Associations of imbalance of intestinal flora with severity of disease, inflammatory factors, adiponectin, and vascular endothelial function of hypertension patients

1 INTRODUCTION

Hypertension is one of the cardio-cerebrovascular diseases affecting human health, and also the most common cardiovascular disease in China.1, 2 The morbidity of hypertension is up to 33% in China, with an increasing trend.3 The damage of hypertension to body is not only the stimulation of the persistently elevated blood pressure against the vascular wall, but also the structural and functional disorders of important visceral organs, such as the heart, kidney and brain, thereby threatening the life of patients.4 The changes in various processes, including enhanced immune and inflammatory responses, and increased blood volume and metabolic level are involved in the onset of hypertension.5, 6 The intestinal flora refers to the microbial population colonized in normal body, which is important for keeping the normal function of digestive tract.7 The disorders of intestinal microorganisms are related to the progression of various diseases, such as chronic heart failure,8 refractory epilepsy9 and spinal cord injury.10 Some preclinical studies have shown that the pro-inflammatory flora of intestinal flora can be transferred into animals, causing systemic inflammation and aggravating atherosclerosis.11 Recent studies have shown that the intestinal flora of hypertensive patients and animals has significantly changed, including the decrease of short-chain fatty acid producing bacteria, and the increase of pro-inflammatory bacteria and opportunistic pathogens.12, 13 Blood pressure in healthy animals can be significantly increased by transplantation of intestinal flora from hypertensive patients or hypertensive animals.14, 15 Some probiotics or prebiotics can also reduce blood pressure in hypertensive patients or animals.13, 16 We hypothesis these characteristic pathological changes of hypertension patients can well indicate the development and severity of hypertension, and intestinal flora may affect the progression of hypertension. It is of great significance to search and identify the changes in various indexes of hypertension patients.

In this study, in total of 60 patients with hypertension of varying degrees (Grades 1, 2, and 3) were selected as the research subjects, to analyze whether there was imbalanced intestinal flora in them, the changes in peripheral blood inflammatory factors, serum immunoglobulin, adiponectin (ADPN), and vascular endothelial function were detected, and the characteristic changes in patients with hypertension of varying degrees were explored.

2 SUBJECTS AND METHODS 2.1 Subjects and grouping

The retrospective analysis included 85 patients with primary hypertension in our hospital in recent years. Blood samples were taken from 60 patients. For the final statistical analysis, data from 60 patients were used, including 32 males and 28 females aged 55.34 ± 4.98 on average. The hypertensive patients, aged from 20 to 87, presented at the cardiology outpatient clinic, and received office blood pressure (OBP) over 140/90 at least twice at different times. All participants were placed on 24 h ambulatory blood pressure monitoring (ABPM).

Inclusion criteria: Primary hypertension; blood pressure was measured three times on the same day, systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg; without receiving any antihypertensive drugs and antibiotic application before sample collection.

Exclusion criteria: Secondary hypertension; long-term use of antibiotics; symptomatic peripheral arterial occlusive disease; aortic insufficiency or stenosis (>stage I); hypertrophic obstructive cardiomyopathy; congestive heart failure (>NYHA II); uncontrolled cardiac arrhythmia with hemodynamic relevance; systolic office BP ≥180 mmHg with the indication of unstable coronary artery disease; bedridden patients for a long term. Written informed consent was obtained from all subjects.

According to the grading criteria for hypertension, 60 patients were divided into Grade 1 group (systolic pressure of 140–159 mmHg or diastolic pressure of 90–99 mmHg), Grade 2 group (systolic pressure of 160–179 mmHg or diastolic pressure of 100–109 mmHg), and Grade 3 group (systolic pressure ≥ 180 mmHg or diastolic pressure ≥ 100 mmHg). The general and clinical data of patients in each group were collected, and the hypertension was diagnosed via the measurement of blood pressure for several times. The patients with other severe diseases such as heart failure and myocardial infarction were excluded.

All experimental procedures conformed with institutional guidelines,the experiment was approved by the Ethics Committee of Qiqihar First Hospital, and all patients in this study have provided written informed consent in accordance with the Helsinki Declaration. The approval number is HQ-3c132.

