Gut dysbiosis induced by a high-salt diet aggravates atherosclerosis by increasing the absorption of saturated fatty acids in ApoE-deficient mice

Gut dysbiosis induced by a high-salt diet aggravates atherosclerosis by increasing the absorption of saturated fatty acids in ApoE-deficient mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gut dysbiosis induced by a high-salt diet aggravates atherosclerosis by increasing the absorption of saturated fatty acids in ApoE-deficient mice Takashi Yoshimura, Takuro Okamura, Hiroki Yuge, Yukako Hosomi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2759933/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background In this study, we investigated the mechanism by which excessive salt intake aggravates atherosclerosis by evaluating the changes in the gut microbiota, the expression of nutrient transporters in the gut, and the fatty acid composition in atherosclerosis. Results Sixteen-week-old male ApoE-deficient mice were either fed a high-fat, high-sucrose diet (HFHSD) or HFHSD, high-salt diet (HFHSD + 4%NaCl) for 8 weeks. The HFHSD + 4%NaCl group showed progression of atherosclerosis, and gut microbiota analysis revealed that this group had a reduced abundance of Allobaculum spp., Ruminococcaceae family, Lachnospiraceae family, and Alphaproteobacteria class compared to the HFHSD group. Furthermore, Cd36 gene expression levels were increased in the small intestine of the HFHSD + 4%NaCl group compared to those in the HFHSD group. The concentration of saturated fatty acids in serum and atherosclerotic lesions, was remarkably increased in the HFHSD + 4%NaCl group. Conclusions Dysbiosis induced by excessive salt intake increases the expression of long-chain fatty acid transporters in the intestinal tract, which increases the influx of saturated fatty acids into the body. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Atherosclerosis is an inflammatory disease caused by the deposition of oxidized low-density lipoprotein (LDL) in the vascular intima, endothelial damage, inflammatory cytokine expression, macrophage migration, intimal thickening, and plaque formation [ 1 , 2 ]. Atherosclerosis is associated with the development of cardiovascular diseases (CVDs), which are the leading cause of death worldwide, accounting for approximately 32% of all deaths in 2019 [ 3 ]. Additionally, excessive salt intake is a major cause of hypertension which is highly associated with atherosclerosis [ 4 ]. Excessive salt intake is also associated with disturbances in the composition of gut microbiota (dysbiosis) which disrupt the intestinal epithelial barrier and induce inflammatory cytokines. In a human study, fecal salt concentration was significantly associated with the presence of obesity, as well as depletion of Akkermansia muciniphila and Bifidobacterium , particularly Bifidobacterium longum and Bifidobacterium adolescentis [ 5 ]. In addition, a previous study comparing the gut microbiota of hypertensive patients and healthy controls have shown higher abundance of the genus Barnesiella in hypertensive patients [ 6 ]. Furthermore, animal studies have reported that excessive salt intake alters the composition of the intestinal microbiota in mice, exacerbating colitis and decreasing the relative abundance of lactobacilli and levels of butyrate [ 7 ]. Chronic inflammation caused by inflammatory cytokines leads to the development of atherosclerosis [ 1 , 8 ]. In fact, the risk of myocardial infarction, stroke, and heart failure is 2- to 4-fold higher in patients with inflammatory bowel disease (IBD). For example, data from Danish and other European cohort studies have shown an association between ischemic heart disease and IBD [ 9 – 11 ]. These previous reports indicate that chronic intestinal inflammation is closely related to atherosclerosis; however, how dysbiosis and intestinal inflammation by excessive salt intake is associated with atherosclerosis remains unclear. LDL cholesterol is a risk factor for atherosclerosis and CVDs. The consumption of food rich in saturated fatty acid increases serum LDL cholesterol levels, which promotes inflammatory triggers, abnormal lipid metabolism, and obesity [ 12 , 13 ]. The apolipoprotein E (ApoE) reduces plasma cholesterol levels and has anti-inflammatory effects. ApoE also inhibits the progression of atherosclerosis, and ApoE-deficient mice are prone to lipid abnormalities and atherosclerosis [ 14 ]. In addition, excessive intake of saturated fatty acids has been reported to be associated with the development of atherosclerosis [ 15 ]. Saturated fatty acids are rapidly taken up by cultured macrophages, potentially leading to a greater accumulation of arterial lipids [ 16 ]. Innate lymphocytes (ILCs) are immune cells that lack antigen receptors and play a role in innate immunity. ILCs are associated with several lifestyle-related diseases, as well as infectious and allergic diseases, and ILC2 have been reported to protect against atherosclerosis [ 17 ]. Moreover, we have previously shown that dysbiosis induces inflammation in the intestinal tract, which promotes the gene expression of saturated fatty acid transporters in the epithelium of the small intestinal, leading to increased absorption of saturated fatty acids and exacerbation of fatty liver disease [ 18 ]. Based on these reports and the results of our previous study, we hypothesized that dysbiosis caused by a high-salt diet may exacerbate atherosclerosis by altering the expression of saturated fatty acid transporters in the intestinal tract. Therefore, the present study aimed to determine the mechanism by which excessive salt intake promotes atherosclerosis and chronic inflammation by evaluating innate immune changes and dysbiosis using a mouse model of atherosclerotic disease. Materials and Methods Mice All animal experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (M2021-56, 2021 − 107). Male B6.129P2-Apoetm1Unc/J (ApoE KO) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and bred in a specific pathogen-free room at this university. Gene disruption was performed by deleting parts of exon 3 and intron 3 of the mouse ApoE locus and inserting a neomycin resistance gene cassette from an E. coli transposon (expressed under a polyomavirus enhancer sequence, an artificial translation start site sequence, and a thymidine