2Department of Cardiology, Faculty of Medicine, Çanakkale Onsekiz Mart University, Çanakkale, Türkiye
Abstract
Background: The increase in aortic stiffness is a significant parameter of cardiovascular diseases (CVDs), posing a substantial global health challenge and economic burden. The gut microbiota and its homeostasis, directly and indirectly, influence CVD. This study investigated the extent to which alterations in the gut microbiota can affect aortic parameters in a rat model through the administration of VSL#3.
Methods: Twelve male Wistar rats were divided into VSL#3-treated and control groups. Cardiac function, aortic systolic, and diastolic values were assessed via echocardiography on day 0 and day 42, and fecal specimens were simultaneously collected from each rat. The formation and composition of the gut microbial flora were profiled using 16S rDNA gene sequencing.
Results: Differences in bacterial density, as indicated by Chao analysis, exhibited statistical significance (P = .037) between the 2 groups. Additionally, in the VSL#3-treated group, significant improvements were observed in aortic systolic and diastolic diameters, as well as in aortic strain parameters, compared to the control group.
Conclusion: This research highlights the potential of gut microbiome modulation, specifically through VSL#3 administration, as a promising strategy to improve aortic parameters, suggesting a novel avenue for cardiovascular health interventions.
Highlights
- This study investigates the effects of gut microbiota modulation on aortic parameters in a rat model.
- VSL#3 supplementation resulted in significant alterations in the gut microbial community composition.
- Statistically significant changes were observed in aortic systolic and diastolic diameters, as well as in aortic strain parameters following VSL#3 treatment.
- The findings support the potential of gut microbiome modulation as a novel approach for improving cardiovascular health.
Introduction
The alteration in aortic diameter represents a critical clinical condition commonly observed in medical practice, with its pathogenesis attributed to various factors, including inflammation of the aortic wall, initiation of muscle cell apoptosis, breakdown of the matrix, formation of plaques, oxidative stress, and restructuring of the vasculature.1,
The main clinical term for the components of arteriosclerosis and atheromatosis is arterial stiffness (AS).8 Arterial stiffness, indicative of large arteries’ capacity to respond to pulse pressure by expanding, is affected by several established atherosclerotic risk elements like aging, smoking, high cholesterol levels (hypercholesterolemia), diabetes mellitus (DM), and hypertension (HT).9-
We aimed to investigate the extent to which VSL#3, a probiotic mixture, can induce changes in the composition of the gut microbiota and its potential effects on aortic parameters in a rat model.
Methods
Animal Experiment Design and Sample Colleciton
A total of 12 male Wistar albino rats (250-350 g) were supplied from the Experimental Research Center. The rats were kept in a controlled environment at 22°C, with a 12-hour light-darkness cycle and free availability of food and tap water. The rats were divided into 2 groups: a control group (n = 6) and an experimental group that received VSL#3 (ACTIAL Farmaceutica SRL, 450 billion CFU per sachet. Available in box of 10 sachets or 30 sachets) via the gavage method. VSL#3, which was dissolved in distilled water, contained the following bacterial species:
Metagenomic Analysis
16S Amplicon Sequencing
The extracted DNA was subjected to amplification using the 16S rDNA V3-V4 primer set (314F-860R). The 16S rDNA in prokaryotic cells’ small ribosomal subunit has 10 conserved regions and 9 hypervariable regions. Conserved areas stay uniform across bacteria, while hypervariable sections are specific to genera or species. Hence, 16S rDNA acts as a unique genetic code for species identification and serves as the prime marker for bacterial phylogeny and classification. As a result, 16S rDNA amplicon sequencing is crucial for analyzing microbial communities in environmental samples.
Library preparation involved the use of the Nextera XT DNA Library Preparation Kit and specific Illumina indices. To ensure high-quality data, the generated libraries were cleaned by size selection according to the manufacturer’s recommendations (AMPure XP, Beckman Coulter). After library preparation, sequencing was performed using the MiSeq system (Illumina).
Bioinformatics Analysis
Paired-end Illumina reads (2 × 250) were loaded into the Qiime2 system.33 Initially, it was determined that all samples had a similar depth of coverage at approximately 100X, and no samples were excluded at this stage. Quality filtering and chimera detection were performed using the DADA2 algorithm in Qiime2 (via q2-dada2).34 Regions with quality scores mostly below 30 were excluded, generating amplicon sequence variants (ASVs). The resulting ASVs were aligned with the GreenGenes (/greengenes.lbl.gov) database to generate taxonomic tables.35,
Statistical Analysis
For alpha diversity, KW analysis was employed, while PERMANOVA was utilized for beta diversity assessment using the adonis function in the vegan R package. Alpha diversity indices (Shannon, Simpson, and Chao1) were calculated using the phyloseq and microbiome packages in R. Beta diversity indices, including PCoA, were generated using the phyloseq package, while Adenism and Adenosis indices were calculated using the ade4 package in RStudio. Kruskal–Wallis and Wilcoxon tests, along with LDA, were applied for LEfSe analysis. The LEfSe analysis in this study was performed using the microbiomeMarker package in R with the following parameters: discriminant score threshold 2.0,
Results
Cardiac Parameters
Significant changes were observed in the aortic diameters in the VSL#3 group compared to the control group on the 42nd day (
Similarly, aortic systolic diameter was also significantly different between the groups on day 42. The control group showed an increase from 2.22 ± 0.10 mm to 2.66 ± 0.22 mm (
These findings suggest that VSL#3 may influence vascular parameters, particularly by limiting the extent of changes in both aortic systolic and diastolic diameters. The changes in these parameters are critical, as they reflect the vascular remodeling process that can contribute to cardiovascular disease progression. VSL#3 may thus have a protective effect on vascular health, possibly reducing the risk of conditions such as hypertension and atherosclerosis.