2.2 Detection of intestinal flora

Frozen-stored mid-posterior-segment fecal samples (about 3–5 g) of each subject were collected, from which the bacterial deoxyribonucleic acid (DNA) was extracted using Microbiome DNA Purification Kit (A29790, Thermo) for polymerase chain reaction (PCR) amplification. The partial DNA fragments of bacterial 16S rRNA genes were amplified by PCR. The V3–V4 hypervariable regions of 16S RNA Gene were amplified (primers: 341F/785R) and sequenced using the Illumina MiSeq platform (2 × 300). The microbial diversity was analyzed and classified using Mothur v.1.36.0 and the database RDP (v9) with a 0.03 cutoff level. The Shannon index was estimated with Mothur. The alpha diversity (diversity within the samples) was estimated with the Shannon index.

2.3 Blood sample collection

About 5 ml of peripheral blood was collected in each group, and was immediately centrifuged at 3000 rpm for 5 min. Then the upper-layer plasma was transferred from anti-coagulation tubes into new centrifuge tubes, and was stored at −20°C for later detection of inflammation-associated protein.

2.4 Inflammatory factors and serum immunoglobulin

The levels of inflammatory factors interleukin-2 (IL-2) (550,611, BD), IL-4 (550,614, BD), tumor necrosis factor-α (TNF-α) (560,923, BD) and IL-1β (557,953, BD), as well as of serum immunoglobulin G (IgG) (554,009, BD) and IgM (554,031, BD) were determined using enzyme-linked immunosorbent assay (ELISA) kits strictly according to the instructions. The supernatant of peripheral blood frozen was detected, with three replicates in each group. The optical density (OD) value was measured at 450 nm using a Model 680 Microplate Reader (Bio-Rad), and was converted into the levels of biomarker and laboratory assessment.

2.5 Quantitative detection of ADPN

The level of ADPN was detected using the quantitative detection kit (SEA605Ra, Cloud-Clone Corp) strictly according to the instructions, briefly as follows: The serum was diluted with buffer. After being placed into the sample wells, the serum was shaken, incubated, washed, and added with enzyme conjugate solution, substrate and stop buffer. Then the OD value was measured at 450 nm, and was converted into the level of ADPN.

2.6 Detection of vascular endothelial function

The vascular endothelial function-related endothelin-1 (ET-1) (EIAET1, Invitrogen), nitric oxide (NO) (MX4732-50T, MKBio), vascular endothelial growth factor (VEGF) (CMK0019, Jiamay Biotech) and intercellular adhesion molecule-1 (ICAM-1) (ECM335, Millipore) were detected using commercial kits strictly according to the instructions.

2.7 Statistical analysis

SPSS Statistics 23.0 software (SPSS Inc, Chicago, Illinois) was used for statistical analysis. Measurement data were expressed as mean ± SD (−χ ± s). Data were analyzed for normal distribution by Kolmogorov Smirnov test. Multivariable-adjusted regression models for alpha-diversity were measured for microbial community composition, and for individual taxa. We controlled for antihypertensive medication use in multivariable-adjusted regression models, as medications may alter the gut microbiota.17, 18 Continuous parameters were normally distributed and were presented as the mean plus standard deviation. Differences between the randomization groups were tested using the one-way analysis of variance (ANOVA) for continuous parameters. Reported p values were corrected for multiple comparisons using the method of Benjamini and Hochberg. p < 0.05 was considered to be statistically significant.

3 RESULTS 3.1 Comparison of general data among the three groups

As shown in Table 1 and Figure 1, there were no significant differences in gender, age, body mass index, the proportion of smokers, diet habit, probiotics and antihypertensive medication use status and number of diabetic cases among groups (p > 0.05).

TABLE 1. Comparison of general data among the three groups Grade 1 group (n = 20) Grade 2 group (n = 20) Grade 3 group (n = 20) p Gender (male/female) 11/9 10/10 11/9 >0.05 Age (Y) 54.23 ± 4.12 56.32 ± 3.29 55.86 ± 5.29 >0.05 BMI (kg/m2) 25.52 ± 3.94 24.85 ± 4.85 25.02 ± 5.74 >0.05 Smoker (n) 5 6 2 >0.05 Diet habit >0.05 Light 7 9 14 Spicy 13 11 6 Probiotics 1 2 0 >0.05 Antihypertensive medication use 7 14 18 >0.05 Diabetic cases (n) 2 1 2 >0.05 Note: ANOVA test was performed for comparing differences among the three groups. image

