Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modification: A Meta-Analysis of Animal Studies.

Leila Khalili, Ann Marie Centner, Gloria Salazar

Meta-Analysis International journal of molecular sciences 2023 18 цитирований

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Study Design

Тип исследования: Meta-Analysis
Популяция: Animal atherosclerosis models

Вмешательство: Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modification: A Meta-Analysis of Animal Studies. Polyphenols, alkaloids, probiotics
Препарат сравнения: Control diet
Первичный исход: Atherosclerosis via gut microbiota modification
Направление эффекта: Positive
Риск систематической ошибки: Moderate

Abstract

Atherosclerosis is a major cause of death and disability. The beneficial effects of phytochemicals and probiotics on atherosclerosis have gained significant interest since these functional foods can improve inflammation, oxidative stress, and microbiome dysbiosis. The direct effect of the microbiome in atherosclerosis, however, needs further elucidation. The objective of this work was to investigate the effects of polyphenols, alkaloids, and probiotics on atherosclerosis using a meta-analysis of studies with mouse models of atherosclerosis. Identification of eligible studies was conducted through searches on PubMed, Embase, Web of Science, and Science Direct until November 2022. The results showed that phytochemicals reduced atherosclerosis, which was significant in male mice, but not in females. Probiotics, on the other hand, showed significant reductions in plaque in both sexes. Berries and phytochemicals modulated gut microbial composition by reducing the Firmicutes/Bacteroidetes (F/B) ratio and by upregulating health-promoting bacteria, including Akkermansia muciniphila. This analysis suggests that phytochemicals and probiotics can reduce atherosclerosis in animal models, with a potentially greater effect on male animals. Thus, consumption of functional foods rich in phytochemicals as well as probiotics are viable interventions to improve gut health and reduce plaque burden in patients suffering from cardiovascular disease (CVD).

Кратко

Consumption of functional foods rich in phytochemicals as well as probiotics are viable interventions to improve gut health and reduce plaque burden in patients suffering from cardiovascular disease (CVD).

Full Text

International Journal of

Molecular Sciences

Article

Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modiﬁcation: A Meta-Analysis of Animal Studies

Leila Khalili 1,†, Ann Marie Centner 1,† and Gloria Salazar 1,2,*

1 Department of Nutrition and Integrative Physiology, College of Health and Human Sciences, Florida State University, Tallahassee, FL 32306, USA
2 Center for Advancing Exercise and Nutrition Research on Aging (CAENRA), Florida State University, Tallahassee, FL 32306, USA

* Correspondence: [email protected] † These authors contributed equally to this work.

Citation: Khalili, L.; Centner, A.M.; Salazar, G. Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modiﬁcation: A Meta-Analysis of Animal Studies. Int. J. Mol. Sci. 2023, 24, 3084. https:// doi.org/10.3390/ijms24043084

Academic Editor: Ida Daniela Perrotta

Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Abstract: Atherosclerosis is a major cause of death and disability. The beneﬁcial effects of phytochemicals and probiotics on atherosclerosis have gained signiﬁcant interest since these functional foods can improve inﬂammation, oxidative stress, and microbiome dysbiosis. The direct effect of the microbiome in atherosclerosis, however, needs further elucidation. The objective of this work was to investigate the effects of polyphenols, alkaloids, and probiotics on atherosclerosis using a meta-analysis of studies with mouse models of atherosclerosis. Identiﬁcation of eligible studies was conducted through searches on PubMed, Embase, Web of Science, and Science Direct until November 2022. The results showed that phytochemicals reduced atherosclerosis, which was signiﬁcant in male mice, but not in females. Probiotics, on the other hand, showed signiﬁcant reductions in plaque in both sexes. Berries and phytochemicals modulated gut microbial composition by reducing the Firmicutes/Bacteroidetes (F/B) ratio and by upregulating health-promoting bacteria, including Akkermansia muciniphila. This analysis suggests that phytochemicals and probiotics can reduce atherosclerosis in animal models, with a potentially greater effect on male animals. Thus, consumption of functional foods rich in phytochemicals as well as probiotics are viable interventions to improve gut health and reduce plaque burden in patients suffering from cardiovascular disease (CVD).

Keywords: berries; polyphenols; alkaloids; berberine; probiotics; atherosclerosis; Akkermansia; mouse; gut microbiota; meta-analysis

1. Introduction

Cardiovascular disease (CVD), in particular atherosclerosis, is one of the leading causes of mortality and remains a signiﬁcant health burden [1]. Over 17 million people die from CVD worldwide, accounting for 31% of all deaths [2]. According to the World Health Organization (WHO), by 2030, over 23 million people will suffer from CVD worldwide [3].

Modiﬁable risk factors for atherosclerosis development include poor lifestyle choices such as high intake of foods rich is saturated fat and sugar and lower intake of foods rich in phytochemicals, such as fruits and vegetables. Poor lifestyle choices are associated with low grade inﬂammation, oxidative stress and elevated low-density lipoprotein (LDL) [4]. Dysbiosis of gut microbiota, characterized by reduced bacterial diversity and increased abundance of disease-promoting bacteria [5], is another important risk factor of atherosclerosis [6]. Gut dysbiosis is also associated with several human diseases, including obesity [7], type 2 diabetes [8], and hypercholesterolemia [9], all of which are risk factors for atherosclerosis development [10]. Furthermore, high fat diet (HFD)-induced

Int. J. Mol. Sci. 2023, 24, 3084. https://doi.org/10.3390/ijms24043084 https://www.mdpi.com/journal/ijms

microbiome dysbiosis correlates with plaque size and circulating cholesterol in Apolipoprotein E-deﬁcient (ApoE / ) mice [11]. Thus, the gut microbiota-derived metabolites have emerged as critical modulators of inﬂammation and lipid metabolism [10,12].

