mmol/L AA (ethanol). n=4. G, Effects of DHA, EPA, and AA and their mixture on the levels of corticosterone produced by C57BL/6 mice colon tissues cultured in vitro. Culture supernatants of colon segments of mice fed with normal diet treated with 0.25 mmol/L ethanol (SnCtrl+EtOH). Culture supernatants of colon segments of HSD-fed mice treated with 0.25 mmol/L ethanol (SnHSD+EtOH), 0.25 mmol/L AA (HSD+AA), 0.25 mmol/L DHA (HSD+DHA), and 0.25 mmol/L EPA (HSD+EPA) and the mixture of the same amount of AA, DHA, and EPA (HSD+mix). n=4. H, The concentrations of AA in medium or culture supernatant assayed by ELISA. M2GSC: culture medium M2GSC; B.f.S: supernatant of M2GSC culture medium in which Bfragilis YCH46 has been cultured; DMEM+fetal bovine serum (FBS): DMEM supplied with 10% FBS for culturing the colon segments; culture supernatants of colon segments of mice fed with normal diet (SnCntrl) or HSD (SnHSD). n=4. I, The concentrations of fecal AA in patients with hypertension (HTN; n=8) and healthy individuals (control; n=8) determined by ELISA. Data presented as median (minimum to maximum). t test adjusted by Benjamini-Hochberg procedure to control false discovery rate (FDR) <5% was used for statistical tests in A. At FDR <5%, +FDR-adjusted P<0.01; #FDR-adjusted P<0.001. One-way ANOVA with Dunnett post hoc test (B, C, and E–H) was used for statistical tests. Kruskal-Wallis test with Dunn post hoc test was used for statistical tests in D. ^Adjusted P<0.05; ^^adjusted P<0.01; ^^^adjusted P<0.001 (HSD+EtOH vs other groups in B–F; SnHSD+EtOH vs other groups in G; B.F.S vs other groups in H). The Wilcoxon rank-sum test was used for statistical tests in I. $P<0.05. Ctrl indicates control; DBP, diastolic blood pressure; and SBP, systolic blood pressure.

Discussion

In this study, we revealed a novel mechanism of BP regulation and hSIH development by intestinal flora. The intestinal flora composition and metabolism were significantly changed during hSIH development, especially Bfragilis and AA decrease and corticosterone increase. Bfragilis YCH46 and its metabolite AA could markedly inhibit HSD-induced intestinal corticosterone production. Salt-induced reduction of Bfragilis YCH46 decreased AA, which increased intestine and serum corticosterone levels.

In recent years, several animal and patient studies have demonstrated that gut dysbiosis is closely related to many diseases including hypertension.21,24 For example, the intestinal Bifidobacterium is significantly reduced, whereas Firmicutes is observably increased in patients and animals with hypertension. The phosphotransferase system pathway is enriched in the intestinal bacteria of patients with hypertension and prehypertension, while the 2-component system is enriched in the healthy individuals, which is consistent with our findings.21 To date, few studies have focused on hSIH and intestinal flora. Mell et al25 have found that the Bacteroidetes family S24-7 and Firmicutes family Veillonellaceae were relatively more abundant in Dahl salt-sensitive than in salt-resistant rats. Recently, Lactobacillus spp. were reported to be significantly decreased in HSD-induced hypertensive FVB/N mice. Moreover, Lactobacillus spp. could inhibit inflammation and decrease BP through its indole metabolites.23 We also found a Lactobacillus spp. decrease (Figure 3D) and immune response activation in HSD group (Figure IIIA through IIID in the Data Supplement). However, we did not find significant changes in indole metabolites. In fact, the inconsistency in significantly changed intestinal bacteria and metabolites exists not only in this study but also in many previous studies. There could be many possible reasons for the inconsistencies. First, the animals used in studies are different, such as Dahl salt-sensitive rats, spontaneously hypertensive rats, DOCA-induced C57BL/6 mice, L-NAME (Nω-nitro-L-arginine methyl ester)/salt-induced FVB/N mice, and outbred albino Wistar rats. The genetic background varies greatly among these animals.19,24 Second, the diets and living conditions of the animals are not the same, such as the feed containing 4% NaCl in the study by Wilck et al and 8% NaCl in this study. It is well known that diet and housing are two of the decisive factors affecting intestinal flora composition and function. Third, the softwares and supporting databases used were different, such as Usearch and Ribosomal Database Project, QIIME2 and GreenGenes reference database. Additionally, the parameters and sensitivity of mass spectrometry and metabolomic softwares used also have significant differences, such as Pegasus IV mass spectrometer, Maui-SILVIA software, AB SCIEX 6600 mass spectrometer, XCMS software, and Metaboanalyst.