kinase gene promoter from herpes simplex virus) by homologous recombination [ 19 ]. The mice were 16-week-old at the beginning of the experimental procedures. They were fed for eight weeks either a high-fat high-sucrose diet (HFHSD; 20% protein, 40% carbohydrate, and 40% fat, coconut oil, 0.3% gm NaCl; D12327, Research Diets, Inc., New Brunswick, NJ, USA) or HFHSD + excessive salt (4% gm NaCl). Paired feeding was performed by supplying an equal amount of feed. The mice were maintained in an environmentally controlled room (temperature, 23 ± 1.5°C; humidity, 40–60%; and a 12-h light/ dark cycle, from 7 a.m. to 7 p.m). Cumulative oral intake was measured for 8 weeks. The manually weighed fresh food was placed in a trough in each cage once every three days at 9 a.m. and the amount of food was then measured after 24 h. The remnants of the chow were discarded. At 24 weeks of age, mice were fasted overnight and were then sacrificed by the administration of a combination of anesthetics: 4.0 mg/kg, midazolam, 0.3 mg/kg of medetomidine, and 5.0 mg/kg of butorphanol [ 20 ]. At the time of sacrifice, blood was collected by puncturing the left ventricle of the mouse. To remove circulating blood from the mice, the mice were slowly perfused with 10 ml of PBS from the left ventricle, then the epididymal adipose tissue, jejunum, and finally the aorta were dissected. Blood Pressure Measurement We assessed systolic (SBP) and diastolic (DBP) blood pressures under anesthesia at 24 weeks of age using a mouse tail-cuff blood pressure monitor (BP-98AL V3.02; Softron Co. Ltd., Tokyo, Japan), according to the manufacturer’s instructions. The mean blood pressure was calculated using the following formula (mean = diastolic pressure + 1/3 of pulse pressure). Biochemistry Blood samples were collected from fasted mice, and serum samples were collected after centrifugation at 14,000 rpm for 10 min at 4°C. The levels of triglycerides (TG), total cholesterol (T-Chol), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol [ 21 ] (Dehghan et al., 2017), and non-esterified fatty acids (NEFAs) [ 22 ] were measured using the enzymatic method. Biochemical examinations were performed using a FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Arteriosclerotic Area Measurement The atherosclerotic foci of the aortic annulus were used to measure the atherosclerotic area. Aortas were refluxed in phosphate-buffered hygienic saline, as previously reported, embedded in a frozen tissue embedding agent, fixed with dry ice, and frozen. Frozen sections were obtained using a cryostat until the aortic valve was visible and then sectioned serially at a thickness of 6 µm [ 23 ]. Ten sections per pull were examined. Tissues were fixed in 60% isopropanol for 15 s and stained with oil red-O (Wako Pure Chemicals) for 30 min at room temperature. After staining, images were taken with BZ-X710 (Keyence Co., Osaka, Japan), and the arterial stiffness area was measured using ImageJ (NIH). Isolation of Mononuclear Cells from Aortas in Mice Using a dissecting microscope equipped with a cold light source (2.5× magnification), fatty tissue adjacent to the adventitia was carefully dissected and removed, leaving the outer aortic membrane intact. Lymph nodes near the aorta were carefully resected. The entire aorta was harvested, and the plaque was detached from the intima. The aortic segment was placed in a 60 mm dish in ice-cold fluorescent activated cell sorting (FACS) buffer and stored until enzymatic digestion. The aortas were transferred from a 60 mm dish to a 1.5 mL Eppendorf tube and 0.5 mL of an enzyme cocktail was added. The enzyme cocktail contained the following: 400 U/mL collagenase type I, 120 U/mL collagenase type XI, 60 U/mL hyaluronidase, 60 U/mL DNase1 (C0130, C765, H3506, and 11284932001, respectively; Sigma-Aldrich, St. Louis, MO, USA); 20 mM HEPES (15630106; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco's phosphate-buffered saline containing calcium (DPBS, Thermo Fisher Scientific). The aortic tissue was cut into small pieces using scissors. The mixture was then transferred to a 50 mL Falcon tube and another 2 mL of the enzyme cocktail was added. The tubes containing the aortic tissue pieces were transferred to a water bath at 37°C for 50 min with slow shaking. After 50 min, the digestion solution was poured into a 100 µm cell strainer placed at the top of a new 50 mL Falcon tube. The remaining aortic tissue was crushed with a syringe plunger, and the cell strainer was rinsed with 5 mL FACS buffer. The filtrates were collected and centrifuged at 300 × g for 10 min at 4°C. The supernatant was carefully removed after centrifugation, and the cell pellet was suspended in 400 µL of FACS buffer [ 24 ]. Tissue Preparation and Flow Cytometry Stained cells were analyzed using FACS Canto II, and the data were analyzed using FlowJo version 10 software (Ashland, OR, USA). For gating of innate lymphoid cells, the following antibodies, purchased from eBioscience (Thermo Fisher Scientific), were used: Biotin-CD3e (100304; clone: 145-2C11; 1/200), Biotin- CD45R/B220 (103204; clone: RA3–6B2; 1/200), Biotin-Gr-1 (108404; clone: RB6-8C5; 1/200), Biotin-CD11c (117304; clone: N418; 1/200), Biotin-CD11b (101204; clone: M1/70; 1/200), Biotin-Ter119 (116204; clone: TER-119; 1/200Biotin-FceRIa (134304; clone: MAR-1; 1/200), FITC-Streptavidin (405202; 1/500), PE-Cy7-CD127 (135014; clone: A7R34; 1/100), Pacific Blue-CD45 (103116; clone: 30-F11; 1/100), PE -GATA-3 (clone: TWAJ, 1/50), APC -RORγ (clone: AFKJS-9, 1/50), and Fixable Viability Dye eFluor 780 (1/400) [ 25 , 26 ]. Additionally, the following antibodies (also purchased from eBioscience) for gating of M1 and M2 macrophages were used: APC- CD45.2 (17045482; clone: 104, 1/50), PE-F4/80 (12480182; clone: BM8, 1/50), APC-Cy7-CD11b (47011282; clone: M1/70, 1/50), FITC-CD206 (MA516870; clone: MR5D3, 1/50), and PE-Cy7-CD11c (25011482; clone: N418, 1/50)[ 27 ]. Quantification of Free Fatty Acids in the Aorta, Sera, and Feces Fatty acid composition in the aorta, sera, and feces of ApoE KO mice was measured using gas chromatography-mass spectrometry (GC-MS) with an Agilent 7890B/7000D instrument (Agilent Technologies, Santa Clara, CA, USA). Fifteen milligrams of aorta and feces and 25 µL of sera were methylated using a fatty acid methylation kit (Nacalai Tesque Inc., Kyoto, Japan). If