Skip to main content
NCBI home page
Liver Research logoLink to Liver Research
. 2025 Feb 19;9(1):57–65. doi: 10.1016/j.livres.2025.02.001

Probiotic Bifidobacterium reduces serum TMAO in unstable angina patients via the gut to liver to heart axis

Zhihong Zhou a,1, Lizhe Sun b,c,d,1, Wei Zhou e,1, Wen Gao a, Xiao Yuan b,c,d, Huijuan Zhou a, Yuzhen Ren a, Bihua Li a, Yue Wu b,c,d, Jianqing She b,c,d,⁎
PMCID: PMC11977283  PMID: 40206430

Abstract

Background and aims

Studies indicate that the gut microbiota and its metabolites are involved in the progression of cardiovascular diseases, and enterohepatic circulation plays an important role in this progression. This study aims to identify potential probiotics for the treatment of unstable angina (UA) and elucidate their mechanisms of action.

Methods

Initially, the gut microbiota from patients with UA and control was analyzed. To directly assess the effects of Bifidobacterium supplementation, 10 patients with UA were enrolled and administered Bifidobacterium (630 mg per intake twice a day for 1 month). The fecal metagenome, serum trimethylamine N-oxide (TMAO) levels, and other laboratory parameters were evaluated before and after Bifidobacterium supplementation.

Results

After supplementing with Bifidobacterium for 1 month, there were statistically significant differences (P < 0.05) in TMAO, aspartate aminotransferase, total cholesterol, and low-density lipoprotein compared to before. Additionally, the abundance of Bifidobacterium longum increased significantly, although the overall abundance of Bifidobacterium did not reach statistical significance. The gut microbiota, metabolites, and gut-liver axis are involved in the progression of UA, and potential mechanisms should be further studied.

Conclusions

Metagenomic analysis demonstrated a reduced abundance of Bifidobacterium in patients with UA. Supplementation with Bifidobacterium restored gut dysbiosis and decreased circulating TMAO levels in patients with UA. This study provides evidence that Bifidobacterium may exert cardiovascular-protective effects through the gut–liver–heart axis.

Clinical trial number

ChiCTR2400093946.

Keywords: Gut–liver–heart axis, Bifidobacterium, Unstable angina pectoris, Trimethylamine N-oxide (TMAO)

1. Introduction

The World Health Organization reports that cardiovascular (CV) diseases (CVDs) are responsible for 17.9 million deaths annually, accounting for 31% of all deaths.1 Of these deaths, 85% are directly related to stroke and heart attack.1 Therefore, significant efforts have been made to investigate and identify new therapeutic risk factors for atherosclerosis that can be exploited to enhance primary and secondary prevention of CVDs. Recent studies have proposed that environmental factors, particularly nutrition and the intestinal microbiota, are important contributors to CVD development.2, 3, 4 Additionally, intestinal dysbiosis, a significant risk factor for CVDs, has been linked to obesity and diabetes development.5,6 Nevertheless, the mechanisms by which the gut microbiota influences the pathogenesis and development of CVDs are still being investigated.

Previous studies have demonstrated that trimethylamine N-oxide (TMAO) plays an important role in digestive system diseases, particularly in fatty liver and metabolic liver diseases.7,8 Moreover, some studies have shown that elevated TMAO levels are associated with the severity of liver diseases.9,10 TMAO is a metabolite linked to the intestinal microbiota, produced jointly by the gut flora and liver.11 The biosynthetic pathway of TMAO involves interactions between diet, gut microbiota, and the liver. Trimethylamine (TMA) is generated by the gut flora through the metabolism of compounds such as choline, phosphatidylcholine, carnitine, gamma-butyrobetaine, betaine, crotonobetaine, and glycerol phosphorylcholine, which are abundant in fish, beef, and eggs. TMA is then absorbed through the portal vein circulation and transported to the liver, where it is oxidized by hepatic flavin monooxygenase to form TMAO. TMAO is both hydrophobic and hydrophilic, regulates protein activity and stability, increases foam cell production, and inhibits cholesterol reverse transport. Given that TMAO production depends on the diversity and composition of the gut microbiota, its levels fluctuate with dysbiosis. A recent study also indicated that liver-generated TMAO might exert a strong effect on CVDs.12 However, the underlying regulatory mechanism remains to be explored.

The interaction between metabolic disease, e.g., metabolic dysfunction-associated fatty liver disease (MAFLD) and CVDs, has elicited increasing research attention. A recent Meta-analysis reported that patients with NAFLD have a nearly 50% higher risk of CV events.13 The risk of adverse CV events increases with the severity of NAFLD. Furthermore, this risk is independent of age, sex, obesity measures, diabetes, and other common myocardial metabolic risk factors for CVDs. Mechanistically, TMAO can modulate the immune system by stimulating TXNIP–NLRP3 inflammasomes, leading to the production of inflammatory markers.14 High TMAO levels also negatively affect lipid profiles and contribute to a 43% increase in the risk of developing coronary artery disease (CAD).15 Moreover, TMAO is associated with high levels of C-reactive protein and endothelial dysfunction caused by increased gut permeability and higher serum levels of lipopolysaccharide endotoxin.16 Recent studies have found that TMAO, as an important liver-regulated metabolite, may play critical roles in disease onset and development.17, 18, 19 However, the underlying mechanisms remain to be elucidated.

