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1 These authors contributed equally to this work and share first authorship.
Han Hao
Footnotes
1 These authors contributed equally to this work and share first authorship.
Affiliations
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
1 These authors contributed equally to this work and share first authorship.
Zhu Li
Footnotes
1 These authors contributed equally to this work and share first authorship.
Affiliations
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
1 These authors contributed equally to this work and share first authorship.
Shi-yang Qiao
Footnotes
1 These authors contributed equally to this work and share first authorship.
Affiliations
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
State Key Laboratory of Pharmaceutical Biotechnology, Department of Cardiology, Affiliated Drum Tower Hospital, Medical School, Nanjing University, No.321, Zhongshan Road, Nanjing, 210008, China
Empagliflozin improves atherosclerosis to some extent by affecting intestinal bacteria.
•
The benefits of Empagliflozin can be delivered through the intestinal flora.
•
It is a novel thought that more drugs that have been used may act in part by influencing intestinal flora.
Abstract
Background and aims
Sodium-glucose cotransporter 2 inhibitor (SGLT2i) has been reported to attenuate atherosclerosis. Further, it has been suggested that intestinal flora influences atherosclerosis progression. Herein we aimed to investigate whether SGLT2i can alleviate atherosclerosis through intestinal flora.
Methods
Six-week-old male ApoE−/− mice fed a high-fat diet were gavaged either empagliflozin (SGLT2i group, n = 9) or saline (Ctrl group, n = 6) for 12 weeks. Feces were collected from both groups at the end of the experiment for fecal microbiota transplantation (FMT). Another 12 six-week-old male ApoE−/− mice were fed a high-fat diet and received FMT with feces either from SGLT2i (FMT-SGLT2i group, n = 6) or from Ctrl (FMT-Ctrl group, n = 6) groups. Blood, tissue, and fecal samples were collected for subsequent analyses.
Results
In comparison with Ctrl group, atherosclerosis was less severe in the SGLT2i group (p < 0.0001), and the richness of probiotic, such as f_Coriobacteriaceae, f_S24-7, f_Lachnospiraceae, and f_Adlercreutzia, was higher in feces. Besides, empagliflozin resulted in a significant reduction in the inflammatory response and altered intestinal flora metabolism. Interestingly, compared with FMT-Ctrl, FMT-SGLT2i also showed a reduction in atherosclerosis and systemic inflammatory response, as well as changes in the component of intestinal flora and pertinent metabolites similar to SGLT2i group.
Conclusions
Empagliflozin seems to mitigate atherosclerosis partly by regulating intestinal microbiota, and this anti-atherosclerotic effect can be transferred through intestinal flora transplantation.
Atherosclerosis (AS) is a major pathological basis of atherosclerotic cardiovascular disease, which is the leading cause of death worldwide, particularly in developed countries. The anti-atherosclerotic effects of sodium-glucose cotransporter 2 inhibitor (SGLT2i) have been demonstrated in several experimental models. Pennig et al. [
] found that empagliflozin treatment reduced not only atherosclerotic plaque areas but also inflammatory cell infiltration in adipose tissue. In our previous study, we found that empagliflozin exerted anti-atherosclerotic effects in non-diabetic states by inhibiting the renin–angiotensin–aldosterone system and sympathetic nerve activity [
] showed that plaques from asymptomatic patients with AS contained more host normal flora (e.g., Porphyromonadaceae and Bacteroidaceae), while those from symptomatic patients with AS displayed noticeably higher abundance of pathogenic bacteria (e.g., Helicobacteraceae and Streptococcaceae). Karlsson et al. [
] proved that altered microbial composition was associated with an increased abundance of genes involved in inflammatory processes in patients. The abundance of intestinal microorganisms, such as Bacteroides, Clostridium, and Lactobacillales, has been proven to be predictive of coronary artery disease [
Characterization of gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism: gut microbiota could be a diagnostic marker of coronary artery disease.