the sample weight was less than 15 mg, the concentration was calculated by dividing by the weight. The final product was loaded onto a Varian capillary column (DB-FATWAX UI, Agilent Technologies). The CP-Sil 88 for the FAME capillary column was used for fatty acid separation (100 m × an inner diameter of 0.25 mm × membrane thickness of 0.20 µm; Agilent Technologies). The column temperature was maintained at 100°C for 4 min and then gradually increased by 3°C/min to 240°C, and held for 7 min. The sample was injected in the split mode with a split ratio of 5:1. Each fatty acid methyl ester was detected in the selected ion monitoring mode. All results were normalized to the peak height of the C17:0 internal standard[ 28 ]. Quantitative Real-Time Polymerase Chain Reaction (q-RT-PCR) of the Aorta and Jejunum Gene expression was analyzed using q-RT-PCR. Each aorta and jejunum sample was homogenized in ice-cold QIAzol Lysis reagent (Qiagen, Hilden, Germany), and total RNA was isolated using the RNeasy MinElute Cleanup Kit (Qiagen), according to the manufacturer’s instructions. Total RNA (0.5 µg) was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) for first-strand cDNA synthesis, using an oligonucleotide dT primer and random hexamer primers according to the manufacturer’s recommendations. The reverse transcription reaction was performed for 120 min at 37°C, and the enzyme was then inactivated by incubation at 85°C for 5 min. RT-PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems), according to the manufacturer’s instructions. The following PCR conditions were used: one cycle of 2 min at 50°C and 20 s at 95°C, followed by 40 cycles of 1 s at 95°C and 20 s at 60°C. Total RNA extracted from the aorta and jejunum was diluted to 5 ng/µL for all samples in DNase-RNase-free water after concentration measurement using Thermo Scientific™ NanoDrop Lite (Thermo Fisher Scientific). The relative expression levels of each target gene in the aorta ( Ccl2 , Il1b , Ifng , Tnfa , Il33 , and Fasn ), the jejunum ( Cd36 , Il22 , and Il6 ), and epididymal white adipose tissue (eWAT) ( Ccl2 , Il1b , Ifng , and Tnfa ) were normalized to the Gapdh threshold cycle (CT) value and quantified using the comparative threshold cycle 2 − ΔΔ CT method as previously described [ 29 ]. We focused on the increase or decrease in fatty acid absorption. Since fatty acid absorption occurs mainly in the small intestine, we evaluated Cd36 and Il22 gene expressions in the jejunum instead of the colon in this study. Signals from HFHS-fed ApoE KO mice were assigned a relative value of 1.0. Six mice from each group were examined, and RT-PCR was performed in triplicates for each sample. 16S rRNA Sequencing Three 24-week-olds of middle weight in the group were selected, and microbial DNA was extracted from frozen fecal samples using the QIAamp DNA Feces Mini Kit (Qiagen), following the manufacturer’s instructions. The V3-V4 region of the 16S rRNA gene was amplified from the DNA using a bacterial universal primer set (341F and 806R). PCR was performed with 20 ng of genomic DNA as a template in a 30 µL reaction mixture using EF-Taq (SolGent, Daejeon, South Korea) for the following cycles: activation of Taq polymerase at 95°C for 2 min, followed by 35 cycles at 95°C, 55°C, and 72°C for 1 min each, finishing with 10 min at 72°C. Amplification products were purified using a multiscreen filter plate (MilliporeSigma, Burlington, MA, USA). 16S rRNA sequencing was performed using a MiSeq sequencer (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions (Macrogen, Seoul, Korea). QIIME version 1.9.1 was used to filter sequences for quality[ 30 ]. Scores less than 75% and mismatches in the barcode or primers were eliminated from the files. The number of operational taxonomic units (OTUs) was determined using the UCLUST algorithm at 97% similarity[ 31 ]. Taxonomic assignment of 16S rRNA was performed with the Greengenes core-set-aligned with UCLUST and UNITE sequence sets for ITS using BLAST (UNITE, 2017). The relative abundance of the phenotypic categories of the taxonomic groups was predicted using METAGENAssist, which is a statistical tool for comparative metagenomics [ 32 ]. Data filtering was based on interquartile range, row normalization by sum, and column normalization based on autoscaling. In addition, the Firmicutes/Bacteroidetes ratio was calculated. Alpha diversity was defined as the diversity within an individual sample using the Shannon index [ 33 ], Chao1 [ 34 ], and Gini-Simpson index [ 35 ]. Moreover, differences in microbial communities between the two groups were investigated using the phylogeny-based weighted UniFrac distance metric and principal coordinate analysis (PCoA) plots, and non-hierarchical K -means cluster analysis was performed with the number of clusters to be generated pre-specified as two using the Tinn-R Gui version 1.19.4.7, R version 1.36[ 36 ]. The relative abundance of phyla in the groups was evaluated by unpaired t -test using the JMP version 13.2 software (SAS Institute Inc., Cary, NC, USA). Furthermore, the relative abundance of bacterial genera between groups was evaluated using linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) ( http://huttenhower.sph.harvard.edu/lefse/ ) [ 37 ]. Using a normalized relative abundance matrix, LEfSe showed taxa with significantly different abundances, and the effect size of the feature was estimated using LDA. A p -value threshold of 0.05 (Wilcoxon rank-sum test) and an effect size threshold of 2 were used for all biomarkers discussed in this study. Statistical Analysis Data were analyzed using JMP ver. 13.0 software (SAS, Cary, NC, USA). Differences between the two groups were compared using Student’s t -test. Statistical significance was set at p < 0.05, and asterisks were indicated in the figures as follows to express statistical significance; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Statistical significance of body weight and oral intake were analyzed using two-way repeated measures ANOVA followed by Bonferroni's tests. Figures were generated using GraphPad Prism software (version 9.0; San Diego, CA, USA). Results Effects of HFHSD With High-salt Loading on Body Weight, Serum Lipids ApoE KO mice were either fed with HFHSD + 4%NaCl or with HFHSD only, and their body weights, blood pressure, and serum lipid