A previous study found that the gut microbiota can directly affect hypercholesterolemia and CAD development by producing metabolites such as bile acids, indoles, and short-chain fatty acids.20 Furthermore, the gut microbiota can contribute to CAD development through indirect mechanisms, such as immune system modulation.21 Dietary patterns may modulate gut microbiota composition and regulate the production of secondary metabolites. Choline, betaine, phosphatidylcholine, lecithin, and L-carnitine, primarily found in red meat, contribute to the production of TMAO,22, 23, 24, 25, 26 which has been established as a factor that can affect CV health in various ways. However, the interaction between the gut microbiota and TMAO generation, as well as their effects on the pathogenesis and progression of CAD, are still unexplored.

Herein, this study aimed to identify potential probiotics for patients with CVDs by exploring alterations in gut microbiota. Through metagenomic analysis of the gut microbiota in the exploration cohort, we identified Bifidobacterium as a potential probiotic in patients with unstable angina (UA). To further validate the clinical application of Bifidobacterium in these patients, a validation cohort of 10 patients who received Bifidobacterium supplementation along with standard secondary prevention medications was established. By integrating gut microbiota and serum TMAO alterations, we discovered that Bifidobacterium can serve as a probiotic that can lower serum TMAO levels and promote long-term CV benefits. This study provides insights into the potential of utilizing Bifidobacterium as a probiotic therapy for patients with CVDs.

2. Materials and methods

2.1. Ethical approval

All procedures involving human participants were conducted in accordance with the ethical standards of the institutional or national research committee, as well as the Helsinki Declaration and its later amendments or comparable ethical standards. This pilot study was registered with the Chinese Clinical Trial Register (https://www.chictr.org.cn/indexEN.html, registration number: ChiCTR2400093946). This study was approved by the Ethics Committee of Bayannur City Hospital (approval number: 20200117002) and the First Affiliated Hospital of Xi’an Jiaotong University (approval number: XJTU1AF2018LSL-025), and written informed consent was obtained from each participant.

2.2. Study population

A total of 30 patients with UA and 30 healthy individuals from Bayannur City Hospital and the First Affiliated Hospital of Xi’an Jiaotong University were recruited in the exploration cohort from August 2020 to May 2022. The validation cohort consisted of 10 patients with UA who received Bifidobacterium supplementation along with standard secondary prevention medications. The present study is a pilot study. Bifidobacterium triple live bacteria enteric-coated capsules (Jincheng Haise Pharmaceutical Co., Ltd., Shanxi, China) were administered twice daily at a dose of 630 mg per intake for 1 month (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Schematic of the study design. A total of 101 patients diagnosed with UA were screened, of whom 41 were excluded for various reasons. The exploration cohort comprised 30 patients with UA and 30 healthy individuals. The validation cohort consisted of 10 patients with UA who were administered Bifidobacterium along with standard secondary prevention medications. Baseline laboratory parameters, fecal metagenome, and serum TMAO levels were evaluated for all participants in the exploration cohort and patients with UA in the validation cohort, both before and after Bifidobacterium supplementation. Abbreviations: TMAO, trimethylamine N-oxide; UA, unstable angina.

The inclusion criteria were as follows: (i) patients newly diagnosed with UA who were undergoing coronary angiography and had a Gensini score of ≥40, (ii) age 45–75 years, (iii) Han ethnicity without blood relationship, (iv) relatively stable long-term residence, (v) regular lifestyle and dietary habits without significant changes, and (vi) normal bowel movements and absence of recent diarrhea, constipation, or changes in bowel frequency or habits. The exclusion criteria were as follows: (i) history of diabetes, hyperlipidemia, hyperuricemia, cerebral infarction, and cerebral hemorrhage; (ii) severe liver diseases, renal dysfunction, and malignant tumors; (iii) digestive system diseases; (iv) intake of traditional Chinese medicines, formulas, antibiotics, and other medications that may have affected the gut microbiota within the past months; and (v) individuals with poor compliance and those currently participating in other clinical trials.

The samples were selected by voluntary participation, and the validation cohort was distinct from the exploration cohort.

2.3. Sample collection

Both fasting venous blood and stool samples were collected from the same individual in the exploration cohort for the analysis of baseline laboratory parameters and fecal metagenome. In the validation cohort, fasting venous blood and stool samples were obtained from each participant before and after 1 month of Bifidobacterium supplementation to analyze laboratory parameters, fecal metagenome, and TMAO levels. Peripheral venous blood samples were obtained in the morning while participants were hospitalized or enrolled. Collected samples were centrifuged at 1000×g for 10 min at 4 °C and stored at −80 °C until further analysis. Stool samples were collected on the same day, snap-frozen with liquid nitrogen, and stored at −80 °C until further testing.