] found an inverse relationship between the relative depletion of butyric acid-producing flora (e.g., Roseburia and Eubacterium) and progression of AS. Meanwhile, some studies have demonstrated that SGLT2i improves the pattern of intestinal flora in model mice. Hata et al. [
] found that treatment with luseogliflozin enhanced the abundance of intestinal microbes involved in the synthesis of short-chain fatty acids (SCFAs), leading to improved amino acid metabolism. In addition, Yang et al. [
] showed that treatment with dapagliflozin increased the abundance of probiotics in the intestine of a type 2 diabetic rat model.
Considering that it remains unclear whether SGLT2i exerts anti-atherosclerotic effects via intestinal flora modulation, in this study, an experimental model of AS was established to explore whether oral SGLT2i treatment modulates intestinal flora to alleviate AS.
2. Materials and methods
2.1 Experimental animals
Fifteen ApoE−/− male mice (from GemPharmatech Co., Ltd, Jiangsu, China) were housed in a specific pathogen-free (SPF) environment, kept under a 12:12-h light–dark cycle, and provided ad libitum access to a high-fat diet (HFD) and water. The animals were treated with 10 mg/kg empagliflozin (SGLT2i group, n = 9) [
] by gavage and control group (Ctrl group, n = 6) received an equivalent amount of saline once a day, 5 days a week for 12 weeks. At the age of 18 weeks, mice were sacrificed, and blood and tissue samples were collected for analyses. Mice were measured random blood glucose levels at the beginning and end of the experiment.
2.2 Fecal sample collection and fecal microbiota transplantation (FMT)
Feces from the SGLT2i and Ctrl groups were collected separately and immediately stored at −80 °C. Fecal samples were then subjected to macrogenomic and metabolomic analyses in the final step of the experiment. All fecal samples were collected at 12th week after gavage. For FMT [
], feces were diluted and homogenized in sterile phosphate buffered saline (PBS) (100 mg feces in 2 mL buffer) for 5 min. The supernatant was collected by agitation for 1 min and centrifugation at 800 rpm for 3 min. This supernatant was administered to 6-week-old male ApoE−/− mice every 2 weeks for 12 weeks. Mice in FMT-SGLT2i (n = 6) were treated with the supernatant from the SGTL2i, and mice in FMT-Ctrl (n = 6) were treated with the supernatant from the Ctrl.
2.3 Histological analysis
Following euthanasia, the thoracic cavity of mice was opened and perfused with PBS. After periarterial connective tissue and fat were removed, the artery was separated from the aortic root to the iliac bifurcation and fixed in 4% paraformaldehyde for over 24 h. The blood vessels were rinsed with PBS to clear the fixative and then longitudinally dissected. The arteries were dehydrated in isopropanol for 3 min before staining with oil red O in darkness. Then, the arteries were washed three times with 60% isopropanol for 3 min each time, followed by a PBS wash. Finally, arterial lipids were observed and photographed.
2.4 Intestinal metabolite assay
Before the end of the experiment, feces were obtained by massaging the hypogastrium and perineum of mice in SPF environment. Each sample weight was >2.0 g. Fecal samples obtained from the four groups of mice were kept at −80 °C and then delivered to Shanghai Prof Leader Biotech Co. Ltd. For the analysis of bile acids (BAs), SCFAs, and trimethylamine N-oxide (TMAO).
2.5 Multiplex enzyme-linked immunosorbent assay
Cytokine levels were analyzed using LEGENDplex™ Th Cytokine and Inflammation panels. The dilution of the serum of the whole blood and standard curve was performed in Matrix C reagent and dyed in accordance with manufacturer instructions (BioLegend, San Diego, CA). Bead–antibody–protein complexes were fixed and run on an LSR II flow cytometer using FACSDiva acquisition software. Data were analyzed using the software provided with the LEGENDplex™ kit.
2.6 Statistical analyses
GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analyses. Values represent mean ± standard deviation or standard error of mean. Student's unpaired t-test or Wilcoxon (Mann–Whitney U) test were used to compare two groups, followed by Welch's t-test when standard deviation was not the same. When two or more groups were involved, we used one-way analysis of variance (ANOVA), followed by Tukey test. Non-parametric multi-group comparisons (Kruskal–Wallis) were performed for non-normally distributed variables. Intestinal microbiota analysis was performed by the genescloud tools, a free online platform for data analysis (https://www.genescloud.cn). p < 0.05 indicated statistically significant differences.