In the human microbiome, Firmicutes and Bacteroidetes are the most abundant phyla. The Firmicutes/Bacteroidetes (F/B) ratio has been used as a predictor of disease, as this ratio increases in obesity [13], with HFD in mouse models [14] and even during aging [15]. In contrast, this ratio decreases in certain conditions such as inﬂammatory bowel disease (IBD) [16]. Thus, improving microbiome health may reduce the risk of or improve CVD and has overall become an attractive target for therapeutic disease interventions, in particular through diet manipulations.

Bioactive compounds, including ﬁber and phytochemicals, are of particular interest as ﬁber is metabolized by the gut bacteria producing health-promoting short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate [17]. Phytochemicals, including polyphenols and alkaloids (berberine) have shown promising effects in reducing atherosclerosis and its risk factors. For example, polyphenols found in fruits, vegetables, nuts, teas, olive oil and spices improve microbiome health by diverse mechanisms including antibacterial activities against disease-promoting bacteria and improving healthy bacterial growth in the gut as well as prebiotic activities [18,19]. Berberine, an alkaloid found in medicinal plants and used for thousands of years in traditional Chinese medicine has shown potent effects against insulin resistance, oxidative stress, hypertension, hyperlipidemia, and inﬂammation [5–7].

Berries are good sources of phytochemicals, including polyphenols and alkaloids as well as ﬁber. We demonstrated that blackberry polyphenols reduced oxidative stress and senescence in vascular smooth muscle cells (VSMCs) in vitro [20] and that blackberry supplementation ameliorated atherosclerosis in ApoE−/− male mice in vivo [21]. Females, however, were resistant to the plaque-lowering effects of this berry [21]. We also demonstrated that gallic acid, a polyphenol enriched in blackberry, mimicked the effect of this berry by reducing plaque in males, but not in female ApoE /− mice. The effect of gallic acid was associated with the restoration of Eubacterium ﬁssicatena and Turicibacter levels and by the upregulation of Akkermansia in the gut [22]. Similarly, the reduction in plaque by berberine in ApoE / male mice was also associated with the upregulation of Akkermansia [23], suggesting that this bacterium may be a common target of phytochemicals. These ﬁndings provide insights into the beneﬁts of berry consumption in the improvement of CVD as berries are a rich source of phytochemicals that can partially restore microbiome dysbiosis and improve cardiometabolic health.

Many studies assessing the effects of berries in atherosclerosis and the microbiome use only one sex; thus, the role of sex in microbiome modulation by diet is not fully understood. In light of this, we analyzed the results of studies evaluating the efﬁcacy of polyphenols, alkaloid (berberine), and berries, as well as probiotics, that have direct effects on gut microbiota composition and plaque size in mice models of atherosclerosis. The ﬁndings revealed a signiﬁcant sex-dependent effectiveness in plaque reductions. Phytochemicals were more effective in males, while probiotics reduced atherosclerosis in both sexes. Furthermore, increases in Akkermansia muciniphila abundance correlated with reduced plaque in several studies, suggesting that increasing the abundance of this strain in the gut is critical for the beneﬁcial effects of phytochemicals in cardiovascular health.

2. Results

The initial search on diverse databases yielded 845 articles. After excluding duplicates, reviews, books, clinical trials, randomized control trials and meta-analyses, 94 articles were selected for potential inclusion. In total, 33 manuscripts contained original research testing the effect of polyphenols, berries, berberine or probiotics on microbiome composition and plaque size in mice. After reviewing the titles and abstracts, 7 studies were removed due to insufﬁcient data and 26 studies were selected for the meta-analysis based on the inclusion criteria (Figure 1).

Figure 1. PRISMA flow diagram of the search strategy. Studies were identified through database searches in PubMed, Embase, Web of Science, and Science Direct until November 2022. The search included studies investigating the effects of berry, polyphenols, alkaloids, berberine and probiotics on gut microbiota and atherosclerosis plaque in ApoE or LDLR mice.

For mouse models, inclusion criteria included ApoE−/− and low-density lipoprotein receptor deﬁcient (LDLR / ) animals, of which ApoE / is the most commonly used animal model of atherosclerosis. ApoE deﬁciency induces plaque accumulation over time, which is accelerated by HFD [24] and presents the most features of cardiometabolic syndrome [25,26].

For mouse models, inclusion criteria included ApoE−/− and low-density lipoprotein receptor deficient (LDLR−/−) animals, of which ApoE−/− is the most commonly used animal model of atherosclerosis. ApoE deficiency induces plaque accumulation over time, which is accelerated by HFD [24] and presents the most features of cardiometabolic syndrome [25,26].

