Endurance exercise ameliorates Western diet–induced atherosclerosis through modulation of microbiota and its metabolites

The World Health Organization determined cardiovascular disease to be the leading cause of death globally; atherosclerosis is the primary cause of the high morbidity and mortality rates. Regular physical activity is an effective strategy for maintaining endothelial health and function to prevent the development of atherosclerosis. Obesity is also a crucial risk factor for atherosclerotic progression in combination with various complications and systemic inflammation. Physiological homeostasis is modulated by the intestinal microbiota, but the mechanisms through which exercise attenuates atherosclerosis through the microbiota have not been elucidated. Therefore, we investigated the effects of endurance exercise on atherosclerosis induced by a Western diet (WD) and apolipoprotein E (ApoE) knockout in terms of microbiota parameters and metabolites. Genetically modified ApoE knockout mice (C57BL/6-Apoeem1Narl/Narl, ApoEKO) and wild-type mice (C57BL6/J) were divided into the following four groups (n = 6), namely, wild-type mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX), for a 12-week intervention. The WD significantly induced obesity and atherosclerotic syndrome in the ApoE WD group. Severe atherosclerotic lesions and arterial thickness were significantly elevated and accompanied by increases in VCAM-1, MCP-1, TNF-α, and IL-1β for immune cell chemotaxis and inflammation during atherosclerotic pathogenesis in the ApoE WD group. In addition, dysbiosis in the ApoE WD group resulted in the lowest short-chain fatty acid (SCFA) production. Endurance exercise intervention (ApoE WD EX) significantly alleviated atherosclerotic syndrome by reducing obesity, significantly inhibiting VCAM-1, MCP-1, TNF-α, and IL-1β expression, and increasing the production of SCFAs. Modulation of the microbiota associated with inflammation, such as Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge, and increased SCFA production, particularly through an abundance of Rikenellaceae and Dubosiella, were also observed after exercise intervention. Endurance exercise can alleviate WD-induced atherosclerosis through the amelioration of obesity, inflammation, and chemotaxis signaling, which are modulated by the microbiota and derived SCFAs.


Scientific Reports
| (2022) 12:3612 | https://doi.org/10.1038/s41598-022-07317-x www.nature.com/scientificreports/ a constant temperature (24 °C ± 2 °C) and humidity (50-60%). To confirm the animals' welfare, a veterinarian monitored their behavior and health status daily. After 1 week of acclimatization, the mice were allocated into the following four groups (n = 6, each): WT mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX). The WD (D12079B, Research Diets, New Brunswick, NJ, USA) was 17% protein, 43% carbohydrate, and 40% fat, totaling 4.67 kcal/g, and was used to induce atherosclerosis in the ApoEKO mice. The experiment, including the WD diet and exercise intervention, was conducted over a 3-month period. At study completion, the mice were anesthetized with isoflurane, and blood samples were collected from the orbital sinus for blood biochemical assessment. Transcardial perfusion was performed using phosphate buffered saline (pH 7.3) through the left ventricle after sacrifice by 95% CO2 asphyxiation. The aorta was sampled immediately and quenched in liquid nitrogen and 10% formaldehyde until further analysis.
Endurance exercise protocol. The ApoE WD EX group underwent a 3-month endurance exercise program comprising swimming sessions performed 5 days per week in a water bath maintained at 35 °C-36 °C. The daily swimming duration was initially 30 min to allow the mice to acclimate to exercise and was increased to 40 min after 2 weeks; this intervention program was slightly modified from a similar study procedure 19 . The exercise was performed at nearly the same time daily (between 2:00 and 4:00 pm). The mice were towel-dried after swimming and placed near an infrared heater to warm up before being returned to their cages.
Plasma biochemistry and inflammation analysis. The sampled blood was centrifuged at 3000 rpm for 10 min at 4 °C for plasma collection. Total cholesterol, triacylglycerol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were analyzed using a Modular P800 Analyzer (Roche Diagnostics, Indianapolis, IN, USA). Plasma tumor necrosis factor alpha (TNF-α) was quantified using a monoclonal antimouse enzyme-linked immunosorbent assay kit (430907, BioLegend, San Diego, CA, USA).