3. Results
3.1 Empagliflozin retarded the progression of AS
To determine whether empagliflozin affects AS progression, we first treated the SGLT2i group with empagliflozin and the Ctrl group with an equivalent amount of saline. Relative to Ctrl group, SGLT2i group showed considerably less aortic plaque area (percentage of aortic plaque area: SGLT2i, 18.98% ± 1.049% vs. Ctrl, 30.55% ± 1.897%, p < 0.0001). However, random blood glucose levels did not show a significant change before and after the experiment (p > 0.05, Supplementary Fig. 5). Further, to verify whether empagliflozin acts by affecting intestinal microbiota, we analyzed plaque areas in FMT-SGLT2i and FMT-Ctrl groups and found that FMT-SGLT2i group showed less aortic plaque area than FMT-Ctrl group (percentage of aortic plaque area: FMT-SGLT2i, 24.59% ± 1.321% vs. FMT-Ctrl, 29.01% ± 2.230%, p < 0.05) (Fig. 1). These findings indicated that empagliflozin reduces AS progression, which seems to be partly achieved by affecting intestinal microbiota.
Fig. 1Representative aortic plaque of mice from each group
(A) Diagram of the experimental design. (B) Aortic oil red staining. (C) Percentage of aortic plaque area (SGLT2i, Ctrl, n = 5; FMT- SGLT2i, FMT-Ctrl, n = 3, ****: p < 0.0001; *: p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.2 Empagliflozin affected the structure of intestinal flora
To further explore the influence of empagliflozin on intestinal bacteria, we assessed intestinal flora in feces collected from Ctrl and SGLT2i groups by 16S ribosomal RNA (16S rRNA) gene sequencing. The average sequencing depth per sample was approximately 60,000 reads/sample, which was saturated to detect intestinal bacteria (Fig. 2A). The Firmicutes/Bacteroidetes (F/B) ratio in SGLT2i group was much lower than that in Ctrl group (Fig. 2B–D). Although Shannon's index, Simpson's index, and Pielou's evenness did not substantially differ between the groups (Fig. 3A), ASV-based principal coordinate analysis (PCoA) revealed considerable differences in intestinal flora between the groups (Fig. 3B). On analysis of similarities (ANOSIM) analysis, we found a remarkable variation in intestinal flora composition between SGLT2i and Ctrl groups (Fig. 3C). Further, metagenomeSeq analysis indicated significantly higher levels of Coriobacteriaceae, S24-7 (also known as Muribaculaceae), and Lachnospiraceae in SGLT2i group (Fig. 3D). LDA (Linear discriminant analysis) effect size analysis showed that at the genus level, Lactobacillus, Subdoligranulum, and Clostridium were considerably enriched in feces of mice treated with empagliflozin than in their counterparts. In contrast, pathogenic bacteria [e.g., g_Dorea [
(A) Species rarefaction curves in stool samples from mice. The slope of the curve for each sample is close to zero when the sequencing depth is sufficient to cover most of the intestinal flora. (B) Firmicutes/Bacteroidetes ratio of stool (F/B), Wilcoxon rank-sum test was used for statistical tests, *: p < 0.05 (SGLT2i, n = 10; Ctrl, n = 5). (C and D) Pie chart of the five most abundant bacterial phyla, SGLT2i group and Ctrl group.