2.4. Metagenomic and 16S rRNA gene sequencing

The TMAO and its related metabolites’ standards and 2 stable isotope-labeled standards were obtained from ZZ Standard Co., Ltd. (Shanghai, China). Ammonium acetate was of analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (Optima LC-MS) was purchased from Thermo-Fisher Scientific (Fair Lawn, NJ, USA) for metagenomic sequencing. Sequencing was performed on the Illumina NovaSeq platform. The abundance and diversity of intestinal flora were determined by Illumina HiSeq sequencing (Novogene Co., Ltd., Beijing, China), after amplification and purification of the V3–V4 region of bacterial 16S rRNA genes. More details were described previously.27

2.5. TMAO measurement

Serum levels of TMAO-related metabolites and amino acids were measured via isotope dilution liquid chromatography–tandem mass spectrometry.

2.6. Statistical analyses

GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics version 22.0 (IBM Corp, Armonk, NY, USA) were used for statistical analyses. Data are presented as frequencies or percentages for categorical variables and means ± SEM for continuous variables. Shapiro-Wilk test was used to determine the normality of the data distribution. To compare differences between two independent groups, a simple t-test was used for normally distributed continuous variables, the Chi-square test for categorical data, and the Mann–Whitney U test for non-normally distributed continuous data. For paired comparisons (e.g., before and after Bifidobacterium supplementation), the Wilcoxon signed rank test was used for non-normally distributed variables. Correlations were conducted using Spearman’s test or Pearson’s correlation analysis. KEGG enrichment analysis was performed using R 3.5.1 and iPath 3 (https://pathways.embl.de/). Advanced Diff Redundancy analysis and LEfSe were performed using OmicStudio tools (https://www.omicstudio.cn/tool). The significance of differences was determined using a two-tailed test. P < 0.05 was considered statistically significant.

3. Results

3.1. Metagenomic analysis revealed a reduced abundance of Bifidobacterium in patients with UA

As shown in Table 1, statistically significant differences in red blood cell count and hemoglobin were observed between the UA and the control groups after excluding drug interference, whereas no differences existed in age and renal functions. Furthermore, metagenome sequencing showed distinct microbiome composition between the UA and control groups (Fig. 2A). No significant difference was observed in the alpha diversity of the gut microbiome of the UA and control groups, indicating that the UA group had similar bacterial diversity to the control group (Fig. 2B). Interestingly, abundance of Bifidobacterium was reduced in the UA group (Fig. 2C and D). These observations suggest that Bifidobacterium may serve as a potential probiotic in patients with UA.

Table 1.

Baseline characteristics of the exploration cohort.

Characteristics Control (n = 30) Unstable angina (n = 30) P-value
Age (years) 56.43 ± 8.49 57.97 ± 8.25 0.481
Female, n (%) 19 (63.3) 11 (30.0) 0.009
WBC (× 109/L) 6.13 ± 1.67 6.32 ± 1.32 0.351
Neu (× 109/L) 4.23 ± 1.29 3.97 ± 1.30 0.438
Ly (× 109/L) 1.98 ± 0.52 1.91 ± 0.66 0.678
RBC (× 1012/L) 4.52 ± 0.44 4.88 ± 0.46 0.004
Hb (g/L) 136.77 ± 15.70 146.27 ± 14.78 0.019
Plt (× 109/L) 227.9 ± 51.25 200.33 ± 40.21 0.866
ALT (U/L) 19.83 ± 11.52 27.2 ± 12.04 0.019
AST (U/L) 19.67 ± 7.97 21.3 ± 10.59 0.494
TC (mmol/L) 4.32 ± 0.93 4.36 ± 1.22 0.888
HDL (mmol/L) 1.15 ± 0.26 1.24 ± 0.62 0.470
TG (mmol/L) 1.71 ± 0.97 1.60 ± 0.73 0.631
LDL (mmol/L) 2.63 ± 0.69 2.66 ± 1.01 0.882
VLDL (mmol/L) 0.34 ± 0.19 0.61 ± 0.80 0.073
sLDL (mmol/L) 0.72 ± 0.41 0.73 ± 0.49 0.948
eGFR (mL/min/1.73 m2) 92.50 ± 14.13 96.00 ± 46.13 0.695
Cre (μmol/L) 64.45 ± 14.48 65.45 ± 13.27 0.900
Open in a new tab

Data are expressed as means ± SEM or n (%). P < 0.05 for equality between control and CAD. Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; Cre, creatinine; eGFR, estimated glomerular filtration rate; Hb, hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Ly, lymphocyte; Neu, neutrophil; Plt, platelet; RBC, red blood cell; sLDL, small dense low-density lipoprotein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; WBC, white blood cell.

Fig. 2.

Fig. 2

Open in a new tab

Metagenomic analysis indicated a reduced abundance of Bifidobacterium in patients with UA. (A) PLS-DA shows the metabolites of patients in the UA and con groups. (B) ɑ-diversity and (C) the abundance of Bifidobacterium metabolites are compared between the UA and Con groups. (D) A stamp plot displays the Bifidobacterium genus and species. ∗P < 0.05; ns, not significant. Abbreviations: Con, control; PLS-DA, partial least squares discriminant analysis; UA, unstable angina.