levels were compared. At 21 weeks of age, the HFHSD + 4%NaCl or group had significantly lower body weights than the HFHSD group (Fig. 1 A). As shown in Fig. 1 B, both groups were pair-fed with similar food intake. The systolic, diastolic, mean blood pressure and pulse pressure were not different between the HFHSD and HFHSD + 4%NaCl groups (Fig. 1 C-F). Serum lipid levels, such as TG, T-chol, LDL-chol, HDL-chol, and NEFA, were investigated. The HFHSD + 4%NaCl group had higher serum TG, T-chol, LDL-chol, and NEFA levels but lower serum HDL-cholesterol levels than the HFHSD group (Fig. 1 G-K). Effects of HFHSD With High-salt Loading on Atherosclerotic Foci of the Aortic Annulus Next, the area of arterial atherosclerosis in the aortic annulus was measured (Fig. 1 L). The atherosclerosis area in the HFHSD + 4%NaCl group was larger than that in the HFHSD group (Fig. 1 M). Effects of HFHSD With High-salt Loading on the weight of visceral fat mass To assess visceral fat mass, epididymal fat was adopted in this study and weighed (Fig. 1 N and O ). Absolute and relative weight of epididymal fat weight of the HFHSD + 4%NaCl group was lower than those of the HFHSD group. Dynamics of Inflammatory and Anti-inflammatory Cells in the Aorta Flow cytometric analysis was performed to determine the number of cells involved in innate immunity in the aorta in the two groups. The M1/M2 macrophage ratio in the aorta of the HFHSD + 4%NaCl group was higher than that of the HFHSD group (Fig. 2 A). The percentage of ILC1 + CD45 + cells of the aorta in the HFHSD + 4%NaCl group was higher than that in the HFHSD group (Fig. 2 B), whereas that of ILC2 in CD45 + cells was lower in the HFHSD + 4%NaCl group (Fig. 2 C). Saturated Fatty Acids in Feces, Sera, and Aorta The concentrations of saturated fatty acids, such as lauric, myristic, palmitic, and stearic acids, in feces, sera, and aortas were investigated by GC/MS. The concentration of saturated fatty acids in the feces of the HFHSD + 4%NaCl group was lower than that of the HFHSD group (Fig. 3 A-D). On the other hand, the concentrations of saturated fatty acids in the sera and aorta of the HFHSD + 4%NaCl group were higher than those in the HFHSD group (Fig. 3 E-L). Effects of HFHSD With High-salt Loading on the Expression of Genes Related to Inflammatory Cytokines in the Aorta and With Anti-inflammatory Cytokines and Fatty Acid Transporters in the Jejunum Gene expression of inflammatory cytokines and fatty acid transporters was analyzed using q-RT-PCR to determine the effect of HFHSD with high-salt loading on chronic inflammation. The expression levels of genes related to inflammatory cytokines, such as Ccl2 , Il1b , Ifng , and Tnfa , in the aorta of the HFHSD + 4%NaCl group, were higher than those in the HFHSD group (Fig. 4 A-D). In addition, the expression level of Il33 , which is one of the factors that activate ILC2, in the aorta of the HFHSD + 4%NaCl group was lower than that of the HFHSD group (Fig. 4 E). On the other hand, the expression levels of Fasn , a gene related to fatty acid metabolism, was not different between the two groups (Fig. 4 F). The Cd36 expression level in the jejunum of the HFHSD + 4%NaCl group was higher than that of the HFHSD group (Fig. 4 G), whereas the Il22 expression level in the HFHSD + 4%NaCl group was lower than that in the HFHSD group (Fig. 4 H). In addition, Il6 gene expression in the jejunum of the HFHSD + 4%NaCl group was higher than that of the HFHSD group (Fig. 4 I). Finally, gene expression in visceral adipose tissue was also evaluated based on the assumption that inflammation in visceral fat may have been exacerbated as the cause of the decrease in visceral fat mass despite the development of atherosclerosis in the HFHSD + 4%NaCl group. In eWAT, Ccl2 , Il1b , Ifng , and Tnfa expressions in the HFHSD + NaCl4% group were higher than those in the HFHSD group (Fig. 4 J-M). Effects of HFHSD With High-salt Loading on the Gut Microbiota We performed 16s rRNA sequencing to determine the effect of high-salt load HFHSD on the gut microbiota. The most abundant phylum was Bacteroidetes in both groups (HFHSD: 47.6 ± 8.1%, HFHSD + 4%NaCl: 52.1 ± 7.4%). The second and third most abundant phyla in the HFHSD group were Firmicutes and Proteobacteria, respectively, whereas those in the HFHSD + 4%NaCl group were Proteobacteria and Firmicutes, respectively (Fig. 5 A). P -values analyzed using the t -test are shown in Fig. 5 B. Next, the Firmicutes/Bacteroidetes phyla (F/B) ratio, whose increase or decrease has been reported to be associated with various diseases, was calculated [ 38 – 40 ]. The F/B ratio in the HFHSD + 4%NaCl group was lower than that in the HFHSD group (Fig. 5 C). The Shannon index, Chao1 index, and Gini-Simpson index were used to assess the diversity of the gut microbiota, which revealed that the HFHSD + 4%NaCl group had lower diversity than the HFHSD group (Fig. 5 D-F). PCoA plots of unweighted and weighted UniFrac distances were constructed to compare the two groups (Fig. 5 G and H ). The clustering results showed that the HFHSD and HFHSD + 4%NaCl groups belonged to different clusters. Additionally, we used the LEfSe algorithm to identify specific taxa that were variably distributed between the two groups. Four taxa (including the class Bacteroidia, order Bacteroidales , and genus Barnesiella ) were over-represented, and five taxa (including the class Alphaproteobacteria, genus Streptococcus , family Lachnospiraceae , family Ruminococcaceae , and genus Allobaculum ) were under-represented in the HFHSD + 4%NaCl group compared to the HFHSD group (Fig. 5 I). Discussion In this study using ApoE KO mice, we evaluated the effects of excessive salt intake on atherosclerosis development and showed that excessive salt intake alters the gut microbiota, increases the gene expression levels of long-chain fatty acid transporters in the small intestine, and the absorption of palmitic acid, a saturated fatty acid, thereby increasing serum saturated fatty acid levels, expanding atherosclerotic foci, and reducing anti-inflammatory innate cells. Previous studies have reported that excessive intake of saturated fatty acids is associated with the development of atherosclerosis [ 15 ], and that saturated fatty acids are rapidly taken up by cultured macrophages, potentially leading to a greater accumulation of