(A) α diversity index. (B) PCOA analysis. (C) Anosim analyses, *: p < 0.05 (SGLT2i, n = 45; Ctrl, n = 50). (D) MetagenomeSeq analysis: the horizontal coordinate is the order of ASV/OTU according to its English taxonomic information (from phylum to species); the ordinate is the -log10 value. The dotted line separates significant differences (above) from non-significant ASV/OTU, and the points of significant differences are marked with colored dots or rings. The color of the dot identifies its gate level name and is marked at the bottom of figure. (E) The ordinate represents the taxa with significant differences between groups, while the ordinate shows the logarithmic score of LDA analysis of each taxa in a bar graph. (F) LDA effect size analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fecal samples collected from FMT-SGLT2i and FMT-Ctrl groups were also subjected to 16S rRNA gene sequencing to determine if changes in intestinal flora reduced AS progression. The average sequencing depth remained at 60,000 reads/sample; this depth reached saturation, and no more intestinal bacteria were detectable (Fig. 4A). Similar to the results presented above, relative to FMT-Ctrl group, FMT-SGLT2i group showed a significant reduction in the F/B ratio (Fig. 4B–D); however, there was no statistical difference in Shannon's index, Simpson's index, and Pielou's evenness (Fig. 5A). In contrast, PCoA revealed a clear distinction in intestinal flora between the groups (Fig. 5B). Meanwhile, ANOSIM analysis demonstrated a statistically significant difference in intestinal flora composition between FMT-SGLT2i and FMT-Ctrl groups (Fig. 5C). MetagenomeSeq analysis indicated similar changes in feces from both the groups; for example, at the family level, a notable rise was observed in the abundance of Coriobacteriaceae, S24-7, Lachnospiraceae, and Ruminococcaceae (Fig. 5D). At the genus level, Adlercreutzia, Subdoligranulum, Oscillospira, Coprococcus, Clostridium, and Anaerotruncus were much more prevalent in the FMT-SGLT2i group than in the FMT-Ctrl group. The abundance of pathogenic bacteria (e.g., Helicobacteraceae_Helicobacter) (Fig. 5E) also particularly appeared to increase [
Helicobacter hepaticus induce colitis in male IL-10(-/-) mice dependent by cytolethal distending toxin B and via the activation of Jak/Stat signaling pathway.
]. In addition, we determined the overall composition of intestinal flora in the SGLT2i, Ctrl, FMT-SGLT2i, and FMT-Ctrl groups, the performance of five bacteria with relatively high abundance at the phylum level over time, and heat maps at the genus and family levels (Supplementary Figs. 1–2).
Fig. 4Fecal microbiota transplantation produced similar results
(A) Species rarefaction curves in stool samples from mice transplanted with faecal bacteria. The slope of the curve for each sample is close to zero when the sequencing depth is sufficient to cover most of the intestinal Flora. (B) Firmicutes/Bacteroidetes ratio of stool (F/B), Wilcoxon rank-sum test was used for statistical tests, **: p < 0.01 (n = 5). (C and D) Pie chart of the five most abundant bacterial phyla, FMT-SGLT2i and FMT-Ctrl groups.
(A) α diversity index. (B) PCOA analysis. (C) Anosim analyses, *: p < 0.05 (FMT-SGLT2i, n = 6; FMT-Ctrl, n = 16). (D) MetagenomeSeq analysis: differences between FMT-SGLT2i and FMT-Ctrl at family level. (E) MetagenomeSeq analysis: differences between FMT-SGLT2i and FMT-Ctrl at genus level.
3.3 Empagliflozin regulated BA metabolism of the intestinal flora
Empagliflozin was found to alter intestinal flora profile. Next, we aimed to elucidate whether empagliflozin regulates intestinal flora metabolism. Feces from the four groups were analyzed. In comparison with Ctrl group, we found a considerable decrease in the level of cholic acid (CA) (CA: SGLT2i, 0.013 ± 0.021 μg/mg vs. Ctrl, 0.18 ± 0.21 μg/mg, p < 0.05) in fecal BAs in the SGLT2i group, as well as a trend of decrease in the level of lithocholic acid (LCA) (LCA: SGLT2i, 0.010 ± 0.004 μg/mg vs. Ctrl, 0.023 ± 0.010 μg/mg, p < 0.05) and deoxycholic acid (DCA) (DCA: SGLT2i, 0.18 ± 0.12 μg/mg vs. Ctrl, 0.41 ± 0.21 μg/mg, p < 0.05). Similarly, as shown in Supplementary Figs. 3A–C, CA (CA: FMT-SGLT2i, 0.0029 ± 0.0030 μg/mg vs. FMT-Ctrl, 0.11 ± 0.06 μg/mg, p < 0.01), LCA (LCA: FMT-SGLT2i, 0.004 ± 0.002 μg/mg vs. FMT-Ctrl, 0.011 ± 0.004 μg/mg, p < 0.05), and DCA (DCA: FMT-SGLT2i, 0.06 ± 0.03 μg/mg vs. FMT-Ctrl, 0.19 ± 0.10 μg/mg, p < 0.05) levels were lower in the FMT-SGLT2i group than in the FMT-Ctrl group.