2.1. Characteristics of Included Studies

The characteristics of the included studies are shown in Table 1. Of the 26 studies, the majority used ApoE−/− mice (23 studies), and only three studies used LDLR−/− mice. Considering sex, 9 studies used males, 10 used females, only 2 included males and females and 5 did not report the sex of the mice. Considering the type of intervention, 17 studies tested berries, polyphenols or berberine [22,23,27–41] and 9 used probiotics [11,42–49]. The intervention duration varied from 4 to 16 weeks of treatment, and all the studies used HFD to induce plaque. With respect to the microbiome, only 10 studies reported F/B ratios. For atherosclerosis quantiﬁcation, all of the included studies, except for one [37], reported aortic plaque size, which was reported for the aortic root (7 studies) [32,33,36,40,47–49], the arch (4 studies) [34,35,42,46], the aortic sinus (11 studies) [11,28–31,38,40,41,43–45] and in the whole aorta (7 studies) [22,23,27,28,35,39,46]. The study by Yang et al. [37] reported a reduction in plaque in the aortic sinus by the intervention, but no quantiﬁcation was provided.

2.1. Characteristics of Included Studies

The characteristics of the included studies are shown in Table 1. Of the 26 studies, the majority used ApoE−/− mice (23 studies), and only three studies used LDLR sidering sex, 9 studies used males, 10 used females, only 2 included males and females and 5 did not report the sex of the mice. Considering the type of intervention, 17 studies tested berries, polyphenols or berberine [22,23,27–41] and 9 used probiotics [11,42–49]. The intervention duration varied from 4 to 16 weeks of treatment, and all the studies used

Table 1. Characteristics of included studies.

F/B Analyzed Aortic

Duration (Week)

Method of Intervention

N ID Genotype Sex Intervention Dose

Section Intervention Control

Berries, Polyphenols, and Alkaloids

1
2 Jiyun Liu, 2022 [33]
3
4
5
6 Feng Wang, 2020 [28]
7 J. Nie, 2019 [29] LDLR / 2 - Quercetin 100 µg/day In diet 12 0.3 1.45 aortic sinus
8 Erika Caro-Gómez, 2019 [30] ApoE / M Green coffee extract 220 mg/kg Oral gavage 14 - - aortic sinus
9 Kaiyang Lin, 2022 [32] ApoE / F Geraniin 80 mg/kg In drinking water 12 0.47 1.33 aortic root
10
11 Shiying Yang, 2021 [37] ApoE / M Procyanidin A2 110 mg/kg In drinking water 12 0.58 0.96 -
12 Ming Gao, 2022 [41] ApoE / - Gypenoside XLIX 30 mg/kg By gavage 6 - - aortic sinus
13
14 Lin Zhu, 2018 [23] ApoE / F Berberine 0.5 g/L In drinking water 14 - - whole aorta
15 Yafei Shi, 2018 [38] ApoE / M Berberine 50 mg/kg
16 Min Wu, 2020 [34]
17 Xingxing Li, 2021 [36]

A ApoE−/− F Berberine 100 mg/kg By gavage 16 aortic root
B ApoE−/− F Berberine 200 mg/kg By gavage 16 aortic root Probiotics

1
2 Frida Fak, 2012 [49] ApoE / - Lactobacillus reuteri DSM 1798 109 cfu In drinking water 12 - - aortic root
3 Andrea Mencarelli, 2012 [48] ApoE / M VSL#3 20 × 109 cfu In drinking water 12 - - aortic root
4 Tianyi Jiang, 2020 [45] ApoE / M Lactobacillus mucosae 109 cfu By gavage 13 - - aortic sinus
5 Jin Li, 2016 [47] ApoE / M Akkermansia muciniphila 5 × 109 cfu Oral gavage 8 - - aortic root
6 Taiji Mizoguchi, 2016 [43] ApoE / F Pediococcus acidilactici 1.8 × 1011 cfu In drinking water 12 - - aortic sinus
7 Yee Kwan Chan, 2016 1 [44] ApoE−/− F VSL#3 2.78 × 1011 cfu In diet 12 - - aortic sinus
8 Yee Kwan Chan, 2016 2 [11] ApoE−/− F Lactobacillus rhamnosus GG 108 cfu In diet 12 - - aortic sinus
9 Adil Hassan, 2016 [42] ApoE / -

Lactobacillus plantarum ATCC 14917 109 cfu In diet 12 - - aortic arch

1 apolipoprotein E (Apoe) knockout; 2 LDL receptor knock-out; 3 colony forming unit.

Considering probiotics, four studies used different strains of Lactobacillus, two studies used VSL#3, a probiotic mixture containing eight strains of bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus delbrueckii subspecies bulgaricus, Streptococcus salivarius subspecies thermophilus, Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium breve). Four studies used one of the following strains Enterobacter aerogenes ZDY01, Lactobacillus reuteri DSM 1798, Pediococcus acidilactici or Akkermansia muciniphila.