Comparison of general data among the three groups (ANOVA test was performed for differences among the study groups. p > 0.05)

3.2 Differences in composition of intestinal flora among the three groups

Results were consistent with the inverse relationship between blood pressure measures and microbial diversity, in particular microbial richness (Figure 2). In multivariable-adjusted regression models, hypertension statistically has a significantly inverse relationship with genera richness, but not with the Shannon diversity index (Table 2).

image

Measures of α-biodiversity including: (A) richness (Chao1) (B) Shannon diversity (Shannon H) in each group (ANOVA test was performed for differences among the study groups. *p < 0.05 vs. other groups)

TABLE 2. Multivariable-adjusted relationship between gut microbial alpha diversity and blood pressure measures Hypertension Systolic blood pressure Group and index Odds ratio (95% CI) p value Beta coefficient (95% CI) p value Shannon index Grade 1 group 0.83 (0.69, 1.00) 0.053 −1.69 (−2.99, −0.28) 0.018 Grade 2 group 0.82 (0.67, 1.00) 0.049 −1.50 (−2.78, −0.22) 0.022 Grade 3 group 0.72 (0.55, 1.03) 0.037 −1.09 (−2.57, −0.0087) 0.049 Richness Grade 1 group 0.72 (0.59, 0.88) 0.0014 −1.75 (−3.19, −0.31) 0.018 Grade 2 group 0.75 (0.60, 0.94) 0.012 −1.52 (−2.92, −0.12) 0.033 Grade 3 group 0.80 (0.62, 1.00) 0.048 −1.32 (−2.54, 0.083) 0.066

As shown in Table 3 and Figure 3, among the four dominant phyla intestinal flora, the composition of Firmicutes (p < 0.05) and Bacteroidetes (p < 0.05) showed obvious differences among the three groups, and their abundance became higher with the increased severity of disease. Grade 1 group had a markedly higher abundance of Bifidobacteria, Grade 2 group had a markedly higher abundance of Pediococci, and Grade 3 group had a markedly higher abundance of Streptococci and Pseudobutyrivibrio.

TABLE 3. Comparison of abundance of intestinal flora among the three groups n Firmicutes Bacteroidetes Actinobacteria Proteobacteria Grade 1 group 20 3.74 ± 1.01 6.82 ± 1.43 7.43 ± 1.93 6.31 ± 2.11 Grade 2 group 20 7.83 ± 1.83 8.64 ± 2.94 6.84 ± 0.35 7.54 ± 2.21 Grade 3 group 20 12.31 ± 2.12 16.32 ± 2.18 9.73 ± 1.23 6.42 ± 1.24 F 23.53 32.12 3.29 3.98 p <0.05 <0.05 0.231 0.247 Note: ANOVA test was performed for comparing differences among the three groups. image

Comparison of intestinal dominant bacteria in each group (ANOVA test was performed for differences among the study groups. *p < 0.05 vs. other groups)

3.3 Comparison of levels of inflammatory factors among the three groups

The levels of IL-2 (p < 0.05), IL-4 (p < 0.05), TNF-α (p < 0.05), and IL-1β (p < 0.05) showed evident differences among the three groups, and they were elevated with the increased severity of disease and increased abundance of Firmicutes and Bacteroidetes (Table 4 and Figures 4 and 8).

TABLE 4. Comparison of levels of inflammatory factors among the three groups n IL-2 (ng/L) IL-4 (ng/L) TNF-α (ng/L) IL-1β (ng/L) Grade 1 group 20 2.31 ± 0.21 3.24 ± 0.88 15.24 ± 2.04 4.02 ± 0.76 Grade 2 group 20 4.21 ± 1.02 5.31 ± 1.12 20.44 ± 3.26 9.88 ± 1.29 Grade 3 group 20 5.84 ± 1.25 5.98 ± 1.61 28.94 ± 5.85 14.21 ± 2.34 F 7.54 6.88 10.21 16.41 p p < 0.05 p < 0.05 p < 0.05 p < 0.05 Note: ANOVA test was performed for comparing differences among the three groups. Abbreviations: IL-1β, Inflammatory factors interleukin-1β; IL-2, Inflammatory factors interleukin-2; IL-4, Inflammatory factors interleukin-4; TNF-α, Tumor necrosis factor-α. image

Comparison of levels of inflammatory factors among the three groups (ANOVA test was performed for differences among the study groups. *p < 0.05, **p < 0.01)

3.4 Comparison of serum immunoglobulins among the three groups

The level of serum IgG was significantly different among the three groups (p < 0.05), and it was the highest in Grade 3 group (Table 5 and Figures 5 and 8).