In addition, SCFAs were detected, but no apparent discrepancy was observed in fecal levels of acetate, propionate, and butyrate, with the exception of valerate, the level of which was markedly lower in the SGLT2i group than in the Ctrl group (valerate content: SGLT2i, 5.646 ± 2.507 μg/g vs. Ctrl, 27.73 ± 21.46 μg/g, p < 0.05); similar results were recorded for the FMT groups (valerate content: FMT-SGLT2i, 1.78 ± 2.809 μg/g vs. FMT-Ctrl, 34.12 ± 7.132 μg/g, p < 0.05) (Supplementary Fig. 3D).
To assess systemic inflammation status, plasma inflammatory factor levels were measured. As shown in Supplementary Fig. 4, relative to Ctrl group, levels of the pro-inflammatory factors TNF-α (TNF-α: SGLT2i, 2.712 ± 1.018 pg/mL vs. Ctrl, 5.796 ± 1.223 pg/mL, p < 0.01) and IL-6 (IL-6: SGLT2i, 3.638 ± 0.568 pg/mL vs. Ctrl, 8.160 ± 1.769 pg/mL, p < 0.001) were dramatically decreased in the SGLT2i group and those of the anti-inflammatory factors IL-4 (IL-4: SGLT2i, 4.738 ± 0.728 pg/mL vs. Ctrl, 2.088 ± 0.413 pg/mL, p < 0.001) and IL-10 (IL-10: SGLT2i, 4.852 ± 1.191 pg/mL vs. Ctrl, 2.208 ± 0.197 pg/mL, p < 0.001) were significantly higher. Similar results were obtained for FMT groups. Relative to the FMT-Ctrl group, the FMT-SGLT2i group showed lower levels of TNF-α (TNF-α: FMT-SGLT2i, 2.278 ± 0.9873 pg/mL vs. FMT-Ctrl, 4.568 ± 0.6895 pg/mL, p < 0.01) and IL-6 (IL-6: FMT-SGLT2i, 4.025 ± 0.483 pg/mL vs. FMT-Ctrl, 6.668 ± 1.558 pg/mL, p < 0.05) and higher levels of IL-4 (IL-4: FMT-SGLT2i, 5.956 ± 1.016 pg/mL vs. FMT-Ctrl, 2.224 ± 0.918 pg/mL, p < 0.001) and IL-10 (IL-10: FMT-SGLT2i, 3.736 ± 0.598 pg/mL vs. FMT-Ctrl, 2.356 ± 0.255 pg/mL, p < 0.01).
4. Discussion
The primary finding of this study was that empagliflozin significantly attenuated AS in HFD-fed ApoE−/− mice, which was accompanied by changes in the structure of intestinal flora and alleviation of systemic inflammation. Moreover, empagliflozin modulated BA metabolism of intestinal flora. We also observed that anti-atherosclerotic effects of empagliflozin could be transmitted through intestinal flora.
We found that AS and systemic inflammatory response were less severe in SGLT2i group. Our results were in line with those of several earlier studies. Kohlmorgen et al. [
] showed that dapagliflozin-mediated atherosclerotic protection was achieved by elevating HDL-C and improving thrombin-platelet-mediated inflammation without interfering with hemostasis. Further, Kim et al. [
] showed that empagliflozin reduced NLRP3-type inflammasome activation and IL-1 secretion, resulting in cardioprotective effects. It has been indicated that the underlying mechanics of cardioprotection by SGLT2i might be related to an improvement in vascular endothelial cell function, inhibition of oxidative stress, suppression of inflammation, and regulation of autophagy, further preventing AS progression [
] suggested that the hypoglycemic effect of empagliflozin could alter the inflammatory state and/or oxidative stress, thereby preventing endothelial dysfunction and AS development. However, several studies and our data suggest that SGLT2i mitigates AS independently of its glucose-lowering effects. As reported by Han et al. [
], in non-diabetic ApoE−/−mice, empagliflozin was more effective in attenuating atherogenesis than another class of an anti-diabetic drug (i.e., glimepiride), suggesting that empagliflozin exerts atheroprotective effects independent of glucose level reduction. There is also partial evidence that the effect of empagliflozin on improving endothelial dysfunction (which is the initial step in AS) is not dependent on its glucose-lowering effect. For example, Mone et al. [
SGLT2 inhibition via empagliflozin improves endothelial function and reduces mitochondrial oxidative stress: insights from frail hypertensive and diabetic patients.