2.2. Berries, Polyphenols, and Alkaloids

2.2.1. The Effect of Berries, Polyphenols, and Berberine in Plaque Burden

A forest plot of individual effect sizes within each study for berries, polyphenols, and berberine in plaque burden is shown in Figure 2. The common standardized mean

difference (SMD) from 17 studies in the 3 groups was −7.31 (95% conﬁdence intervals (CI): −12.61 to −2.02, p-value < 0.05) based on a random effect model, with signiﬁcant heterogeneity between studies (τ2 = 188.60, I2 = 99.99%, H2 = 17091.74, Q(df = 26) = 3374.41, PQ < 0.001). Further investigation by sensitivity analysis detected one study with a wide and unacceptable CI (SMD = −68.51, CI: −72.14 to −64.88) (Ming-liang Chen, 2016 A) that was removed from further analysis. The removal of this study resulted in a change in effect size (SMD = −4.55 (95% CI: −7.01 to −2.09), p-value < 0.05), with a slight decrease in heterogeneity, which remained signiﬁcant (τ2 = 36.24, I2 = 99.97%, H2 = 3416.13, Q(df = 25) = 2012.10, PQ < 0.001). Considering all the studies, the effect of the overall interventions resulted in a signiﬁcant reduction in plaque burden.

6 of 20

Figure 2. Forest plot of individual SMD of plaque size grouped by treatment type. Studies examining the effect of intervention in plaque size were divided in three groups including berberine, whole berries and probiotics. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent findings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

Figure 2. Forest plot of individual SMD of plaque size grouped by treatment type. Studies examining the effect of intervention in plaque size were divided in three groups including berberine, whole berries and probiotics. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent ﬁndings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

2.2.2. Subgroup Analysis by Treatment Type
2.2.3. Subgroup Analysis by Treatment Duration

The forest plot of individual SMD of predetermined subgroup analysis by treatment duration (1 = ≤12 weeks and 2 = >12 weeks) is presented in Figure 3. The results showed a signiﬁcant treatment effect in both groups ≤ 12 weeks (SMD = −5.70, 95% CI = −10.69 to −0.71) and >12 weeks (SMD = −3.33, 95% CI = −5.26 to −1.41) subgroups. Thus, even 4 weeks of treatment is enough to see signiﬁcant reductions in plaque, as seen by Ming-liang Chen, B [40] using 0.4% resveratrol in the diet in female ApoE−/ mice.

The forest plot of individual SMD of predetermined subgroup analysis by treatment duration (1 = ≤12 weeks and 2 = >12 weeks) is presented in Figure 3. The results showed a significant treatment effect in both groups ≤12 weeks (SMD = −5.70, 95% CI = −10.69 to −0.71) and >12 weeks (SMD = −3.33, 95% CI = −5.26 to −1.41) subgroups. Thus, even 4 weeks of treatment is enough to see significant reductions in plaque, as seen by Ming-liang Chen, B [40] using 0.4% resveratrol in the diet in female ApoE−/− mice.

Figure 3. Forest plot of individual SMD of plaque size grouped by treatment duration. Nine studies were conducted for 4–12 weeks (1), and eight studies were conducted for more than 12 weeks (2). The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent findings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

Figure 3. Forest plot of individual SMD of plaque size grouped by treatment duration. Nine studies were conducted for 4–12 weeks (1), and eight studies were conducted for more than 12 weeks (2). The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent ﬁndings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

2.2.4. Subgroup Analysis by Sex
2.2.5 Meta-Regression Results

Figure 4. Forest plot of individual SMD of plaque size grouped by sex. The analysis included six studies conducted in female mice, six studies conducted in male mice, and two studies conducted in both sexes. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent ﬁndings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

2.2.5. Meta-Regression Results

Among our pre-speciﬁed potential moderators, sex of mice (male and female), treatment type (berry, polyphenol, berberine), treatment duration (≤12 weeks and >12 weeks), and study size did not signiﬁcantly moderate the effect (p = 0.586, p = 0.728, p = 0.637, and p = 0.646, respectively). Therefore, these parameters were not a source of heterogeneity.

Among our pre-specified potential moderators, sex of mice (male and female), treatment type (berry, polyphenol, berberine), treatment duration (≤12 weeks and >12 weeks), and study size did not significantly moderate the effect (p = 0.586, p = 0.728, p = 0.637, and p = 0.646, respectively). Therefore, these parameters were not a source of heterogeneity.

2.2.6. Bias Assessment

Funnel plot examining the publication bias of studies evaluating the effect of phytochemical supplementation on atherosclerosis plaque size. Meta-trim, a nonparametric “trim-and-fill” method, was used to estimate the number of possible studies missing from the meta-analysis because of publication bias. No missing studies were found.

Figure 5. Funnel plot examining the publication bias of studies evaluating the effect of phytochemical supplementation on atherosclerosis plaque size. Meta-trim, a nonparametric “trim-and-ﬁll” method, was used to estimate the number of possible studies missing from the meta-analysis because of publication bias. No missing studies were found.

2.2.7 Simple Correlation between Plaque and F/B Ratio

From the eight studies reporting F/B ratios, a positive correlation between plaque and F/B ratio was seen; however, this correlation did not reach significance (r = 0.51, p = 0.087) (Figure 6A). Additionally, a negative correlation was seen for the control group, which was also non-significant (r = -0.01, p = 0.956) (Figure 6B).

2.2.7. Simple Correlation between Plaque and F/B Ratio

From the eight studies reporting F/B ratios, a positive correlation between plaque and F/B ratio was seen; however, this correlation did not reach signiﬁcance (r = 0.51, p = 0.087) (Figure 6A). Additionally, a negative correlation was seen for the control group, which was also non-signiﬁcant (r = −0.01, p = 0.956) (Figure 6B).

Figure 6. Scatterplot of the correlation between plaque size and F/B ratio. A positive correlation between plaque size (%) and F/B ratio was seen in the intervention groups (A), but not in the control groups (B).