Many studies have shown that the plasma cortisol level was obviously decreased, whereas the plasma corticosterone level was significantly increased in patients with hypertension.16,26 Corticosterone was found to increase BP and induce hypertension in rats.15,27 These findings implied that although most of glucocorticoids in human body are cortisol, corticosterone likely also plays important roles in hypertension development. Intestine has been reported to be an important source of bioactive corticosterone. Previous studies have shown that corticosterone produced by intestine could enter circulation.12 We also found that serum corticosterone levels were significantly elevated in rats and patients with hypertension, which might activate MR and contribute to the salt-induced sympathetic excitation and changes in sodium handling and vascular resistance.11,12,15,27 Meanwhile, corticosterone could also promote sodium absorption by activating MR in intestinal epithelial cells.28,29 These intestinal and extraintestinal effects of corticosterone could synergistically elevate BP. Corticosterone can also be transformed to aldosterone, which plays critical roles in RAAS and BP regulation by activating MR. Corticosterone and aldosterone have similar affinity for MR. Under physiological conditions, corticosterone circulates at a concentration 100 to 1000× higher than that of aldosterone, but inactivation by HSD11B2 causes corticosterone to occupy only part of MR, thus allowing MR to be bound and activated mainly by aldosterone.30 Under HSD conditions, the circulating corticosterone and aldosterone levels were significantly increased and decreased, respectively, in rats.30,31 Thence the further excessive corticosterone might saturate HSD11B2 and overload its ability to protect MR against illicit activation, resulting in inappropriate MR activation by corticosterone.32 Both activated MR and hypokalemia caused by excessive corticosterone can inhibit aldosterone synthesis and reduce circulating aldosterone level.33 Therefore, although the circulating aldosterone level was decreased, it still shows an apparent mineralocorticoid excess to a certain extent because corticosterone replaces aldosterone to activate MR. Nonetheless, RAAS is very complicated. The effects and mechanisms of HSD on RAAS and the role of gut microbiota on them should be further investigated in more studies.

At present, the regulatory mechanism of intestinal corticosterone synthesis has not yet been fully elucidated, and little is known about the role of intestinal flora in it.11,12 Our studies in vitro and in vivo revealed for the first time that AA produced by the intestinal Bfragilis YCH46 could reverse corticosterone increase in HSD rat and mouse colon. The findings enriched the understanding of hSIH and glucocorticoid regulation, but the molecular mechanisms behind them remain poorly understood. AA is reported to activate JNK (c-Jun N-terminal kinases)/c-Jun, NF-κB (nuclear factor-kappa B), and p38 MAPK (mitogen-activated protein kinase).34,35 JNK and NF-κB can downregulate Cyp11a1 and Cyp11b1 expressions, whereas p38 can increase Hsd11b2 expression,34,36 thereby leading to a decrease in corticosterone production and activity.12,37 Therefore, AA produced by intestinal bacteria might have the same effects through these mechanisms.

20-HETE—an important metabolite of AA—can not only raise BP by constricting arterial blood vessels or promoting sodium conservation but also reduce sodium and fluid absorption in nephron to decrease BP.38 Therefore, 20-HETE roles in hypertension are sophisticated. Some studies showed that chronic blockade of 20-HETE formation promoted hypertension development,39 whereas some others found that 20-HETE overproduction in kidneys also contributed to BP increase.40 We found that serum 20-HETE level significantly elevated in HSD rats, while renal 20-HETE level increase was statistically insignificant (Figure IVA and IVB in the Data Supplement), which was consistent with previously reported results.39 Moreover, the circulating 20-HETE level significantly correlated with Simpson and Pielou index (Figure IVC and IVD in the Data Supplement). However, there was no significant correlation between circulating or renal 20-HETE level and intestinal Bacteroides abundance (Figure IVE and IVF in the Data Supplement). These results suggest that gut microbiota may closely relate to the Cyp4a-20-HETE axis, but Bacteroides in the gut may not be involved in it. Obviously, more studies are still needed to clarify the mechanism behind these findings.