Morphological analysis of atherosclerotic plaques. The proximal aorta was trimmed, and paraffinembedded tissue samples were sectioned to a 4-μm thickness for hematoxylin and eosin (H&E) staining. Pathological examination was performed with an optical microscope (DM2700M, Leica, Wetzlar, Germany), and the atherosclerotic lesion area and aortic sinus thickness were quantified in Hamamatsu C9600-12 NDP scanning software.
Fecal microbiota analysis. The amplification and library construction for the 16s rRNA gene were performed with reference to Illumina's recommended protocols. The V3-V4 region of the bacterial 16s rRNA gene was amplified using the universal primers 341F (5′-CCT ACG GGNGGC WGC AG-3′) and 805R (5′-GAC TAC HVGGG TAT CTA ATC C-3′) with Illumina overhang adapter sequences in the forward (5′-TCG TCG GCA  GCG TCA GAT GTG TAT AAG AGA CAG-3′) and reverse (5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG  ACAG-3′) primers. Then, a Nextera XT Index Kit (Illumina) was employed to adjust the Illumina sequencing adapters and dual-index barcodes of the targets in the amplicon, and aQsep100 Analyzer (BiOptic, New Taipei City, Taiwan) was used for qualification and quantification of the libraries. Finally, the libraries of indicated samples were normalized to an equimolar ratio and sequenced with an Illumina MiSeq system. The taxonomy of the inferred amplicon sequence variants (ASVs) was assigned using the SILVA ribosomal reference database (v138) with an 80% minimum bootstrap confidence threshold, and multiple sequence alignment of the ASVs was performed using DECIPHER (v2.14.0). A frequency table and taxonomy were used to form a phyloseq object for downstream bacterial community analysis with phyloseq (v1.30.0), and the UniFrac distances were calculated using the R package GUniFrac. Microbiota enrichment and difference analysis were conducted using the linear discriminant analysis effect size (LEfSe) method, and the results were visualized as a cladogram by using GraPhlAn. A linear discriminant analysis (LDA) score greater than 2 was used to identify the gut microbiota succession associated with the effects of endurance exercise on WD-induced atherosclerosis.
Fecal SCFA analysis. Fecal SCFAs were extracted using a method described in a previous study 20 with slight modification. In brief, fecal samples from the various treatment groups were weighed and suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of fecal sample and stored at − 30 °C until further analysis. The samples were thawed and homogenized using an ultrasonic homogenizer (BiOptic) at a 30 W for 2 min. The homogenized samples were centrifuged at 14.8 rpm for 10 min to collect the supernatant. Then, a 300-μL aliquot of ethyl acetate was mixed into the supernatant prior to centrifugation at 14.8 rpm for 10 min. The organic phase was collected and stored at − 30 °C until further analysis. A gas chromatography-mass spectrometry system composed of an Agilent 5977B and 7693A autoinjector (Agilent Technologies, Palo Alto, CA, USA) was used for analysis. The gas chromatograph was equipped with a Nukol™ Capillary GC Column (30 m × 0.25 mm id, 0.25 μm d f , Merck, Germany) with helium as the carrier gas at an injection rate of 1 mL/min. The injection volume is 1 μL and the injection temperature is 250 °C. A blank sample of hexane was arranged after five consecutive fecal sample injections to assess the residual effects. The MS detector was operated in electron impact ionization mode (electron energy, 70 eV) and scanned from 30 to 250 m/z. The temperatures of the ion source, quadrupole, and interface were 230 °C, 150 °C, and 280 °C, respectively. Identification and quantification of the SCFAs were based on the retention time of the standard compounds propionate and butyrate from the NIST 08 and Wiley 7 N libraries. Statistical analysis. All data are presented as the mean ± standard error of the mean (SEM). All variables were qualified using a Kolmogorov-Smirnov test before parametric testing. Statistically significant differences in body weight, food intake, biochemical parameters, microbiota abundance, cytokine and metabolite levels, protein expression, and histological scores among the groups were analyzed using one-way analysis of variance (ANOVA) and validated with a Duncan post hoc test. SPSS v22 (IBM, Armonk, NY, USA) was used for all statistical analyses, and a significant difference was defined as one for which the probability of a type I error was < 0.05.
Ethics approval. All experimental animal procedures were reviewed by Taipei Medical University's Institutional Animal Care and Use Committee (IACUC), and the protocol guidelines LAC-2017-0230 were approved by the ethics committee of the IACUC. The current study was carried out in compliance with the ARRIVE guidelines.