TABLE 5. Comparison of serum immunoglobulins among the three groups n IgG (U/ml) IgM (U/ml) Grade 1 group 20 62.31 ± 4.54 74.21 ± 5.84 Grade 2 group 20 87.41 ± 3.85 72.18 ± 4.64 Grade 3 group 20 102.87 ± 6.35 77.54 ± 5.49 F 8.43 2.31 p <0.05 0.549 Note: ANOVA test was performed for comparing differences among the three groups. Abbreviations: IgG, Immunoglobulin G; IgM. Immunoglobulin M. image

Comparison of serum immunoglobulins among the three groups (ANOVA test was performed for differences among the study groups)

3.5 Comparison of level of serum ADPN among the three groups

The level of ADPN decreased in Grade 3 group than that in Grade 2 group (p < 0.05), while it was also evidently decreased in Grade 2 group than that in Grade 1 group (p < 0.05), which might be related to the changes in the abundance of Firmicutes and Bacteroidetes (Figures 6 and 8).

image

Comparison of level of serum ADPN among the three groups (*p < 0.05)

3.6 Comparison of vascular endothelial function among the three groups

The levels of ET-1 (p < 0.05), NO (p < 0.05), VEGF (p < 0.05), and ICAM-1 (p < 0.05) were different among the three groups, and they showed consistent trends with the changes in the abundance of Firmicutes and Bacteroidetes (Table 6 and Figures 7 and 8).

TABLE 6. Comparison of vascular endothelial function among the three groups n ET-1 (ng/L) NO (μmol/L) VEGF (pg/ml) ICAM-1 (μg/L) Grade 1 group 20 6.23 ± 0.45 45.36 ± 4.37 156.45 ± 12.52 175.95 ± 19.43 Grade 2 group 20 23.41 ± 2.12 67.58 ± 3.45 212.46 ± 16.15 256.19 ± 25.37 Grade 3 group 20 65.73 ± 3.21 92.49 ± 5.84 398.76 ± 23.27 402.24 ± 32.18 F 75.34 94.21 104.21 121.23 p p < 0.05 p < 0.05 p < 0.05 p < 0.05 Note: ANOVA test was performed for differences among the study groups. Abbreviations: ET-1, Vascular endothelial function-related endothelin-1; ICAM-1, intercellular adhesion molecule-1; NO, nitric oxide; VEGF, vascular endothelial growth factor. image

Comparison of vascular endothelial function among the three groups

image

Correlation between levels of inflammatory cytokines, ADPN and vascular endothelial function and abundance of firmicutes and Bacteroidetes. (ANOVA test was performed for differences among the study groups. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

4 DISCUSSION

We showed in this study that that the imbalanced intestinal flora may be related to the inflammatory level and immune state in hypertension patients. In this study, 60 patients with hypertension of varying degrees (Grades 1, 2, and 3) were selected as the research subjects, to analyzed whether there was imbalanced intestinal flora in these patients. Regression results were consistent with an inverse relationship between blood pressure measures and microbial diversity, in particular microbial richness. It was found that among the four major kinds of intestinal flora, the composition of Firmicutes (p < 0.05) and Bacteroidetes (p < 0.05) had obvious differences among the three groups, and the abundance of them became higher with the increased severity of disease. In Grade 1 group, the abundance of Bifidobacteria was significantly higher, in Grade 2 group, the abundance of Pediococci was observably higher, and in Grade 3 group, the abundance of Streptococci and Pseudobutyrivibrio was visually higher. The above findings indicate that the imbalanced intestinal flora is significantly associated with the severity of hypertension. Besides, the levels of IL-2 (p < 0.05), IL-4 (p < 0.05), TNF-α (p < 0.05), and IL-1β (p < 0.05) showed evident differences among the three groups, and they elevated with the increased severity of disease and abundance of Firmicutes and Bacteroidetes. The level of serum IgG was significantly different among the three groups (p < 0.05), and it was the highest in Grade 3 group.