3.2. Supplementation with Bifidobacterium restored gut dysbiosis in patients with UA

To directly test the effects of Bifidobacterium supplementation, 10 patients with UA were enrolled and administered Bifidobacterium capsules twice daily for 1 month (Fig. 3A). The 10 UA patients were all male with an average age of 57.36 ± 9.06. Plasma levels of total cholesterol and low-density lipoprotein decreased in the UA group after Bifidobacterium supplementation (P = 0.042 and 0.036, respectively); however, no significant difference in the levels of other lipids, including high-density lipoprotein and triglycerides, was found. Notably, AST levels increased post-intervention (Table 2).

Fig. 3.

Fig. 3

Open in a new tab

Effect of Bifidobacterium supplementation on gut microbiota in patients with unstable angina. (A) Flowchart showing Bifidobacterium supplementation in patients with unstable angina. (B) Genus-level evolutionary tree of the study cohort. (C) PCoA of the gut microbiota before and after Bifidobacterium supplementation. (D) Abundance of Bifidobacterium at the genus levels after supplementation. (E) Abundance of Bifidobacterium at the species levels after supplementation. ∗P < 0.05; ns, not significant.

Table 2.

Characteristics of the participants before and 1 month after Bifidobacterium supplementation.

Characteristics Before (n = 10) After (n = 10) P-value
WBC (× 109/L) 7.76 ± 1.62 7.03 ± 1.69 0.342
Neu (× 109/L) 5.23 ± 1.55 4.77 ± 1.78 0.544
Ly (× 109/L) 1.84 ± 0.38 1.62 ± 0.31 0.189
RBC (× 1012/L) 4.92 ± 0.31 4.84 ± 0.39 0.603
Hb (g/L) 151.00 ± 8.83 146.80 ± 10.18 0.338
Plt (× 109/L) 211.30 ± 29.80 231.70 ± 75.19 0.436
ALT (U/L) 23.50 ± 11.62 32.60 ± 13.16 0.119
AST (U/L) 18.00 ± 3.94 22.70 ± 5.69 0.046
TC (mmol/L) 4.15 ± 0.81 3.46 ± 0.57 0.042
HDL (mmol/L) 0.95 ± 0.21 0.94 ± 0.27 0.923
TG (mmol/L) 2.06 ± 0.91 1.91 ± 0.76 0.683
LDL (mmol/L) 2.54 ± 0.76 1.89 ± 0.48 0.036
VLDL (mmol/L) 0.41 ± 0.18 0.38 ± 0.15 0.687
sLDL (mmol/L) 0.74 ± 0.33 0.59 ± 0.23 0.270
eGFR (mL/min/1.73 m2) 92.30 ± 12.86 95.40 ± 6.86 0.510
Cre(μmol/L) 79.80 ± 11.06 77.30 ± 7.39 0.560
Open in a new tab

Data are expressed as means ± SEM or n (%). P < 0.05 for equality between control and CAD. Abbreviations: ALT, alanine transaminase; AST, aspartate aminotransferase; Cre, creatinine; eGFR, estimated glomerular filtration rate; Hb, hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Ly, lymphocyte; Neu, neutrophil; Plt, platelet; RBC, red blood cell; sLDL, small dense low-density lipoprotein; TC, total cholesterol; TG, triglyceride; VLDL, very low-density lipoprotein; WBC, white blood cell.

Furthermore, fecal 16S rRNA gene sequencing analysis was conducted in the UA group before and after Bifidobacterium supplementation (Fig. 3B and C). Figure. 3B shows the results of a phylogenetic tree analysis of the microorganisms before and after Bifidobacterium supplementation. Principal coordinate analysis revealed that the gut microbiota changed after Bifidobacterium supplementation (Fig. 3C). After 1 month of treatment, patients with UA showed an increase in the abundance of Bifidobacterium; however, this change was not statistically significant (Fig. 3D). In contrast, Bifidobacterium longum demonstrated a statistically significant increase (Fig. 3E). These findings suggest that Bifidobacterium may restore gut dysbiosis in patients with UA.

3.3. Bifidobacterium supplementation modulated metabolic pathways and TMAO levels in patients with UA

To better understand the potential mechanisms through which Bifidobacterium may influence the occurrence of UA, we functionally characterized the altered gut microbiome before and after Bifidobacterium supplementation and examined its relationships with serum TMAO levels and other parameters. Functional alterations in the microbiome of patients with UA following Bifidobacterium supplementation were associated with various metabolic categories, including carbohydrate, amino acid, and nucleotide metabolism (Fig. 4A). Additionally, Bifidobacterium abundance was correlated with clinical indicators at both the genus and species levels; of note, negative correlation between TMAO and bifidobacterium bifidum and bifidobacterium dentium and positive correlation between TMAO and bifidobacterium longum and bifidobacterium pseudolongum were identified (Fig. 4B).

Fig. 4.

Fig. 4

Open in a new tab

Bifidobacterium supplementation altered metabolic pathways and was correlated with clinical indicators in patients with unstable angina. (A) KEGG enrichment analysis shows marked enrichment in the carbohydrate metabolism pathway. (B). A correlation network heatmap was created to illustrate the relationship between Bifidobacterium and clinical indicators. Abbreviations: Hb, hemoglobin; Ly, lymphocyte; Neu, neutrophil; Plt, platelet; RBC, red blood cell; TMAO, trimethylamine N-oxide; WBC, white blood cell.