] showed that empagliflozin significantly attenuated mitochondrial Ca2+ overload and the subsequent increase in ROS production in human endothelial cells and improved endothelial cell permeability, which went beyond its effects attributed solely to lowering blood glucose or counteracting insulin resistance. Further studies are warranted to explain this contradiction.
The effects of intestinal flora have been extensively validated in recent years. Intestinal flora profile is influenced by multiple variables, including area of residence, diet, lifestyle, and pharmacological interventions [
]. We found that empagliflozin significantly altered intestinal flora structure in HFD-fed ApoE−/− mice, which is supported by several previous studies. Lee et al. [
] also observed that empagliflozin improved intestinal microbiota structure in patients with type 2 diabetes. The ratio of F/B is reflective of intestinal health status [
Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related gut-liver axis activation.
]. In our experiments, the F/B ratio was significantly lower in mice treated with SGLT2i, suggesting that SGLT2i contributes to a change in intestinal microecology toward a healthy state. Besides, it has been shown that empagliflozin alters the composition of intestinal microbiota and promotes the richness and diversity of intestinal microbiota in type 2 diabetes mellitus mice [
]. Therefore, in this study, to compare intestinal flora composition among the four groups of mice, we performed α- and β-diversity tests. The former revealed no significant differences, indicative of no statistical differences in intestinal flora diversity, richness, or homogeneity. However, on β-diversity analysis, PCoA revealed marked differences between SGLT2i and Ctrl groups and between FMT-SGLT2i and FMT-Ctrl groups, indicating that intestinal flora was composed of distinct types of bacteria. Similarly, Du et al. [
Potent sodium/glucose cotransporter SGLT1/2 dual inhibition improves glycemic control without marked gastrointestinal adaptation or colonic microbiota changes in rodents.
] found that SGLT1/2 dual inhibition improved glycemic control in rodents without significant colonic microbiota changes. This discrepancy in results can be attributed to different lysis techniques, which can affect α-diversity at both quantitative and qualitative levels [
]. Therefore, we believe that empagliflozin does not significantly alter the total number of strains or species but rather the type of bacteria in intestinal flora. Further studies are needed to validate this hypothesis.
Herein in mice treated with empagliflozin, we found a notable improvement in the abundance of Coriobacteriaceae, S24-7, and Lachnospiraceae at the family level and in that of Lactobacillus, Subdoligranulum, and Clostridium at the genus level; it is notable that they are major flora and probiotics in the healthy intestine [
]. Similar results were obtained for the FMT-SGLT2i group: the prevalence of Coriobacteriaceae, S24-7, Lachnospiraceae, and Ruminococcaceae was higher at the family level and that of Adlercreutzia, Subdoligranulum, Oscillospira, Coprococcus, Clostridium, and Anaerotruncus was higher at the genus level. The latter six are associated with butyrate as well as cholinesterase production [
Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related gut-liver axis activation.
Potent sodium/glucose cotransporter SGLT1/2 dual inhibition improves glycemic control without marked gastrointestinal adaptation or colonic microbiota changes in rodents.
Helicobacter hepaticus induce colitis in male IL-10(-/-) mice dependent by cytolethal distending toxin B and via the activation of Jak/Stat signaling pathway.
Faecal microbiota transplantation alters gut microbiota in patients with irritable bowel syndrome: results from a randomised, double-blind placebo-controlled study.
]. On performing FMT in ApoE−/− mice fed a high-fat diet, we found that compared to FMT-Ctrl group, FMT-SGLT2i showed less severe AS and inflammation. Several previous studies have also confirmed the effectiveness of FMT. Zhang et al. [
] transplanted young microbiota into aged rats to restore intestinal structure and NLRP3-inflammasome activity in the atria and to inhibit atrial fibrillation associated with ageing. Further, Li et al. [
] demonstrated that the therapeutic effects of dicyclomine on AS could be delivered by FMT. Our results suggested that empagliflozin partly reduces AS by altering intestinal flora profile; moreover, this anti-atherosclerotic effect could be transmitted through intestinal flora.