Regarding aortic strain, a significant improvement was observed in the VSL#3 group compared to the control group after 42 days of treatment. At baseline, aortic strain was similar between the 2 groups (2.3 ± 0.3 in the control group and 2.7 ± 0.4 in the VSL#3 group,
The observed increase in aortic strain is particularly important because it reflects the arterial walls’ ability to expand and contract in response to blood flow. Enhanced aortic strain is associated with better vascular health and lower cardiovascular disease risk. This result further supports the potential of VSL#3 in improving cardiovascular parameters, especially by promoting arterial elasticity and reducing the risk of conditions such as hypertension and atherosclerosis.
Gut Microbiota Composition
DNA extraction was successfully carried out, yielding uniform DNA concentrations ranging from 18.5 to 212.5 ng/μL for all samples. The bacterial sequencing preparation (V4) was also successful, with read counts ranging from 31 574 to 221 386 after quality control and bioinformatic processing.
The gut microbiota structure was analyzed using the Illumina MiSeq PE250 sequencing platform. A total of 24 fecal samples were collected from the control and VSL#3 groups on days 0 and 42. Taxonomic composition at the phylum level revealed that
A significant variation in bacterial density, as assessed by Chao analysis, was found between the 2 groups (
Discussion
The microbiota residing in the gut significantly influence host physiology, contribute to pathological conditions, and help maintain the delicate balance of the intestinal immune system.44 In this study, alterations in the structure and composition of the intestinal microbial community due to VSL#3 probiotic treatment in a rat model were determined using 16S rDNA genomic sequencing. Furthermore, the impact of these changes on aortic elasticity parameters was investigated.
The primary composition of the gut microbiota community involves 5 principal bacterial phyla:
The notion that increasing beneficial bacteria in the host’s gut can yield favorable outcomes has led to the application of probiotics. Following probiotic administration, a meta-analysis of 14 studies published between 2002 and 2019, involving 846 cases diagnosed with hypertension, noted decreases in arterial pressure and blood sugar levels.44 Another study conducted on spontaneously hypertensive rats demonstrated that probiotics such as
In patients with Type 2 DM, it has been found that probiotic and synbiotic supplements have beneficial effects on systolic and diastolic blood pressure as cardiovascular health factors.47 A study suggests that probiotic supplementation, including
It has been reported that long-term kefir supplementation leads to improvement in the gut, resulting in the alleviation of high blood pressure.51 Furthermore, in apoE-/- mice fed a fat-rich diet, supplementation with
Through genomic sequencing and metagenomic analysis methods, the potential effects of the gut microbiota on CVDs are being revealed.25,
Study Limitations
This study was conducted in a rat model, which provides valuable insights into the potential role of gut microbiota modulation in cardiovascular health. However, inherent species differences between rats and humans limit the direct translatability of these findings to clinical practice. While the results offer preliminary evidence, additional investigations are required to confirm their applicability in human populations. Future studies with larger cohorts would help to establish more robust conclusions. Moreover, this study focused primarily on aortic diameter and strain values, without evaluating other critical cardiovascular parameters such as heart rate, vascular resistance, or left ventricular function. These aspects are important for a comprehensive understanding of the effects of VSL#3 on cardiovascular physiology. Lastly, the study was conducted in a healthy rat model, without incorporating a disease model that mimics human cardiovascular pathologies. Exploring the impact of VSL#3 in disease conditions such as HT, DM, or atherosclerosis would provide a deeper understanding of its therapeutic potential. Future research addressing these limitations will be essential to fully elucidate the role of gut microbiota modulation in cardiovascular health and its clinical implications.
Conclusion
Cardiovascular diseases continue to represent a significant threat to public health, with rising prevalence and mortality rates contributing to a substantial economic burden. In this study, the effect of VSL#3 probiotic intervention on gut microbiota composition in a rat model was investigated, aiming to reduce inflammation and enhance cardiovascular health. Although many probiotic studies report no significant changes in bacterial diversity or abundance, these findings emphasize the intricate and dynamic nature of the gut microbiota, where even small changes in specific bacterial populations can trigger cascading effects that impact the broader microbial ecosystem. Despite the absence of direct inflammatory marker analyses in this study, the observed changes in aortic parameters, particularly AS, suggest that gut microbiota modulation via probiotics may have a beneficial impact on cardiovascular health. This conclusion aligns with existing research suggesting that gut microbiota, through its effects on inflammation, may be linked to cardiovascular disease progression.
This study is original in its approach, as there is restricted comprehensive analysis addressing the effects of probiotic intervention on cardiovascular parameters mediated by gut microbiota in a mammalian model. These results provide novel insights into the positive impact of VSL#3 on aortic parameters, which are key indicators of cardiovascular health. By demonstrating that the reconfiguration of the gut microbiota through probiotic treatment can influence these parameters, it was suggested that probiotic interventions could play a role in preventing or mitigating the risk of cardiovascular diseases. The findings of this study contribute valuable data to the emerging field of gut microbiome research, providing a foundation for future investigations into gut microbiota modulation as a potential therapeutic target for cardiovascular diseases. Further research, particularly with larger sample sizes, disease models, and direct inflammatory assessments, is essential to fully understand the clinical implications of these findings and their potential for human application.
Supplementary Materials
Footnotes
References
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