Figure 6. Scatterplot of the correlation between plaque size and F/B ratio. A positive correlation between plaque size (%) and F/B ratio was seen in the intervention groups (A), but not in the control groups (B).

Scatterplot of the correlation between plaque size and F/B ratio. A positive correlation between plaque size (%) and F/B ratio was seen in the intervention groups (A), but not in the control

2.3. Probiotics 2.3.1. Aortic Plaque Size

acidilactici [43], and VSL#3 [44] and for male ApoE mice treated with Lactobacillus muco-

2.3. Probiotics 2.3.1. Aortic Plaque Size

2.3.2. Subgroup Analysis by Treatment Duration
2.3.3. Subgroup Analysis by Sex

Nine studies treating ApoE / mice with speciﬁc bacteria (Table 1, probiotics) showed a common SMD of −3.98 (95% CI: −6.29 to −1.68, p-value < 0.05) based on a random effect model, with signiﬁcant heterogeneity between studies (τ2 = 11.17, I2 = 99.64%, H2 = 277.25, Q(df = 9) = 395.72, PQ < 0.001) (Figure 7). Sensitivity analysis detected no study with unacceptable CI. Thus, the forest plot of individual effect sizes within each study showed a signiﬁcant reduction in plaque burden in the intervention group compared with controls. The most signiﬁcant effects were seen for female ApoE−/− mice treated with Pediococcus acidilactici [43], and VSL#3 [44] and for male ApoE−/− mice treated with Lactobacillus mucosae [45].

Figure 7. Forest plot of individual SMD of plaque size grouped by sex. Four studies were conducted in female mice, and 3 studies were conducted in male mice. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent ﬁndings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

2.3.2. Subgroup Analysis by Treatment Duration

Probiotic studies were also analyzed by treatment duration for studies of ≤12 weeks (group 1) and >12 weeks (group 2) treatments (Figure 7). The results of the forest plot of individual SMD showed a signiﬁcant treatment effect in both ≤12 weeks (SMD = −3.52, 95% CI = −6.54 to −0.50) and >12 weeks (SMD = −4.81, 95% CI = −6.47 to −3.16) subgroups. The shortest duration tested in the included studies was for 8 weeks. This study treated male ApoE / mice with Akkermansia muciniphila [47].

In terms of sex (F = female, M = male, Figure 8), a significant treatment effect was seen for both male (SMD = −1.95, 95% CI = −2.75 to −1.15) and female (SMD = −5.75, 95% CI = −9.57 to −1.93) subgroups.

2.3.3. Subgroup Analysis by Sex

In terms of sex (F = female, M = male, Figure 8), a signiﬁcant treatment effect was seen for both male (SMD = −1.95, 95% CI = −2.75 to −1.15) and female (SMD = −5.75, 95% CI = −9.57 to −1.93) subgroups.

Figure 8. Forest plot of individual SMD of plaque size sub-grouped by sex. Four studies were conducted on females, and three studies were conducted on males. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent ﬁndings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

Forest plot of individual SMD of plaque size sub-grouped by sex. Four studies were conducted on females, and three studies were conducted on males. The common effect size was calculated as SMD and 95% CI for each study outcome. Squares represent findings for individual studies; red diamonds represent the overall result for each sub-group analysis and the green diamond the overall effect of the meta-analysis for all the studies.

2.3.4. Meta-Regression
2.3.5. Bias Assessment

2.3.4. Meta-Regression
2.3.5. Bias Assessment Small-study bias, as measured by Egger’s and Begg’s tests, showed no significant

Small-study bias, as measured by Egger’s and Begg’s tests, showed no signiﬁcant results (p = 0.079, and p = 0.371; >0.05). However, visual assessment of the funnel plot showed slight evidence of publication bias. The nonparametric “trim and ﬁll” results showed one missing study to be included. The re-estimation of the overall SMD after adding the “missing” studies, still resulted in a signiﬁcant SMD (observed + imputed SMD = −3.56 (95% CI: −5.88 to −1.25)) (Figure 9).

Int. J. Mol. Sci. 2023, 24, 3084 ing the “missing” studies, still resulted in a significant SMD (observed + imputed SMD =11 of 19 −3.56 (95% CI: −5.88 to −1.25)) (Figure 9).

3. Discussion

Functional foods, such as berries, have shown multiple health beneﬁts in reducing CVD and its risk factors in both animal and clinical studies. Many of the beneﬁts of these foods and their components (polyphenols, alkaloids, and ﬁber) are associated with improvements in microbiome heath, assessed, in part, by reductions in the F/B ratio. However, the direct effect of the microbiome on plaque burden needs further elucidation. The present meta-analysis analyzed animal studies that assessed the effects of berries (as whole food), polyphenols, berberine (as an alkaloid), and probiotics on atherosclerotic plaque size and microbiome modulation in mouse models of atherosclerosis (ApoE−/− and LDLR−/−). The results showed that polyphenols, berberine, and probiotic interventions signiﬁcantly reduced atherosclerotic plaque size in mice. The effect of berries was not statistically signiﬁcant as there were limited studies that measured the effect of berries on the microbiome and atherosclerosis.