arterial lipids[ 16 ]. In clinical practice, the current cardiovascular guidelines recommend reducing saturated fatty acids and replacing them with unsaturated fatty acids[ 41 ]. However, recent epidemiological studies have failed to reveal an increased risk of CVDs associated with diets high in saturated fat [ 21 ]. Furthermore, several studies have found no association between saturated fat intake and carotid intima-media thickness [ 42 ]. In contrast, high serum saturated fatty acid levels have been shown to increase the risk of coronary artery disease[ 43 ]. Taken together, these studies suggest that increased serum saturated fatty acid levels might be more strongly associated with atherosclerosis than saturated fatty acid intake. In the present study, although there was no difference in caloric intake between the HFHSD and HFHSD + 4%NaCl groups after pair feeding, serum lipid levels were significantly higher in the HFHSD + 4%NaCl group than in the HFHSD group. Based on these results, we evaluated the expression levels of genes related to inflammation in the small intestine to determine whether inflammation caused by a high-salt diet alters the dynamics of nutrient absorption from the intestinal tract. Il22 mRNA levels in the small intestine of the HFHSD + 4%NaCl group were lower than those in the HFHSD group. IL22 is an important cytokine that plays a role in the thickening of the mucin layer of the small intestine and protects against intestinal inflammation by enhancing the expression of antimicrobial proteins in epithelial cells [ 44 ]. In contrast, Cd36 mRNA levels, which are long-chain fatty acid transporters, increased following the administration of excessive salt in this study. In our previous study, we reported that dysbiosis caused by excessive intake of saturated fatty acids, sucrose, or trans-fatty acids causes inflammation in the intestinal tract, which in turn increases Cd36 expression levels in the small intestine [ 45 ]. Dysbiosis is difficult to define precisely, but this study used changes in the Firmicutes/Bacteroidetes (F/B) ratio and reduced diversity as markers. Increased F/B ratio has been reported to be associated with obesity [ 39 ], whereas decreased the ratio has been reported to be associated with type 2 diabetes [ 40 ] and inflammatory bowel disease [ 38 ]. Moreover, Morgan, et al. reported that the phylum Proteobacteria increased in IBD patients compared to healthy subjects. In this study, F/B ratio in the HFHSD + 4%NaCl group was lower than that in HFHSD group and the abundance of phylum Proteobacteria in the HFHSD + NaCl4% group was higher than that in the HFHSD group. In summary, the excessive salt intake group in this study showed the gut microbiota more similar to IBD than to the gut microbiota that cause obesity and glucose intolerance. Recently, other groups have reported that Cd36 expression in the small intestine is increased by dysbiosis and intestinal inflammation. Shi, et al. have reported that the supplementation of Bacteroides fragilis , which is reported to be related with obesity [ 46 ], did not only deteriorated metabolic dysfunction but also increased the expression of Cd36 in small intestine [ 47 ]. In the present study, there was no difference in the expression of Fasn , a gene that promotes the conversion of lauric acid to myristic acid and myristic acid to palmitic acid in the aorta between the two groups. In summary, the study suggested that the increase in saturated fatty acids in arteries may be due to increased fatty acid absorption from Cd36, rather than changes in the metabolism of saturated fatty acids in aorta. The present study also suggested that inflammation in the intestine caused by a high-salt diet may have increased the gene expression of long-chain fatty acid transporters in the intestinal tract, resulting in increased saturated fatty acid absorption. The expression of Il6 in the small intestine was also increased by the high salt diet in this study. As microorganisms rely on dietary substrates in the gut, the gut microbiota is often proposed as a mediator of the pro-and anti-inflammatory effects of diet. Similar to other nutrients, animal studies have demonstrated that salt-rich foods induce inflammation and autoimmunity through microbial mechanisms, such as the induction of T-helper 17 cells [ 48 ]. Moreover, type 3 innate lymphoid cells have been reported to have a function homologous to that of Th17, are very abundant in the intestinal mucosa, and play a role in intestinal homeostasis by producing interleukin (IL)-22 and IL-17 in response to IL-23 and IL-1β [ 49 ]. In the 16s rRNA analyses of gut microbiota, the phylum Bacteroidetes, class Bacteroidia, order Bacteroidales , and genus Barnesiella were more abundant in HFHSD + 4%NaCl mice than in HFHSD mice. Barnesiella , a genus of the family Porphyromonadaceae and order Bacteroidales , is one of the most abundant genera detected in the mouse intestine. Several studies have revealed that the abundance of the phylum Bacteroidetes, class Bacteroidia, order Bacteroidales , family Porphyromonadaceae , and genus Barnesiella in diabetic ( db/db ) mice was higher than that in lean mice [ 50 , 51 ]. A previous study comparing the microbiota of patients with hypertension to healthy individuals has revealed that the abundance of the genus Barnesiella was higher in patients with hypertension [ 6 ]. In addition, the abundance of the genus Barnesiella increased in IL-22 deficient mice[ 52 ]. The function of the genus Barnesiella remains unclear; however, several previous studies and the present study suggest that the high-salt diet-induced increase in the genus Barnesiella is associated with dysbiosis, which might be related to a decrease in IL-22 in the intestinal tract. Although the present study showed that a high-salt diet can promote the development of atherosclerosis, no obvious increase in blood pressure was detected. One possible reason for this observation is that in other studies of high-salt diet-induced atherosclerosis in ApoE KO mice, the dietary salt concentration was 7–8% gm and the administration period was 12 weeks[ 53 ]. In contrast, in our previous study, a high-salt diet of 8% gm resulted in extremely low food intake and frequent sudden death of mice during the 12-week treatment period; therefore, we reduced the salt concentration