3.4. Bifidobacterium supplementation decreased circulating TMAO levels in patients with UA

Finally, circulating TMAO levels were evaluated in 10 patients with UA before and after Bifidobacterium supplementation. Bifidobacterium significantly reduced serum TMAO levels in patients with UA (Fig. 5A). Additionally, TMAO levels were significantly correlated with clinical indicators both before and after supplementation (Fig. 5B). Overall, the composition and function of the gut microbial community differed between the control and UA groups, and Bifidobacterium supplementation may lower TMAO levels and exert a CV protective effect (Fig. 5C).

Fig. 5.

Fig. 5

Open in a new tab

Bifidobacterium supplementation decreased circulating TMAO levels in patients with unstable angina. (A) Circulating TMAO levels before and after Bifidobacterium supplementation. (B) RDA shows significant correlations between Bifidobacterium and clinical indicators at different time points. (C) Proposed mechanism. ∗∗P < 0.01. Abbreviations: Hb, hemoglobin; Ly, lymphocyte; Neu, neutrophil; Plt, platelet; P-vals, P-values; RBC, red blood cell; RDA, redundancy analysis; TMA, trimethylamine; TMAO, trimethylamine N-oxide; WBC, white blood cell.

4. Discussion

In this study, metagenomic analysis of the exploration cohort revealed a decreased abundance of Bifidobacterium in patients with UA. However, ethnic or regional differences must be considered during sample enrollment, which could become confounding factors that affect the internal and external validity of the present results. We also examined differences in gut microbiota and TMAO levels between patients who received Bifidobacterium supplementation and the control group. The findings showed that an increase in Bifidobacterium concentration correlates with a reduction in TMAO levels. The mediation of liver-generated TMAO by the intestinal microbiota has emerged as a promising therapeutic target for atherosclerosis treatment. This study provides novel evidence that Bifidobacterium potentially confers CV protection via the gut–liver–heart axis.

The gut microbiota plays a pivotal role in host metabolism and indirectly affects lipid metabolism by influencing cholesterol and bile acid metabolism. For instance, certain probiotics can increase the conversion of cholesterol to bile acids, promote cholesterol excretion, and reduce cholesterol levels in the blood. The gut microbiota also supports the integrity of the gut barrier, preventing the leakage of toxins into the bloodstream. Additionally, an imbalance in the gut microbiota can result in inflammation that is associated with insulin resistance and metabolic syndrome. Understanding the intricate relationship between gut microbiota and host metabolism is critical for developing therapeutic strategies for diabetes, cardiometabolic diseases, and other metabolic disorders. This study showed a decreased abundance of Bifidobacterium in patients with UA. Bifidobacterium supplementation restored gut dysbiosis and reduced plasma total cholesterol and low-density lipoprotein levels in patients with UA. These findings provide evidence for the potential of Bifidobacterium as a probiotic adjuvant therapy for UA.

The liver-generated metabolite TMAO plays a critical role in CVDs by modulating intestinal flora. Probiotics are ingestible microorganisms that reach the intestinal lumen, where they can play functional roles in host physiology.28 The oral administration of adequate amounts of probiotics has been reported to provide CV benefit.29,30 Moreover, the treatment of apolipoprotein E (ApoE)-deficient mice with Bacteroides vulgatus and Bacteroides dorei, two species potentially exhibiting lower abundance in patients with CAD, inhibited the formation of atherosclerotic plaques.31 This study underscores the potential of TMAO therapy to exert beneficial effects on CAD prevention and treatment.

This study provides further evidence that changes in gut microbiota after Bifidobacterium supplementation lowered TMAO levels in patients with UA. Bifidobacterium indirectly influences immune homeostasis by regulating the intestinal environment and microbial community structure. Bifidobacterium’s bile salt hydrolase can hydrolyze bile salts into free bile acids, thereby enhancing its antibacterial activity against species such as Bacteroidetes, Bacillus, Ruminococcus, Clostridium, and Escherichia coli.32, 33, 34 These bacteria further metabolize bile components into secondary bile acids. The transformation of primary bile acids into secondary bile acids is a pivotal intestinal process that affects metabolism and immune function, thereby helping maintain intestinal homeostasis.35,36 Furthermore, five species, namely, Eubacteria, Anaeroplasma, Roseburia, Oscillospira, and Dehalobacteria, were found to be effective in preventing atherosclerosis in a previous animal study.37 Similarly, experiments performed by Stepankova et al.38 demonstrated the protective effects of intestinal bacteria on the progression of atherosclerotic lesions.