Polyphenols have shown promising effects in the treatment of chronic diseases as they exert powerful anti-inﬂammatory, anti-antioxidative, and cholesterol-lowering effects [50]. In human studies, a mixture of strawberries, bilberries, chokeberries, and black currants decreased systolic blood pressure and LDL, while upregulating HDL [51]. In another study, strawberry alone reduced LDL cholesterol as well as vascular cell adhesion molecule-1 (VCAM-1) levels in patients with metabolic syndrome [52]. In animal studies, blueberry [53], blackberry [21], and prunes [54] reduced plaque. Comprehensive reviews of nutritional interventions for CVD can be found in recent reviews [55–57]. However, only a limited number of studies in mice using berries as a whole food have measured plaque together with microbiome composition. We identiﬁed only two studies which used lingonberry as the intervention to reduce plaque. In one study by Matziouridou et al. [27], plaque size showed a positive correlation with Bilophila, Mucispirillum, Turicibacter, and Lactococcus and two unclassiﬁed bacterial genera in Clostridiaceae and Peptostreptococcaceae, while lingonberry increased the abundance of Akkermansia muciniphila, Blautia producta, Clostridum difﬁcile, and Eubacterium dolichum. The second study by Liu et al. [31] showed a positive correlation between plaque and Mucispirillum, Streptococcus, Peptococcaceae and Bilophila genera. Similar to the previous study, lingonberry increased the abundance of Akkermansia. Thus, for these studies increases in Mucispirillum seems to be associated with plaque in both male and female mice, while Akkermansia was upregulated by lingonberry in both sexes. Major changes in the abundance of bacteria associated with interventions is shown in Table 2.

Table 2. Summary of changes in the microbiome associated with interventions.

Effect on Plaque Berries, Polyphenols, and Alkaloids

Major Change in Bacteria by Intervention

Metabolites

N ID Genotype Sex Intervention Microbiome

Yafei Shi, 2018 [38]

TMAO

Verrucomicrobia Proteobacteria

ApoE−/− M Berberine Feces

Reduced

Table 2. Cont.

Effect on Plaque Berries, Polyphenols, and Alkaloids

Major Change in Bacteria by Intervention

Metabolites

N ID Genotype Sex Intervention Microbiome

LachnospiraceaeNK4A136, Bacteroidales S24-7 (unclassiﬁed), Eubacterium, Marvinbryantia, Clostridiales unclassiﬁed, Ruminiclostridium 5, PrevotellaceaeNK3B31, Biﬁdobacterium

Xingxing Li, 2021 [36]

ApoE−/− F Berberine Cecum

TMAO Reduced

Probiotics

Bacteroides, Bacteroidaceae, Parabacteroides and Tannerellaceae Desulfovibrionaceae, Lachnospiraceaeand Ruminococcaceae

Lactobacillus plantarum ATCC 14917

Adil Hassan,

ApoE−/− -

Feces

ND Reduced

2016 [42]

N: refers to the number of the study in Table 1; ND: not determined; garnet color indicates increase and blue color indicates reduction.

The genus Akkermansia, which belongs to the Verrucomicrobia phylum, includes mucindegrading bacteria, with the major species being Akkermansia muciniphila, a producer of acetate and propionate [58]. Akkermansia muciniphila abundance is upregulated with interventions improving metabolic disturbances. For example, its levels were elevated in obese mice treated with prebiotics, which correlated with improved metabolic status [59]. Upregulation of Akkermansia by metformin improved glucose homeostasis in diet-induced obesity in mice [60]. Additionally, the abundance of these bacteria is inversely associated with inﬂammatory diseases of the gut, such as IBD and Crohn’s disease [61].

Several of the studies we identiﬁed in this meta-analysis showed a negative correlation of Akkermansia abundance with plaque. For example, in our previous study using gallic acid, upregulation of Akkermansia correlated with a reduction in plaque in male mice. The lack of effect of gallic acid in females was associated with a reduction in Akkermansia [22]. Similarly, the plaque-reducing effects of resveratrol [40], quercetin [29], geraniin [32], procyanidinA2 [37], and berberine [23] were also associated with the upregulation of Akkermansia (Table 2). Another study using berberine [38] analyzing the microbiome at the phylum level reported an upregulation of Verrucomicrobia. Moreover, among these studies, resveratrol, geraniin and berberine supplementation [38] led to a reduction in TMAO levels. Consistent with the production of propionate, two studies using lingonberry in which Akkermansia was upregulated, reported increases in propionate [27,33].

Not all studies measured SCFAs to draw strong conclusions of the role the microbiome composition in the levels of SCFAs; however, these data suggest that Akkermansia reduces plaque burden, at least in part, by reducing the TMAO in circulation. Further evidence in favor of the protective effect of this genus in cardiovascular health was provided by Li et al. [47] using Akkermansia muciniphila, as a probiotic, to reduce plaque in male ApoE−/− mice. Interestingly, the effects of Akkermansia muciniphila were associated with reduced endotoxemia-induced inﬂammation which was independent of changes in the lipid proﬁle. More speciﬁcally, Akkermansia muciniphila reduced macrophage inﬁltration and the expression of intracellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis alpha (TNFα) in the plaque. In circulation, MCP-1 and interleukin 1 beta (IL-1β) were also reduced by treatment. Thus, the anti-atherogenic effects of the nutritional interventions reviewed here are likely mediated by anti-inﬂammatory mechanisms driven by the microbiome, in particular by Akkermansia muciniphila.