Results
Effects of endurance exercise on WD-induced obesity, hyperlipidemia, and inflammation. The body weight and blood lipid profiles of the WT CD, ApoE CD, ApoE WD, and ApoE WD EX groups exhibited significant differences after 3 months (Table 1); significant differences in the initial body weight, cholesterol, and triacylglycerol were also observed among groups (F(3,20) = 4.25, P = 0.018; F(3,20) = 29.1, P < 0.0001; F(3,20) = 10.1, P < 0.0001, respectively) before the WD dietary and exercise intervention. The body weight in the ApoE CD group was significantly higher than those in the WT CD and ApoE WD groups before intervention, but no significant difference was noted among the WT CD, ApoE WD, and ApoE WD EX groups. The levels of the lipids cholesterol and triacylglycerol in the ApoE CD, ApoE WD, and ApoE WD EX groups were significantly higher than those in the WT CD group before intervention as a result of ApoE knockout. After 12 weeks of WD diet and exercise intervention, a significant difference was observed among the groups in body weight and cholesterol and triacylglycerol levels (F(3,20) = 36.9, P < 0.001; F(3,20) = 76.0, P < 0.0001; F(3,20) = 18.1, P < 0.0001, respectively). The body weight of the WD groups (ApoE WD and ApoE WD EX) was significantly higher than that of the normal chow diet groups (WT CD and ApoE CD), but the exercise significantly ameliorated the WD- Effects of endurance exercise on development of atherosclerotic plaques. H&E staining of aortic sinus sections from the WT CD, ApoE CD, ApoE WD, and ApoE WD EX groups was used to visualize the pathological syndromes of atherosclerosis as representative photomicrographs; we quantified these images to determine vascular thickness and plaque area (Fig. 1). The arrowheads in the images of the ApoE CD, ApoE WD, and ApoE WD EX groups depicted in Fig. 1a indicates atheromatous plaques. Atherosclerotic plaques were found in the ApoE knockout mice under WD and normal chow diet conditions, and the WT CD group was regarded as a negative reference. Furthermore, WD-induced endothelium thickening in the aortic root resulted in the formation of fibrous plaques, which accelerated the development of atherosclerosis. Aortic thickness and plaque area exhibited significant differences among the groups (F(3,20) = 32.1, P < 0.001 and F(3,20) = 30.6, P < 0.0001, respectively). The ApoE knockout and WD (ApoE WD) group had significantly greater aorta thickness than the chow diet groups, but exercise (ApoE WD EX) significantly mitigated the vascular thickening caused by the ApoE knockout and WD (Fig. 1b). The ApoE knockout groups (ApoE CD, ApoE WD, and ApoE WD EX) exhibited pathological plaque with mild to severe degrees; the WD (ApoE WD) also exacerbated this pathological syndrome as compared with a normal chow diet (ApoE CD). The exercise intervention led to significantly less plaque area in the ApoE WD EX group than in the ApoE WD group. Thus, endurance exercise protects against cholesterol-driven plaque formation and vascular thickening in the aortic root during the pathogenesis of atherosclerosis.

Endurance exercise alleviates WD-driven dysbacteriosis.
To determine the effects of endurance exercise on gut microbiota composition, we conducted Illumina-assisted 16S rRNA amplicon sequencing for 20 samples (n = 5 per group) to compare microbial abundance, composition, and population among the groups (Fig. 2). Intragroup and intergroup α-diversity was assessed using observation and Chao1 indexes, and β-diversity was measured through weighted and unweighted UniFrac principal coordinate analysis (PcoA). As presented in Fig. 3, the α-diversity of the WD groups (ApoE WD and ApoE WD EX) was significantly lower than www.nature.com/scientificreports/ that of the normal diet groups (WT CD and ApoE CD); thus, diet crucially affected intragroup microbial diversity. The distance between microbial communities (intergroup diversity) was computed from the constructed OTUs using UniFrac analysis with PcoA plots, revealing significant differences in the microbiota composition among groups (P = 0.001 and P = 0.001 in the Adonis test, respectively), with homogeneous dispersions (P = 0.54 and P = 0.41, respectively). Exercise intervention modulated the microbiota under the conditions of hyperlipidemia and a high-fat diet, acting as a physiological regulator. LEfSe and LDA assays were used to characterize  www.nature.com/scientificreports/ the microbial communities and identify significant differences in abundance (Fig. 2); the WT CD group encompassed most of the bacterial community. In the ApoE WD EX group, the Rikenellaceae and Erysipelotrichaceae families and their associated genera, Rikenella, Rikenellaceae RC9, Dubosiella, and Faecalibaculum, were significantly abundant.