The major metabolites of intestinal flora include, for example, TMAO, SCFAs, and BA. Increasing studies exist on TMAO and SCFAs, but BAs have not been extensively studied. Yaribeygi et al. showed that SGLT2 inhibitors regulate lipid metabolism via at least five cellular pathways: lipid biogenesis (lipogenesis and lipolysis), lipid peroxidation, lipid transport, cholesterol biosynthesis, and fatty acid β-oxidation [
], with cholesterol metabolism involving BAs. We focused on BAs in feces of mice. In the feces of mice treated with empagliflozin or FMT from mice treated with empagliflozin, there was less excretion of BAs. Such a reduction in fecal BA levels has been reported to be related to intestinal farnesoid x receptor (FXR) activation. The activation of FXR promotes reverse cholesterol transport by macrophages, which has an anti-atherosclerotic effect [
]. Thus, according to our data, empagliflozin seems to regulate FXR activity by affecting BA levels, thereby modulating reverse cholesterol transport. Further studies are warranted to verify these results.
In addition, we found that valeric acid were significantly lower in the SGLT2i and FMT-SGLT2i groups. Valeric acid is known to promote immune response through GPR41/43 and is currently only detectable in feces [
The multiple effects of fecal microbiota transplantation on diarrhea-predominant irritable bowel syndrome (IBS-D) patients with anxiety and depression behaviors.
]. The level of valeric acid is significantly elevated in feces of patients with inflammatory bowel disease, which seems to play a role in promoting intestinal inflammation [
]. Therefore, treatment with empagliflozin may lead to a reduction in pathogenic bacteria associated with valeric acid synthesis, resulting in amelioration of intestinal and systemic inflammation.
To our knowledge, this is the first study to elucidate the anti-atherosclerotic effect of SGLT2i from the perspective of intestinal flora. Nevertheless, this study has some limitations. As the anti-atherosclerotic effects of estrogen have been widely demonstrated [
], only male ApoE−/− mice were used in this study. Therefore, further studies in female counterparts are needed to explore the anti-atherosclerotic effects of empagliflozin through the regulation of intestinal flora. Besides, future studies should focus on exploring the molecular mechanisms of which empagliflozin affects AS through intestinal flora regulation.
4.1 Conclusions
Our results revealed that empagliflozin can partly retard AS progression through regulating intestinal flora, it can also influence intestinal flora metabolism and reduce inflammation. All these benefits could be transmitted upon FMT. To our knowledge, this is the first study to investigate the protective effects of SGLT2i against atherosclerosis in the context of intestinal flora. Future studies should focus on analyzing the characteristics of intestinal flora in healthy individuals so that new drugs can be developed that conform to the laws of self-regulation in humans and provide novel ideas for cardiovascular disease treatment.
Financial support
This research was funded and sponsored by the Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (Grant Numbers: ZKX20018). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors thank everyone in the team for collecting and analyzing the data for this study.
Appendix A. Supplementary data
The following is/are the supplementary data to this article:
Characterization of gut microbiota profiles in coronary artery disease patients using data mining analysis of terminal restriction fragment length polymorphism: gut microbiota could be a diagnostic marker of coronary artery disease.
Helicobacter hepaticus induce colitis in male IL-10(-/-) mice dependent by cytolethal distending toxin B and via the activation of Jak/Stat signaling pathway.
SGLT2 inhibition via empagliflozin improves endothelial function and reduces mitochondrial oxidative stress: insights from frail hypertensive and diabetic patients.
Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related gut-liver axis activation.
Potent sodium/glucose cotransporter SGLT1/2 dual inhibition improves glycemic control without marked gastrointestinal adaptation or colonic microbiota changes in rodents.
Faecal microbiota transplantation alters gut microbiota in patients with irritable bowel syndrome: results from a randomised, double-blind placebo-controlled study.
The multiple effects of fecal microbiota transplantation on diarrhea-predominant irritable bowel syndrome (IBS-D) patients with anxiety and depression behaviors.
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