The changes in Akkermansia muciniphila were observed in different sections of the gastrointestinal tract including the cecum [27,29,33], colon [32] and feces [22,23,37]. In terms of other similarities among studies, millet shell polyphenols [31] and quercetin [29] increased Ruminococcus, and resveratrol [40] and gypenoside [41] increased Lactobacillus.

Probiotics using different strains of Lactobacillus [11,42,45,49] reported reductions in plaque, except for Lactobacillus reuteri DSM 1798. Berberine [34] and Enterobacter aerogenes ZDY01 [46] increased Turicibacter abundance, while gallic acid [22] and Lactobacillus Mucosae [45] reduced its levels. Blautia was increased by gallic acid [27] and by berberine [34] in ApoE−/− mice.

Among the probiotic studies, only four out of nine measured microbiome composition and only one measured TMAO. Several changes in the microbiome were observed. For example, the study by Hassan et al. [42] showed that treatment with Lactobacillus plantarum upregulated Bacteroides, Bacteroidaceae, Parabacteroides and Tannerellaceae and downregulated Desulfovibrionaceae, Lachnospiraceae and Ruminococcaceae, suggesting that changes in other bacteria, and not the probiotic itself, may mediate the reduction in plaque.

Alkaloids are naturally occurring compounds that have shown a wide range of protective effects, including reductions in inﬂammation, oxidative stress, and atherosclerosis [62]. For example, berberine, found in medicinal plants, has been used for centuries in the treatment of inﬂammatory diseases [63]. Similar to polyphenols, all berberine studies reduced plaque through modulation of gut microbiota composition and gut barrier function. Of the four berberine studies, three reported reductions in TMAO.

According to the results of the present study, the consumption of berries and polyphenols shows a positive correlation with the F/B ratio [22,29,31–33,37]; however, this correlation did not reach signiﬁcance since not all studies reported this ratio. Only two berberine studies reported the F/B ratio, which was not improved by the intervention [34,38].

In terms of barrier function, only one study using curcumin reported a reduction in plasma LPS levels and improved intestinal barrier function [35]. Unfortunately, this study did not report speciﬁc changes in the microbiome.

In terms of sex-dependent effects, only two studies used male and female mice, including our previous study testing gallic acid and the study by Liao et al. [39] testing tea polyphenols. As mentioned before, Akkermansia abundance showed a negative correlation with plaque since Akkermansia was increased in males (decreased plaque) and reduced in females (no effect on plaque) with gallic acid treatment. For the tea polyphenols study, only Biﬁdobacterium was measured. Thus, it is unknown if other bacteria genera were affected by treatment. However, overall sex difference analysis showed that berries, polyphenols, and berberine were more effective in male mice, while probiotic supplementation was effective in both sexes. Studies using both sexes are needed to evaluate the role of sex in the effects of the microbiome in atherosclerosis. It is possible that diet composition (fat and sugar in HFD), age, and hormone status may play a role in the effectiveness of dietary interventions in females.

Duration sub-group analysis showed that both ≤12 weeks and >12 weeks interventions were effective. The minimum duration examined in the analyzed studies was 4 weeks for phytochemicals and 8 weeks for probiotics, suggesting that this time is sufﬁcient to induce signiﬁcant changes in the microbiome and plaque burden.

The present study has some limitations. First, the number of studies evaluating the effect of whole berries supplementation on atherosclerosis plaque was low. Second, different types of polyphenol and probiotics supplementation have been used in different studies, although it is generally accepted that different types of polyphenols and probiotics have cardioprotective efﬁcacy. Third, signiﬁcant heterogeneity was found in most of the analyzed parameters, and the source of the heterogeneity was not explored further. Only random-effect models were used to address heterogeneity, which may have affected the strength and extrapolation of our conclusions. Forth, the F/B ratio was reported only in eight studies, which were conducted for different treatment durations. Fifth, there is insufﬁcient data to evaluate the reasons female animals were not signiﬁcantly affected by the supplementations that were effective in male animals. Additionally, only two studies used both sexes to assess the effect of the intervention. Further, in the probiotic studies, analyses of microbiome composition and gut-derived metabolites were needed to identify health-promoting bacteria. More studies are needed to identify the effective phytochemical types and probiotic strains as well as the effective dose to be proposed for therapeutic interventions. Finally, fecal transplantation studies are needed to demonstrate that microbiome modulation is the driver of the protective effects seen in the berry, polyphenols and berberine studies, as demonstrated by Li et al. [47]

In summary, this meta-analysis suggests that modulation of the microbiota by lingonberry, polyphenols, berberine and probiotics reduces plaque by a mechanism mediated, in part, by the upregulation of Akkermansia and a reduction in TMAO. We propose that a probiotic aimed to reduce plaque in patients suffering from cardiovascular disease should contain Akkermansia muciniphila, and other bacteria upregulated by different types of intervention, such as Blautia (lingonberry and berberine), Turicibacter (berberine and Enterobacter aerogenes) and different strains of Lactobacillus (plantarum, rhamnosus and mucosae).

4. Materials and Methods

4.1. Search Strategy

A search in databases including PubMed, Web of Science, Science Direct, and Embase was performed until November 2022 for studies investigating the effects of phytochemical and probiotic supplementation on gut microbiota and atherosclerosis plaque size in mice.