Previous studies have reported that the antibiotic treatment can effectively decrease the BP of refractory patients with hypertension, and the antihypertensive effect can still be maintained for 6 months after stopping antibiotic therapy.41 We also found that BP of the hSIH rats was significantly decreased by quadruple antibiotic treatment and further decreased after receiving fecal microbiota from healthy rats. However, the BP of healthy rats was elevated after quadruple antibiotic treatment and further elevated after receiving intestinal flora from the hSIH rats. These results indicated that the effect of antibiotics on BP should be closely related to the composition of intestinal flora,42 and the same antibiotics may have an opposite effect if the intestinal flora composition is different. The harmful bacteria are predominant in the intestinal flora of hypertensive rats, and, therefore, the impacts of antibiotics on harmful bacteria are more superior, and they mainly exert antihypertensive effect. The transplantation of intestinal flora from healthy rats further improved gut dysbiosis of the hypertensive rats, resulting in harmful bacteria decrease, beneficial bacteria increase, and further BP reduction. However, the beneficial bacteria are dominant in the intestinal flora of healthy rats; hence antibiotics mainly influence them and destroy the homeostasis of intestinal flora with increasing BP. The transplantation of intestinal flora from hypertensive rats further disturbs the intestinal flora balance, namely the decrease of beneficial bacteria with antihypertensive action and the increase of harmful bacteria with prohypertensive effect, resulting in further BP elevation.

In addition to corticosterone, other metabolites including acetylcholine, prostaglandin E2, and leukotriene B4 have also been found to be significantly associated with BP and some intestinal bacteria such as Flexispira, Odoribacter, Bacteroides, and Lactobacillus in this study. Acetylcholine can mediate vasodilation in patients with hypertension and improve endothelial dysfunction.43 Prostaglandin E2 and its receptor EP1 (prostaglandin E2 receptor 1) are key players in angiotensin II–dependent hypertension and related end-organ damage.44 Leukotriene B4 is an effective chemoattractant related to the inflammatory response, and blocking its receptor can lower arterial BP in spontaneously hypertensive rats.45 The mechanisms by which the intestinal flora and their metabolites regulate BP are very complicated. In addition to the mechanisms found in this and previous studies, other mechanisms may be involved. Further studies are needed to reveal whether the abovementioned intestinal bacterial metabolites also contribute to hSIH development.

The function of intestinal bacteria and the pathogenesis of hypertension are extremely sophisticated. Inflammatory immune response is one of the critical mechanisms for hypertension development. This study reveals a novel mechanism by which intestinal flora regulates BP and is involved in hSIH (Figure 7). Namely, HSD reduces the abundances of Bfragilis and its metabolite AA in the intestine, thereby attenuating the inhibitory effect of AA on intestinal corticosterone production, resulting in increased corticosterone level. Our findings indicate that salt intake has profound effects on hormonal systems by influencing the gut bacteria—a salt-sensitive compartment. This study also suggests that the gut microbiota may serve as a potential target to counteract hSIH.

Figure 7. Schematic diagram representing the mechanism of high salt–induced hypertension via modulating gut microbiota and gut-derived hormone. AA indicates arachidonic acid; B fragilis, Bacteroides fragilis; CHOL, cholesterol; Cor, cortisone; CORT, corticosterone; ENaC, epithelial sodium channel; JNK, c-Jun N-terminal kinase; MR, mineralocorticoid receptor; and NF-kB, nuclear factor-kappa B.

Nonstandard Abbreviations and Acronyms AA arachidonic acid BP blood pressure DBP diastolic blood pressure FDR false discovery rate HSD high-salt diet Hsd11b2 hydroxysteroid 11-beta dehydrogenase 2 hSIH high salt–induced hypertension MR mineralocorticoid receptor NF-κB nuclear factor-kappa B PICRUSt phylogenetic investigation of communities by reconstruction of unobserved states RAAS renin-angiotensin-aldosterone system SBP systolic blood pressure

Acknowledgments

We would like to thank Xinjie Zhang for her help in the experiments.

Sources of Funding This work was supported by the National Natural Science Foundation of China (No. 81570712, 81670247, and 91439111) and the Natural Science Outstanding Youth Foundation of Shandong Province (No. JQ201519), Major Science and Technology Innovation Project of Shandong Province (No. 2018CXGC1218), and Jinan Clinical Medical Science and Technology Innovation Program (No. 201805055).

Disclosures None.

Supplemental Materials

Expanded Materials & Methods

Major Resources Table

Online Tables I–III

Online Figures I–IV

References 21,46–55

Footnotes