Furthermore, a correlation was found between exogenous supplementation of Bifidobacterium and TMAO levels. Long-term dietary habits can alter the composition of the intestinal flora, ultimately affecting TMAO generation. Studies have indicated that TMAO plays a significant role in the onset and progression of various diseases, including coronary heart disease and fatty liver, and dietary interventions can notably reduce CV risk.39,40 A high-fiber diet has been reported to increase the proportion of acetate-producing microbiota, reduce blood pressure, and alleviate heart hypertrophy and fibrosis41, 42, 43, 44 However, an ongoing debate exists regarding whether TMAO acts as a trigger for the onset and progression of human diseases or simply serves as a potential pathological marker. Interestingly, our study revealed that different species of Bifidobacterium exhibit distinct correlations with TMAO. The potential reasons are speculated as follows: Bifidobacterium bifidum and Bifidobacterium dentium may reduce TMAO levels by competitively inhibiting other TMA-producing microorganisms or by directly metabolizing TMA. As the gut microbiota is a complex ecosystem, the interactions among different Bifidobacterium species as well as with other microbes may influence TMAO levels. Bifidobacterium longum and Bifidobacterium pseudolongum may have synergistic effects with other TMA-producing microorganisms, or their presence may not be sufficient to counteract the TMAO-promoting effects of other microbes. Further research is required to elucidate the relationship between TMAO and bifidobacteria. If the full potential of TMAO can be realized, it could emerge as a novel therapeutic target for improving patient prognosis.

This study has several limitations. First, the sample size was relatively small, including only 10 patients with UA who were administered Bifidobacterium. This limitation may constrain the identification of potential risk factors in microbial community analysis through 16S rRNA gene sequencing. Therefore, large-scale, multicenter randomized controlled trials with long-term follow-up are needed to determine how Bifidobacterium might exert its long-term CV benefits in patients with CAD. Second, inconsistencies in sex composition might have led to variations in certain indicators. Although this study enrolled patients diagnosed with UA, which has a higher prevalence in men, this issue of sex composition could not be easily avoided and should be considered a confounding factor. Third, alterations in the patients’ gut microbiota and TMAO levels may not be solely attributed to Bifidobacterium. These changes could also be influenced by variations in gut permeability following dietary changes, coronary stent implantation, and modifications in blood flow. Therefore, further in-depth studies are needed to elucidate the potential mechanisms by which Bifidobacterium lowers circulating TMAO levels.

5. Conclusions

This study evaluated changes in the gut microbiota and TMAO levels in patients with UA. We found that the abundance of Bifidobacterium was significantly decreased in patients with UA. Probiotic intervention via Bifidobacterium supplementation decreased the levels of the liver-generated metabolite TMAO, which could represent a new strategy for improving UA outcomes in the future.

Authors’ Contribution

Zhihong Zhou, Lizhe Sun, and Wei Zhou contributed equally to this paper and should be considered co-first authors. Zhihong Zhou: Visualization, Software, Methodology, Investigation. Lizhe Sun: Writing – original draft, Software, Methodology. Wei Zhou: Formal analysis, Data curation. Wen Gao: Data curation. Xiao Yuan: Investigation, Data curation. Huijuan Zhou: Methodology. Yuzhen Ren: Data curation. Bihua Li: Data curation. Yue Wu: Writing – review & editing, Project administration. Jianqing She: Writing – review & editing, Supervision, Project administration, Funding acquisition. All of the authors approved the final manuscript.

Data availability

The study protocol, standard operating procedures, and data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgements

This study was supported by the Inner Mongolia Autonomous Region Science and Technology Plan Project (No. 2020GG0278), Bayannur City Science and Technology Plan Project (No. K202331), the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (No. XJTU1AF2021CRF-007) and Key Research and Development Program of Shaanxi (Program No. 2022JQ-797). We thank Novogene Co., Ltd. for their assistance during targeted metabolomic analysis. Schematic diagrams were generated by Biorender.com.

Footnotes

Peer review under the responsibility of Editorial Office of Liver Research.