Intestinal microorganisms such as Lactobacillus, Bifidobacterium, Bacteroides and Eubacterium play an important role in keeping normal digestive function.17 The Firmicutes/Bacteroidetes (F/B) ratio is widely accepted to play an important role in maintaining normal intestinal homeostasis.18 Under a pathological state, there are certain changes in the composition or abundance of intestinal flora, so that digestive and absorptive functions of normal intestinal are affected, or the local microenvironment and systemic inflammatory state are changed, ultimately affecting the progression of disease.19 At the same time, it has also been found that intestinal microorganisms can regulate blood pressure of body, and the short-chain fatty acids they produced may affect the progression of hypertension.20 Low levels of inflammation, such as obesity, may be the result of reduced microbial diversity. One study in 2005 described the correlation between the F/B ratio, they analyzed cecal bacteria from homozygous obese (ob/ob), heterozygous obese (ob/+), or homozygous lean (+/+) mice, finding that F/B ratio was increased in obese mice and decreased in lean mice.21 Most of studies also support a relationship between increased F/B ratio and obesity in humans.22 Individuals with a F/B ratio of ≧1 were 23% more likely to be overweight than those with a F/B ratio of <1.23 As the important factors for obesity, changed F/B ratios were also found in inflammatory bowel disease (IBD). In contrast to obesity with increased F/B ratios, decreased F/B ratios have been observed in IBD.24, 25 The intestinal microbiome may reduce the synthesis of inflammatory markers through metabolizing plant polyphenols into uroliths, thereby reducing PAI-1 that is secreted by intestinal epithelial cells and aortic endothelial cells.26, 27 Therefore, it was pridicted that the imbalanced intestinal flora may be related to the severity of hypertension.

As an important substance in metabolic pathways such as fatty acid oxidation and glucose absorption, ADPN has been proved to be associated with high blood pressure in the body.28 In this study, the results revealed that ADPN was closely related to the severity of hypertension. The level of ADPN decreased in Grade 3 group than that in Grade 2 group (p < 0.05), while it was also evidently decreased in Grade 2 group than that in Grade 1 group (p < 0.05), which might be related to the changes in the abundance of Firmicutes and Bacteroidetes. It can be inferred that the imbalanced intestinal flora may have a correlation with the changes in ADPN level of hypertension patients.

In addition to inflammatory state, the progression of hypertension is also closely related to the changes in metabolic level in the body. Persistently high blood pressure greatly does harm to endothelial cells, thereby damaging their normal function. With the increased severity of disease, the vascular endothelial cell functions are changed irreversibly. In this study, the levels of ET-1 (p < 0.05), NO (p < 0.05), VEGF (p < 0.05), and ICAM-1 (p < 0.05) were different in three groups, and they showed consistent trends with the changes in the abundance of Firmicutes and Bacteroidetes. In recent years, several studies on animal and patients have shown that intestinal dysregulation is closely related to many diseases, including hypertension.29, 30 For example, in patients with hypertension and animal models, the number of Bifidobacterium decreased significantly, while that of Firmicutes increased significantly, which is consistent with our study.15, 31 To sum up, the imbalanced intestinal flora may also be related to the impaired vascular endothelial cell function during the progression of hypertension.

Monitoring indexes in hypertension patients is conducive to the early prediction of the severity and development of disease. This study explored the correlation between inflammatory cytokines, adiponectin, endothelial function and hypertension, further suggesting that the imbalanced intestinal flora composition is associated with inflammatory cytokine levels, and hypertension severity. It is of great significance for the search of hypertension-related factors, the treatment of hypertension with symptoms, condition judgment, prognosis analysis, and mechanism research.

5 LIMITATION

This study has several limitations, including the need to expand the sample size. In addition, the impact of intestinal flora on inflammatory factors, AD, and endothelial function has not been fully elucidated so far, and thus its relevant mechanism cannot be further elucidated in this study.

6 CONCLUSION

Overall, our results suggest that gut microbial composition is associated with inflammatory cytokine levels, and hypertension severity. Intestinal microbial imbalance may lead to changes in some inflammatory factors and endothelial cell function of patients with hypertension. The regulation of the microbiome is an attractive therapeutic option to improve health outcomes.

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