to 4% gm and shortened the treatment period to 8 weeks. Additionally, hypertension has long been known to increase the prevalence of coronary artery disease and the extent and severity of atherosclerosis in both humans [ 54 , 55 ] and animals [ 56 – 58 ]. In other words, we examined the effects of increased saturated fatty acid absorption on increased arterial stiffness while excluding the blood pressure component. In this study, we investigated innate immune cells in the aorta. Previous studies have reported that ILC2 protects against atherosclerosis [ 17 , 59 ]. In addition, ILC2 transfer to ApoE KO mice reduced the lipid content of the atherosclerotic lesions [ 17 ]. In the present study, the number of ILC2 cells and the expression level of Il33 in the aorta of the HFHSD + 4%NaCl group were lower than those of the HFHSD group. IL-33, which is secreted by ILC2 cells [ 60 ], has been reported to play a protective role against the development of atherosclerosis [ 61 ]. The decrease in arterial ILC2 associated with excessive salt intake may exacerbate atherosclerosis by reducing the secretion of IL-33, which has anti-arterial effects. As a limitation of this study, we did not reveal significant difference of blood pressure by excessive salt intake. However, a previous study showed that blood pressure did not change with high salt diet in the inactive phase but did in the active phase [ 62 ]. Therefore, re-measuring blood pressure during the active phase could show an increase in blood pressure due to the high-salt diet. In addition, in this study, the body weight of the HFHSD + 4% NaCl group was significantly lower than that of the HFHSD group. Furthermore, absolute and relative epididymal fat weights were lower than those of the HFHSD group. Previous studies have shown an inverse correlation between salt intake and body weight gain in mice fed a high-fat diet [ 63 , 64 ]; DeClercq et al. reported that excess salt intake increased epididymal fat weight in male mice, and Pitynski-Miller et al. reported that excess salt intake in female rats. In summary, the effects of high-salt diets on body weight and visceral fat mass may vary among various backgrounds and require further study. On the other hand, in this study, excessive salt intake induced the inflammation in eWAT. In a previous study, expression of the inflammatory adipocytokines have been reported to increase in a dose-dependent manner upon salt treatment [ 65 ]. Furthermore, the inflammation in adipose tissue induces adipose tissue dysfunction such as decrease in fatty acid intake [ 66 ]. It has been reported that decreased fatty acid uptake into visceral fat increases ectopic fat accumulation, whereas visceral adipose tissue weight decreases. Since atherosclerosis, a form of ectopic fat accumulation, was advanced in this study, the decrease in body weight in the HFHSD + NaCl4% group may have been due in part to a reduction in visceral fat weight caused by inflammation within visceral fat. Moreover, we did not evaluate the glucose tolerance. Additional data on blood glucose levels could have explained the weak effect of high salt load on the arterial stiffness region. Lastly, we did not have the data of the characterization of the initial fecal sample prior feeding. If we had the data, we could compare the data with the baseline data and more accurately determine the changes in the gut microbiota due to differences in the diet. In conclusion, this study revealed the mechanism by which an HFHSD that mimics the modern diet, loaded with excess salt promotes the development of atherosclerosis. We revealed that excessive salt intake causes alterations in the gut microbiota, induces inflammation in the gut, increases the expression of long-chain fatty acid transporters in the small intestine, and increases the influx of saturated fatty acids into the body, thereby worsening atherosclerosis. This study offers insights into the mechanism of aggravation of atherosclerosis which could not be explained by an increase in saturated fatty acid intake alone. Declarations Ethics approval and consent to participate All animal experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (M2021-56, 2021-107). All methods were performed in accordance with relevant guidelines and regulations. All methods were reported according to the ARRIVE guidelines for reporting animal experiments (https://arriveguidelines.org/) Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests Takashi Yoshimura has no competing interest. Takuro Okamura has no competing interest. Hiroki Yuge has no competing interest. Yukako Hosomi has no competing interest. Emi Ushigome has received grants from the Japanese Study Group for Physiology and Management of Blood Pressure, the Astellas Foundation for Research on Metabolic Disorders (Grant number: 4024). Donated Fund Laboratory of Diabetes therapeutics is an endowment department, supported with an unrestricted grant from Ono Pharmaceutical Co., Ltd., and received personal fees from AstraZeneca plc, Astellas Pharma Inc., Daiichi Sankyo Co., Ltd., Kyowa Hakko Kirin Company Ltd., Kowa Pharmaceutical Co., Ltd., MSD K.K., Mitsubishi Tanabe Pharma Corp., Novo Nordisk Pharma Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd., outside the submitted work. Naoko Nakanishi received grant support from Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 19K23999) (JSPS KAKENHI Grant Number 20K16158) and The Japan Food Chemical Research Foundation, and received personal fees from Kowa Pharmaceutical Co., Ltd., and Novo Nordisk Pharma Ltd. Ryoichi Sasano has no competing interest. Takehiro Ogata has no competing interest. Masahide Hamaguchi has received grants from Asahi Kasei Pharma, Nippon Boehringer Ingelheim Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novo Nordisk Pharma Ltd., and Eli Lilly Japan K.K., outside the submitted work. Michiaki Fukui has received grants from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Co, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Hakko Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd. Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Terumo Co., Teijin Pharma Ltd., Nippon Chemiphar Co., Ltd., and Johnson & Johnson K.K. Medical Co., Abbott Japan Co., Ltd., and received personal fees from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharma Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., Mochida Pharma Co., Ltd., Abbott Japan Co., Ltd., Medtronic Japan Co., Ltd., Arkley Inc., Teijin Pharma Ltd. and Nipro Cor., outside the submitted work. Funding Ushigome Emi received grant support from the Japan Society for the Promotion of Science (grant numbers 18K15897 and 22K08108). Authors’ contributions M.H. originated and designed the study, researched the data, and wrote the manuscript. T.Y., T.O., and M.F. originated and designed the study, researched the data, and reviewed the manuscript. D.Y., Y.H., T.K., E.U., and N.N. researched the data and contributed to the discussion. R.S. and T.O. supported the techniques of research. H.M. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors were involved in the writing of the manuscript and approved the final version of this article. Acknowledgments We would like to thank Editage (www.editage.com) for English language editing. References Duttaroy AK. Role of Gut Microbiota and Their Metabolites on Atherosclerosis, Hypertension and Human Blood Platelet Function: A Review. Nutrients. 2021;13:1–17. Marchio P, Guerra-Ojeda S, Vila JM, Aldasoro M, Victor VM, Mauricio MD. Targeting Early Atherosclerosis: A Focus on Oxidative Stress and Inflammation. Oxid Med Cell Longev. 2019;2019. 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Int J Obes (Lond). 2017;41:1685–92. Lee M, Sorn SR, Lee Y, Kang I. Salt Induces Adipogenesis/Lipogenesis and Inflammatory Adipocytokines Secretion in Adipocytes. Int J Mol Sci. 2019;20. Longo M, Zatterale F, Naderi J, Parrillo L, Formisano P, Raciti GA, et al. Adipose Tissue Dysfunction as Determinant of Obesity-Associated Metabolic Complications. Int J Mol Sci. 2019;20. Additional Declarations Competing interest reported. akashi Yoshimura has no competing interest. Takuro Okamura has no competing interest. Hiroki Yuge has no competing interest. Yukako Hosomi has no competing interest. Emi Ushigome has received grants from the Japanese Study Group for Physiology and Management of Blood Pressure, the Astellas Foundation for Research on Metabolic Disorders (Grant number: 4024). Donated Fund Laboratory of Diabetes therapeutics is an endowment department, supported with an unrestricted grant from Ono Pharmaceutical Co., Ltd., and received personal fees from AstraZeneca plc, Astellas Pharma Inc., Daiichi Sankyo Co., Ltd., Kyowa Hakko Kirin Company Ltd., Kowa Pharmaceutical Co., Ltd., MSD K.K., Mitsubishi Tanabe Pharma Corp., Novo Nordisk Pharma Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd., outside the submitted work. Naoko Nakanishi received grant support from Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 19K23999) (JSPS KAKENHI Grant Number 20K16158) and The Japan Food Chemical Research Foundation, and received personal fees from Kowa Pharmaceutical Co., Ltd., and Novo Nordisk Pharma Ltd. Ryoichi Sasano has no competing interest. Takehiro Ogata has no competing interest. Masahide Hamaguchi has received grants from Asahi Kasei Pharma, Nippon Boehringer Ingelheim Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novo Nordisk Pharma Ltd., and Eli Lilly Japan K.K., outside the submitted work. Michiaki Fukui has received grants from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Co, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Hakko Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd. Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Terumo Co., Teijin Pharma Ltd., Nippon Chemiphar Co., Ltd., and Johnson & Johnson K.K. Medical Co., Abbott Japan Co., Ltd., and received personal fees from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharma Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., Mochida Pharma Co., Ltd., Abbott Japan Co., Ltd., Medtronic Japan Co., Ltd., Arkley Inc., Teijin Pharma Ltd. and Nipro Cor., outside the submitted work. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Hamaguchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCRBRkQAimdFE8Wo5k8DAQ5oWxjZ0LfiA7uzmpxt+zkuTs2dvYDYubGOIZmA//IDBcgduLWZ3jpnd7N2WY8zDc4A5eWYbQ24DT5oBg+QZPFpuJJjd4N1WkdgjkcB8mLftf24DQw4Dg2QbPi3p327+nQPXArSF/w0hLTlmt3kbcsBaksFaJAjaklN2W+ZYmjHPmYPNxjPOMeS2STwzOIDfL+nbbr6pSZZjb28+LF1QxpDbz5/88LEknhBDAowN4IhhA+LDkg1EaUGKS8aPxGoZBaNgFIyCkQAA2DhN3Jj3QZkAAAAASUVORK5CYII=","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Masahide","middleName":"","lastName":"Hamaguchi","suffix":""},{"id":198538499,"identity":"1a4a9916-966f-42f4-83d1-f6f1493bb3cb","order_by":10,"name":"Michiaki Fukui","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michiaki","middleName":"","lastName":"Fukui","suffix":""}],"badges":[],"createdAt":"2023-03-31 07:59:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2759933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2759933/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36925737,"identity":"706e6f24-3484-4ef7-a2be-061d98c8a02e","added_by":"auto","created_at":"2023-05-12 03:12:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":540785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in body weight, blood pressure, blood biochemistry, and visceral fat weight in a mouse model of atherosclerosis. (A) \u003c/strong\u003eBody weight changes in 16-week-old and 24-week-old ApoE KO mice fed with HFHSD and HFHSD+4%NaCl (n=6). Two-way repeated measures ANOVA followed by Bonferroni's tests (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003e(B)\u003c/strong\u003eDietary intake (n=6). \u003cstrong\u003e(C)\u003c/strong\u003e Systolic blood pressure (n=6). \u003cstrong\u003e(D)\u003c/strong\u003eDiastolic blood pressure (n=6). \u003cstrong\u003e(E)\u003c/strong\u003e Mean blood pressure (n=6). \u003cstrong\u003e(F)\u003c/strong\u003ePulse pressure (n=6). Serum levels of \u003cstrong\u003e(G)\u003c/strong\u003e TG, \u003cstrong\u003e(H)\u003c/strong\u003e T-Chol, \u003cstrong\u003e(I)\u003c/strong\u003eLDL-Chol, \u003cstrong\u003e(J)\u003c/strong\u003e HDL-Chol, and\u003cstrong\u003e (K)\u003c/strong\u003e NEFA (n=6). \u003cstrong\u003e(L)\u003c/strong\u003eRepresentative histological images of aortic valves stained with oil red-O. \u003cstrong\u003e(M)\u003c/strong\u003eAtherosclerosis area (n=6). (\u003cstrong\u003eN and O)\u003c/strong\u003e Absolute and relative epididymal fat weight (n=6). Data are presented as mean ± SD values and analyzed using unpaired t-tests; *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003cbr\u003e\nHDL-Chol, high-density lipoprotein cholesterol; HFHSD, high-fat high-sucrose diet; HFHSD+4%NaCl, HFHSD and 4% gm NaCl; LDL-Chol, low-density lipoprotein cholesterol; NEFA, non-esterified fatty acid; TG, triglycerides; T-Chol, total cholesterol.