References

  • 1.Timmis A., Kazakiewicz D., Townsend N., Huculeci R., Aboyans V., Vardas P. Global epidemiology of acute coronary syndromes. Nat Rev Cardiol. 2023;20:778–788. doi: 10.1038/s41569-023-00884-0. [DOI] [PubMed] [Google Scholar]
  • 2.Xue H., Chen X., Yu C., et al. Gut microbially produced indole-3-propionic acid inhibits atherosclerosis by promoting reverse cholesterol transport and its deficiency is causally related to atherosclerotic cardiovascular disease. Circ Res. 2022;131:404–420. doi: 10.1161/circresaha.122.321253. [DOI] [PubMed] [Google Scholar]
  • 3.Chen Y., Zhou J., Wang L. Role and mechanism of gut microbiota in human disease. Front Cell Infect Microbiol. 2021;11 doi: 10.3389/fcimb.2021.625913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tousoulis D., Guzik T., Padro T., et al. Mechanisms, therapeutic implications, and methodological challenges of gut microbiota and cardiovascular diseases: a position paper by the ESC Working Group on Coronary Pathophysiology and Microcirculation. Cardiovasc Res. 2022;118:3171–3182. doi: 10.1093/cvr/cvac057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Liu R., Hong J., Xu X., et al. Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat Med. 2017;23:859–868. doi: 10.1038/nm.4358. [DOI] [PubMed] [Google Scholar]
  • 6.Miyauchi E., Shimokawa C., Steimle A., Desai M.S., Ohno H. The impact of the gut microbiome on extra-intestinal autoimmune diseases. Nat Rev Immunol. 2023;23:9–23. doi: 10.1038/s41577-022-00727-y. [DOI] [PubMed] [Google Scholar]
  • 7.Janeiro M.H., Ramírez M.J., Milagro F.I., Martínez J.A., Solas M. Implication of trimethylamine N-oxide (TMAO) in disease: potential biomarker or new therapeutic target. Nutrients. 2018;10 doi: 10.3390/nu10101398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Oh J., Kim J., Lee S., et al. 1H NMR serum metabolomic change of trimethylamine N-oxide (TMAO) is associated with alcoholic liver disease progression. Metabolites. 2024;14:39. doi: 10.3390/metabo14010039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Chen Y.M., Liu Y., Zhou R.F., et al. Associations of gut-flora-dependent metabolite trimethylamine-N-oxide, betaine and choline with non-alcoholic fatty liver disease in adults. Sci Rep. 2016;6 doi: 10.1038/srep19076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.León-Mimila P., Villamil-Ramírez H., Li X.S., et al. Trimethylamine N-oxide levels are associated with NASH in obese subjects with type 2 diabetes. Diabetes Metab. 2021;47 doi: 10.1016/j.diabet.2020.07.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gilbert M.C., Setayesh T., Wan Y.Y. The contributions of bacteria metabolites to the development of hepatic encephalopathy. Liver Res. 2023;7:296–303. doi: 10.1016/j.livres.2022.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wang Z., Klipfell E., Bennett B.J., et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472:57–63. doi: 10.1038/nature09922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Mantovani A., Csermely A., Petracca G., et al. Non-alcoholic fatty liver disease and risk of fatal and non-fatal cardiovascular events: an updated systematic review and meta-analysis. Lancet Gastroenterol Hepatol. 2021;6:903–913. doi: 10.1016/S2468-1253(21)00308-3. [DOI] [PubMed] [Google Scholar]
  • 14.Canakis A., Haroon M., Weber H.C. Irritable bowel syndrome and gut microbiota. Curr Opin Endocrinol Diabetes Obes. 2020;27:28–35. doi: 10.1097/med.0000000000000523. [DOI] [PubMed] [Google Scholar]
  • 15.Allayee H., Hazen S.L. Contribution of gut bacteria to lipid levels: another metabolic role for microbes? Circ Res. 2015;117:750–754. doi: 10.1161/circresaha.115.307409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Al-Obaide M.A.I., Singh R., Datta P., et al. Gut Microbiota-dependent trimethylamine-N-oxide and serum biomarkers in patients with T2DM and advanced CKD. J Clin Med. 2017;6:86. doi: 10.3390/jcm6090086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.McCarthy R.E., Bowen D.G., Strasser S.I., McKenzie C. The dangers of herbal weight loss supplements: a case report of drug-induced liver injury secondary to Garcinia cambogia ingestion. Pathology. 2021;53:545–547. doi: 10.1016/j.pathol.2020.08.021. [DOI] [PubMed] [Google Scholar]
  • 18.Azevedo R.D., Reis F. Transient bilateral striatal lesion associated with varicella infection. Radiol Bras. 2017;50:347–348. doi: 10.1590/0100-3984.2016.0058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Cordier F., Lapeire L., Sys G., Van Dorpe J., Creytens D. A challenging case of a poorly differentiated small cell variant of synovial sarcoma with unusual immunophenotype. Pathology. 2021;53:658–660. doi: 10.1016/j.pathol.2020.08.028. [DOI] [PubMed] [Google Scholar]
  • 20.Mailer R.K.W., Gisterå A., Polyzos K.A., Ketelhuth D.F.J., Hansson G.K. Hypercholesterolemia induces differentiation of regulatory T cells in the liver. Circ Res. 2017;120:1740–1753. doi: 10.1161/circresaha.116.310054. [DOI] [PubMed] [Google Scholar]
  • 21.Sarkar A., Rafiq K. Humoral immunity in heart failure. Cardiovasc Hematol Disord Drug Targets. 2019;19:14–18. doi: 10.2174/1871529x18666180518101527. [DOI] [PubMed] [Google Scholar]