The following search terms were used: (Berry OR Polyphenol OR Alkaloid OR Berberine OR Probiotic) AND (Atherosclerosis) AND (Plaque OR Lesion) AND (Gut microbiota OR Gut microbiome) AND (ApoE / OR LDLR /−) AND (Mice OR Mouse). Only studies published in English were included.

4.2. Inclusion and Exclusion Criteria
4.3. Statistical Analyses

The analyses were performed using STATA17 (StataCorp, College Station, TX, USA). PRISMA was used for the reporting of presented studies, as this is the preferred platform for reporting data for systematic reviews and meta-analyses [64].

Random effect meta-analyses were performed using a restricted maximum likelihood approach [65]. As there might be unknown or unregistered studies that could not be accessed, the random-effect model was used. The heterogeneity between studies was evaluated using the Cochran I-Squared, Tau-squared, and Q tests. A considerable heterogeneity included values higher than 75% for I-squared [66]. The common effect size was reported as SMD (standardized mean difference) and 95% CI (conﬁdence interval) for each study.

Funnel plots and Egger’s [67] and Begg’s [68] tests were conducted to assess publication bias. Meta-trim was used in case of evidence for probable publication bias. This nonparametric “trim-and-ﬁll” method estimates the number of potentially missing studies, imputes them, and re-computes the overall effect-size using the observed and imputed studies [67]. Furthermore, meta-regression analysis was performed to ﬁnd the source of heterogeneity. Meta-regression investigates whether the moderators (sex, treatment type, treatment duration, and study size, in this study) can explain between-study heterogeneity. In addition, sensitivity analyses were conducted using the leave-one-out method, in which, one study is removed each time and the meta-analysis is carried out for the rest of the studies.

The Pearson correlation coefﬁcient was used to assess the correlation between plaque and the F/B ratio.

5. Conclusions

The present meta-analysis suggests that consuming polyphenols, berries (a rich source of polyphenols), berberine (as an alkaloid), and probiotics can improve atherosclerosis plaque size in mouse models of atherosclerosis. Moreover, this study revealed that in addition to their direct effect on cardiometabolic health, polyphenols, berries, and berberine could affect aortic plaque size through modulation of gut microbiota composition, as well as probiotic consumption. The other important ﬁnding of this meta-analysis was that the probiotic supplements reduce plaque in both sexes. In contrast, polyphenols, berries, and berberine showed plaque reducing effects mainly in male mice. In addition, Akkermansia muciniphila emerged as an important strain upregulated by several interventions. These ﬁndings may inform clinical trials using probiotics containing Akkermansia muciniphila to improve human health.

Author Contributions: Conceptualization, G.S., L.K and A.M.C.; methodology, L.K. and A.M.C.; data analysis, L.K. and A.M.C.; formal analysis, L.K. and A.M.C.; investigation, L.K. and A.M.C.; writingoriginal draft preparation, L.K.; writing—review and editing, G.S., L.K. and A.M.C.; supervision, G.S.; project administration, G.S.; funding acquisition, G.S. All authors have read and agreed to the published version of the manuscript.

Funding: This research was supported by the US Department of Agriculture (USDA-AFRI, GRANT12444832) and the Florida Department of Health, James and Esther King Biomedical Research Program (9JK01).

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Figures

Figure 1

Overview of the relationship between gut microbiota, dietary phytochemicals, and atherosclerosis development. The diagram illustrates how berries, polyphenols, and probiotics may modulate microbial composition to influence cardiovascular risk factors.

diagram

Figure 2

PRISMA flow diagram for the meta-analysis of animal studies investigating berries, phytochemicals, and probiotics on atherosclerosis through gut microbiota modification. Study identification, screening, and inclusion criteria are documented.

flowchart

Figure 3

Forest plot summarizing the pooled effect of polyphenol-rich interventions on atherosclerotic plaque area in animal models. Heterogeneity across studies reflects differences in polyphenol type, dosage, and animal model used.

forest_plot

Figure 4

Forest plot of probiotic effects on atherosclerosis endpoints in animal studies, showing individual study contributions and the pooled estimate. Lactobacillus and Bifidobacterium strains are the most frequently tested probiotic interventions.

forest_plot

Figure 5

Publication and editorial information for the meta-analysis of phytochemicals and probiotics on atherosclerosis through gut microbiota modification.

Figure 6

Mechanisms linking gut microbiota dysbiosis to atherosclerosis progression, including TMAO production, bile acid metabolism, and systemic inflammation pathways. The data from this meta-analysis of animal studies support a role for microbiome modulation in cardiovascular protection.

diagram

Figure 7

Classification of berry-derived phytochemicals investigated for anti-atherosclerotic properties, organized by chemical class including anthocyanins, ellagitannins, and proanthocyanidins. The data from this meta-analysis of animal studies support a role for microbiome modulation in cardiovascular protection.

Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modification: A Meta-Analysis of Animal Studies.

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Study Design

Abstract

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Molecular Sciences

Effects of Berries, Phytochemicals, and Probiotics on Atherosclerosis through Gut Microbiota Modiﬁcation: A Meta-Analysis of Animal Studies

Leila Khalili 1,†, Ann Marie Centner 1,† and Gloria Salazar 1,2,*

1. Introduction

2. Results

3. Discussion

Probiotics

4. Materials and Methods

5. Conclusions

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