Effects of exercise and WD on gut microbiota genera. The microbial genera largely belonged to the Bacteroidetes, Firmicutes, and Proteobacteria phyla and demonstrated significant differences in abundance among groups. As described in Fig. 4, the ApoE WD group exhibited significantly higher abundance of Desulfovibrio, Parabacteroides, Bacteroides, Ruminococcaceae spp., Lactococcus, Peptococcaceae, Ruminiclostridium_5, Tyzzerella, and Lachnospiraceae than did the ApoE CD group. However, endurance exercise resulted in significantly lower relative abundance of Desulfovibrio, Parabacteroides, Bacteroides, Lactococcus, Peptococcaceae, Tyzzerella, and Lachnospiraceae in the ApoE WD EX group than in the ApoE WD group, a result consistent with the exercise-induced inhibition of atherosclerotic pathogenesis (Fig. 2). Thus, endurance exercise intervention could potentially prevent hyperlipidemia-induced dysbiosis and maintain the homeostasis of the gut microbial community for disease prevention.
Endurance exercise promotes fecal SCFA production. CVDs and their major risk factors such as atherosclerosis and hypertension may be related by an inflammatory response highly associated with cholesterol metabolism, gut dysbiosis, and microbial metabolites 13 . Therefore, the concentration of fecal SCFAs, the main metabolites produced and fermented by microbiota from dietary fibers and resistant starch, were assessed in this study (Fig. 5). The total SCFAs and propionates and butyrate levels were significantly different among groups  www.nature.com/scientificreports/ between the WT CD and ApoE CD groups in the expression of VCAM-1, MCP-1, IL-1β, or TNF-α (Fig. 6). However, the ApoE WD group exhibited significantly higher VCAM-1, MCP-1, IL-1β, and TNF-α expression than did the chow diet groups (WT CD and ApoE CD). The highest VCAM-1 and MCP-1 expression were found in the ApoE WD group, which could mediate the adhesion and recruitment of leukocytes to the vascular endothelium for migration to inflamed site; VCAM-1 and MCP-1 were also reciprocally activated by the high levels of the aortic proinflammatory proteins IL-1β and TNF-α to cause more severe atherosclerotic lesions, which were observed through pathological examination. Exercise intervention in the ApoE WD EX group significantly lowered the expression of VCAM-1, MCP-1, IL-1β, and TNF-α to mitigate the chemoattraction of immune cells and local inflammation in the aorta, ameliorating atherosclerotic development; we also observed this as milder atherosclerotic lesions in the aortic root (Fig. 2).

Discussion
CVD is the leading cause of death worldwide and can be categorized into coronary heart disease, cerebrovascular disease, peripheral artery disease, and aortic atherosclerosis. As economies have advanced, many jobs have shifted from being physically demanding to being sedentary, and this shift is often accompanied by longer working hours, which may explain the significant and steady increase in CVD incidence in past decades. Physical inactivity and poor diets (high in calories, saturated fats, and sugars) and lifestyle choices can cause obesity-associated metabolic syndromes (hyperglycemia, hyperlipidemia, and hypertension) associated with the development of atherosclerosis 22 . Exercise was determined to be an effective strategy for obesity management and CVD improvement through the regulation of mitochondrial function and myokine secretion, restoration and improvement of the vasculature, and mitigation of inflammation 23 . The microbiota influences a range of physiological processes, including host immunity, metabolism, and pathogenesis 24 , and homeostasis and physiological immunomodulation can be affected by environmental conditions and exercise stimuli 25 . In this study, we employed a murine  www.nature.com/scientificreports/ model with ApoE knockout in combination with a WD to evaluate the effects of endurance exercise on atherosclerotic development. Twelve weeks of endurance exercise ameliorated obesity and atherosclerotic lesions through decreases in chemokines (VCAM-1 and MCP-1) and inflammatory cytokines (IL-1β and TNF-α) as well as the microbiota associated with inflammation (Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge) and SCFA production (Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge). Atherosclerosis, a chronic inflammatory disease, is triggered by lipid retention in the arterial wall, and circulating lipoprotein particles, macrophages, and cholesterol promote plaque formation in the intima. The phagocytic activity of macrophages in lipoprotein ingestion causes the formation of foam cells with lipid droplets and dying macrophages, which constitute the necrotic core in plaques. Furthermore, plaque macrophages and their associated immune cells continually contribute to the local inflammatory response through the secretion of proinflammatory cytokines and chemokines and the offing application of oxidative stress during atherosclerosis 26 . The IL-1β and TNF-α secreted by macrophages, lymphocytes, natural killer cells, and vascular smooth muscle cells are proatherogenic cytokines. A genetic knockout of IL-1β was demonstrated by a significantly decrease atherosclerotic severity in the ApoE-deficient mice, and a simultaneous decrease in aortic VCAM-1 and MCP-1 was also observed 27 . The negative dominance of TNF-α decoy, a recombinant soluble TNF-αp55 receptor, can also ameliorate atherogenesis, endothelial adhesion, and elevation of inflammatory markers 28 . Besides, the VCAM-1 can be also upregulated the interaction between leukocytes and the endothelium with TNF-α activation 29 . Pharmaceuticals such as statins, aspirin, methotrexate, and colchicine may prevent or delay atherogenesis or CVD progression. However, regular exercise induced antiatherogenic effects through the amelioration of inflammation and immune cell chemotaxis in this study (Table 1 and Fig. 6).
The ApoE knockout mice were genetically modified to C57BL/6-Apoe em1Narl /Narl mice by using CRISPR/ Cas9, and their phenotypes were manipulated with a high-cholesterol and high-fat WD to induce pathological atherosclerosis. ApoE knockout mice are widely used as an atherosensitive platform to evaluate the effect of modulator genes on the development and progression of atherosclerosis. The mechanisms of atherosclerotic regulation and therapeutic strategies were extensively investigated using an ApoE knockout model as a study platform 30 . LDL receptor and ApoE knockout in mice induced severe dyslipidemia and liver steatosis as well as atherosclerotic plaque formation; inflammatory cytokine levels and atherosclerotic syndrome were also exacerbated 31 . Our results demonstrated that ApoE knockout with a normal diet resulted in hypercholesterolemia and arterial thickening as compared with WT mice, and the WD aggravated the hyperlipidemia, inflammation, and atherosclerotic lesions in the ApoE knockout mice, as detailed in Table 1 and Fig. 1.
In a previous study of the effects of exercise on ApoE knockout model with a high-fat det, a 12-week swimming protocol (60 min/day) alleviated hyperlipidemia indexes and lipid metabolism through the activation of the PPARγ, CPT1, and MCAD genes 32 . However, the similar 12-week swimming intervention in our study did not affect lipid indexes, possibly because of our lower training volume (40 min/day); however, our chosen intensity ameliorated obesity and atherosclerosis. For physiological adaption, the chronic and acute exercise could contribute the anti-inflammation and pro-inflammation, but the inflammatory effect caused by the high-intensity, frequent, and prolonged exercise could accelerate severity in atherosclerotic athlete. The higher coronary artery calcification among athletes may not associated with an increased risk for cardiovascular events like the general population possible due to plaque stabilization with exercise training effects 33 . Therefore, the regular exercise and training volume could be important factors to alleviate the atherosclerosis. In clinical guideline of exercise to cardiovascular disease, the low to moderate endurance intensity, exercise frequency (> 150 min/week with at least 2 sections/week), resistant exercise and caloric expenditure (1000-2000 kcal/week) could also be considered as secondary prevention through exercise-based cardiac rehabilitation for cardiovascular health outcomes 34 .