  • 22.Adak A., Khan M.R. An insight into gut microbiota and its functionalities. Cell Mol Life Sci. 2019;76:473–493. doi: 10.1007/s00018-018-2943-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhu W., Gregory J.C., Org E., et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell. 2016;165:111–124. doi: 10.1016/j.cell.2016.02.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Crimarco A., Springfield S., Petlura C., et al. A randomized crossover trial on the effect of plant-based compared with animal-based meat on trimethylamine-N-oxide and cardiovascular disease risk factors in generally healthy adults: Study With Appetizing Plantfood-Meat Eating Alternative Trial (SWAP-MEAT) Am J Clin Nutr. 2020;112:1188–1199. doi: 10.1093/ajcn/nqaa203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Saaoud F., Liu L., Xu K., et al. Aorta- and liver-generated TMAO enhances trained immunity for increased inflammation via ER stress/mitochondrial ROS/glycolysis pathways. JCI Insight. 2023;8 doi: 10.1172/jci.insight.158183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Tang W.H., Wang Z., Levison B.S., et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013;368:1575–1584. doi: 10.1056/NEJMoa1109400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.She J., Tuerhongjiang G., Guo M., et al. Statins aggravate insulin resistance through reduced blood glucagon-like peptide-1 levels in a microbiota-dependent manner. Cell Metab. 2024;36:408–421. doi: 10.1016/j.cmet.2023.12.027. (e405) [DOI] [PubMed] [Google Scholar]
  • 28.Abid M.B., Koh C.J. Probiotics in health and disease: fooling mother nature? Infection. 2019;47:911–917. doi: 10.1007/s15010-019-01351-0. [DOI] [PubMed] [Google Scholar]
  • 29.Sadeghzadeh J., Vakili A., Sameni H.R., Shadnoush M., Bandegi A.R., Zahedi Khorasani M. The effect of oral consumption of probiotics in prevention of heart injury in a rat myocardial infarction model: a histopathological, hemodynamic and biochemical evaluation. Iran Biomed J. 2017;21:174–181. doi: 10.18869/acadpub.ibj.21.3.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hmar E.B.L., Paul S., Sharma H.K. An insight into the combination of probiotics and their implications for human health. Endocr Metab Immune Disord Drug Targets. 2024;24:1–12. doi: 10.2174/1871530323666230502141717. [DOI] [PubMed] [Google Scholar]
  • 31.Quigley E.M.M. Prebiotics and probiotics in digestive health. Clin Gastroenterol Hepatol. 2019;17:333–344. doi: 10.1016/j.cgh.2018.09.028. [DOI] [PubMed] [Google Scholar]
  • 32.Wang G., Jiao T., Xu Y., et al. Bifidobacterium adolescentis and Lactobacillus rhamnosus alleviate non-alcoholic fatty liver disease induced by a high-fat, high-cholesterol diet through modulation of different gut microbiota-dependent pathways. Food Funct. 2020;11:6115–6127. doi: 10.1039/c9fo02905b. [DOI] [PubMed] [Google Scholar]
  • 33.Bourgin M., Kriaa A., Mkaouar H., et al. Bile salt hydrolases: at the crossroads of microbiota and human health. Microorganisms. 2021;9:1122. doi: 10.3390/microorganisms9061122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bustamante J.M., Dawson T., Loeffler C., et al. Impact of fecal microbiota transplantation on gut bacterial bile acid metabolism in humans. Nutrients. 2022;14 doi: 10.3390/nu14245200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lugli G.A., Milani C., Duranti S., et al. Isolation of novel gut bifidobacteria using a combination of metagenomic and cultivation approaches. Genome Biol. 2019;20:96. doi: 10.1186/s13059-019-1711-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Yang M., Gu Y., Li L., et al. Bile acid-gut microbiota axis in inflammatory bowel disease: from bench to bedside. Nutrients. 2021;13:3143. doi: 10.3390/nu13093143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Chan Y.K., Brar M.S., Kirjavainen P.V., et al. High fat diet induced atherosclerosis is accompanied with low colonic bacterial diversity and altered abundances that correlates with plaque size, plasma A-FABP and cholesterol: a pilot study of high fat diet and its intervention with lactobacillus rhamnosus GG (LGG) or telmisartan in ApoE-/- mice. BMC Microbiol. 2016;16:264. doi: 10.1186/s12866-016-0883-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Stepankova R., Tonar Z., Bartova J., et al. Absence of microbiota (germ-free conditions) accelerates the atherosclerosis in ApoE-deficient mice fed standard low cholesterol diet. J Atherosclerosis Thromb. 2010;17:796–804. doi: 10.5551/jat.3285. [DOI] [PubMed] [Google Scholar]
  • 39.Guasti L., Galliazzo S., Molaro M., et al. TMAO as a biomarker of cardiovascular events: a systematic review and meta-analysis. Intern Emerg Med. 2021;16:201–207. doi: 10.1007/s11739-020-02470-5. [DOI] [PubMed] [Google Scholar]
  • 40.Zhen J., Zhou Z., He M., et al. The gut microbial metabolite trimethylamine N-oxide and cardiovascular diseases. Front Endocrinol (Lausanne) 2023;14 doi: 10.3389/fendo.2023.1085041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Li D., Lu Y., Yuan S., et al. Gut microbiota-derived metabolite trimethylamine-N-oxide and multiple health outcomes: an umbrella review and updated meta-analysis. Am J Clin Nutr. 2022;116:230–243. doi: 10.1093/ajcn/nqac074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wang M., Li X.S., Wang Z., et al. Trimethylamine N-oxide is associated with long-term mortality risk: the multi-ethnic study of atherosclerosis. Eur Heart J. 2023;44:1608–1618. doi: 10.1093/eurheartj/ehad089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Marques F.Z., Nelson E., Chu P.Y., et al. High-fiber diet and acetate supplementation change the gut microbiota and prevent the development of hypertension and heart failure in hypertensive mice. Circulation. 2017;135:964–977. doi: 10.1161/CIRCULATIONAHA.116.024545. [DOI] [PubMed] [Google Scholar]
  • 44.Gao Y., Lin J., Ye C., Guo S., Jiang C. Microbial transformations of bile acids and their receptors in the regulation of metabolic dysfunction-associated steatotic liver disease. Liver Res. 2023;7:165–176. doi: 10.1016/j.livres.2023.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The study protocol, standard operating procedures, and data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Liver Research are provided here courtesy of Third Affiliated Hospital of Sun Yat-sen University

RESOURCES