The gut microbiota may be crucial to the integrity of the intestinal barrier and its effects on immune system development and immune mediator modulation 35 . Dysbiosis and gut barrier hyperpermeability can result in inflammation associated with diseases such as inflammatory bowel disease and colitis-associated cancers 36 . SCFAs, such as acetate, propionate, and butyrate, can affect intestinal barrier function, inflammatory regulation, oxidative stress, and carcinogenesis through the major signaling mechanisms of histone acetylation promotion and G-protein-coupled receptor activation 37 . In our study, dysbiosis resulted in a higher relative abundance of Desulfovibrio, Parabacteroides, Bacteroides, Ruminococcaceae_UCG_009, Lactococcus, Peptococcaceae_ge, Rumi-noclostridium_5, Ruminococcaceae_ge, Tyzzerella, and Lachnospiraceae_ge. A correlation between significant enrichment of the Lachnospiraceae and Ruminococcaceae families and atherosclerotic lesions was also observed in another study employing ApoE knockout and a WD 38 . The plasma levels of triglycerides and cholesterol were positively correlated with Tyzzerella in a high-fat, high-fructose diet animal model 39 , and increased abundance of Tyzzerella is a risk factor for CVD 40 . Desulfovibrio spp. also exhibited significantly higher prevalence in patients with inflammatory bowel disease than in healthy individuals 41 . However, exercise intervention (ApoE WD EX) significantly decreased the abundance of Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge and elevated the abundance of Rikenella, Dubosiella, and Faecalibaculum through fucoidan supplementation for the amelioration of cyclophosphamide-induced inflammation and for the intestinal integrity in relation to SCFA production 42 . Furthermore, lipocalin 2, an antimicrobial peptide expressed in the intestine, modulated obesity-associated and metabolic dysregulation through SCFA production regulated by Dubosiella and Angelakisella 43 . In addition, enriched Dubosiella in fermented rice bran-treated mice improved insulin sensitivity, antioxidative capacity, and lipid levels 44 . In another study, Faecalibaculum and its homology, Holdemanella biformis, demonstrated intestinal antitumorigenic effects through the production of SCFAs, which controlled the protein acetylation and tumor cell proliferation underlying calcineurin inhibition and NFATc3 activation 45 . SCFAs and their specific G-proteincoupled receptor 43 agonists also exhibited anti-inflammation and oxidative stress inhibition capacity 46 . Thus, the dysbiosis caused by a WD and hyperlipidemia resulted in intestinal hyperpermeability and inflammation alongside lower SCFA production, eventually contributing to the acceleration of atherosclerotic development in the ApoE WD group in our study. The divergence in the microbial composition in the ApoE WD group was www.nature.com/scientificreports/ corresponded to severe atherosclerotic lesion areas in the aortic root, further indicating the diet-dependent effects of the gut microbiota on atherosclerosis.
Moderate-intensity exercise assists with weight loss, fat loss, and cardiorespiratory fitness 47 and can be medically prescribed for atherosclerosis 48 . Exercise can also induce physiological adaptations, including anti-inflammation and antioxidative stress activity, for disease prevention and therapy. The microbiota is a key modulator of health and disease progression, and exercise can affect the populations of beneficial and deleterious microbial species, microflora diversity, energy metabolism, and commensal bacteria growth 49 . Factors such as a high-fat diet could also alter the microbiota composition, reversing changes associated with obesity and preventing subsequent chronic disease development through the improvement of energy harvest and storage and reductions in gut permeability and inflammation 50 . Desulfovibrio, Ruminococcaceae, Tyzzerella, and Lachnospiraceae, associated with obesity-induced inflammation, dyslipidemia, and atherosclerosis, were significantly elevated in the ApoE WD group. In addition, the SCFAs produced by Rikenellaceae and Dubosiella were significantly elevated after exercise intervention. SCFAs can modulate immune and inflammatory responses through the activation of free fatty acid receptors 2 and 3 and the G-protein-coupled receptor 109A and the inhibition of histone deacetylases 51 . Therefore, the SCFAs produced by Rikenellaceae and Dubosiella in the ApoE WD EX group modulated chemotaxis through inflammation suppression in mild aortic atherosclerotic lesions. Overall, the data demonstrate that endurance exercise can modulate the microbiota to result in high SCFA production, alleviating WD-induced atherosclerotic progression through the inhibition of inflammation and the chemotaxis signaling pathway (Fig. 7).

Conclusions
In summary, this study identified the mechanisms underlying the antiatherosclerotic effects of endurance exercise exerted by the microbial community and microbial-derived SCFAs. This is the first study to demonstrate that endurance exercise can modulate microbial populations, including those of Desulfovibrio, Tyzzerella, Lachno-spiraceae_ge, Rikenellaceae, and Dubosiella for anti-inflammatory activity and SCFA production; aortic inflammatory responses significantly decreased after exercise with amelioration of atherosclerotic pathogenesis.