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/9463cb33f301f632121c0868.jpg"},{"id":36924360,"identity":"ae6d81fa-ffd5-46ce-a83c-9694f532a6a2","added_by":"auto","created_at":"2023-05-12 03:07:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInnate immune cells in the aorta. (A)\u003c/strong\u003eM1/M2 macrophages ratio,\u003cstrong\u003e (B) \u003c/strong\u003eILC1s/CD45 positive cells, and \u003cstrong\u003e(C) \u003c/strong\u003eILC2s/CD45 positive cells in the aorta (n=6). Data are presented as mean ± SD values and analyzed using unpaired t-tests, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05.\u003cbr\u003e\nHFHSD, high-fat high-sucrose diet; HFHSD+4%NaCl, HFHSD and 4% gm NaCl; ILC, innate lymphoid cells; Mφ, macrophages.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/eccfc2ba5f384083cc6505a6.jpg"},{"id":36925734,"identity":"0a746493-4fea-492e-a655-dcf8c5e6938b","added_by":"auto","created_at":"2023-05-12 03:12:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":486016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe concentrations of saturated fatty acid in the aorta, sera, and feces.\u003c/strong\u003eThe concentrations of lauric, myristic, palmitic, and stearic acids in the aorta (\u003cstrong\u003eA-D\u003c/strong\u003e), sera (\u003cstrong\u003eE-H\u003c/strong\u003e), and feces (\u003cstrong\u003eI-L\u003c/strong\u003e) of HFHSD and HFHSD+4%NaCl groups. Data are presented as mean ± SD values and analyzed using unpaired t-tests;*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003cbr\u003e\nHFHSD, high-fat high-sucrose diet, HFHSD+4%NaCl, HFHSD and 4% gm NaCl.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/29e380b3b076b3ed927e67e0.jpg"},{"id":36927040,"identity":"4ff6252a-df8c-426a-82c5-c43e5de03bed","added_by":"auto","created_at":"2023-05-12 03:16:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":592155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of genes related to inflammatory cytokines in the aorta and jejunum.\u003c/strong\u003eRelative mRNA expression of (\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eCcl2\u003c/em\u003e, (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eIl1b\u003c/em\u003e, (\u003cstrong\u003eC\u003c/strong\u003e)\u003cem\u003eIfng\u003c/em\u003e, (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eTnfa\u003c/em\u003e, (\u003cstrong\u003eE\u003c/strong\u003e) \u003cem\u003eIl33\u003c/em\u003e, and (\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eFasn\u003c/em\u003ein the aorta, (\u003cstrong\u003eG\u003c/strong\u003e)\u003cem\u003e Il22\u003c/em\u003e, (\u003cstrong\u003eH\u003c/strong\u003e) \u003cem\u003eCd36\u003c/em\u003e, and (\u003cstrong\u003eI\u003c/strong\u003e) \u003cem\u003eIl6\u003c/em\u003ein the jejunum, and (\u003cstrong\u003eJ\u003c/strong\u003e) \u003cem\u003eCcl2\u003c/em\u003e, (\u003cstrong\u003eK\u003c/strong\u003e) \u003cem\u003eIl1b\u003c/em\u003e, (\u003cstrong\u003eL\u003c/strong\u003e) \u003cem\u003eIfng\u003c/em\u003e, and (\u003cstrong\u003eM\u003c/strong\u003e) \u003cem\u003eTnfa\u003c/em\u003e in the eWAT normalized to the expression of \u003cem\u003eGapdh\u003c/em\u003e(n = 6). Data are presented as mean ± SD values and analyzed using unpaired t-tests; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. eWAT, epididymal white adipose tissue; HFHSD, high-fat high-sucrose diet; HFHSD+4%NaCl, HFHSD and 4% gm NaCl.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/47b5e12edf448904a4aba2b2.jpg"},{"id":36923212,"identity":"f02f956d-d273-4c5c-b2ee-a039fe546237","added_by":"auto","created_at":"2023-05-12 03:02:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":514266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e16s rRNA sequencing of the gut microbiota of the two groups of mice. (A)\u003c/strong\u003eThe relative abundance of phyla (%) (n=3). Others included the phylum, such as Actinobacteria, Cyanobacteria, Spirochaetes, Synergistetes, TM7, and Tenericutes. \u003cstrong\u003e(B)\u003c/strong\u003e P-values calculated with unpaired\u003cem\u003e t\u003c/em\u003e-test between the phyla of HFHSD and HFHSD+4%NaCl groups (n=3). \u003cstrong\u003e(C) \u003c/strong\u003eFirmicutes/Bacteroidetes ratio (n=3). \u003cstrong\u003e(D)\u003c/strong\u003e Shannon index (n=3). \u003cstrong\u003e(E)\u003c/strong\u003e Chao1 index (n=3). \u003cstrong\u003e(F)\u003c/strong\u003eGini-Simpson index (n=3). \u003cstrong\u003e(G)\u003c/strong\u003e Unweighted PCoA plots (n=3). \u003cstrong\u003e(H)\u003c/strong\u003eWeighted PCoA plots (n=3). \u003cem\u003eK\u003c/em\u003e-means clustering for gut microbiota is shown. \u003cem\u003eRed\u003c/em\u003e, HFHSD group; \u003cem\u003eBlue\u003c/em\u003e, HFHSD+4%NaCl group. (\u003cstrong\u003eI\u003c/strong\u003e) Linear discriminant analysis (LDA) score (Log10) and LEfSe cladogram of HFHSD and HFHSD+4%NaCl group (n=3). (Red) taxa enriched in HFHSD group; (Green) taxa enriched in HFHSD+4%NaCl group. Only taxa with a significant LDA threshold value \u0026gt;2 are shown. Data are presented as mean ± SD values and analyzed using unpaired t-tests, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003cbr\u003e\nHFHSD, high-fat high-sucrose diet; HFHSD+4%NaCl, HFHSD and 4% gm NaCl; LDA, linear discriminant analysis; LEfSe, LDA coupled with effect size; PCoA, principal coordinates analysis.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/40f969d8fa63f94a8130cb8c.jpg"},{"id":40949259,"identity":"7b4defb1-506c-4523-8323-5d6a50a489a0","added_by":"auto","created_at":"2023-08-02 13:15:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1082853,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2759933/v1/45ac9787-0bf2-420d-bd98-f8354a36fb33.pdf"}],"financialInterests":"Competing interest reported. akashi Yoshimura has no competing interest.\nTakuro Okamura has no competing interest.\nHiroki Yuge has no competing interest.\nYukako Hosomi has no competing interest.\nEmi Ushigome has received grants from the Japanese Study Group for Physiology and Management of Blood Pressure, the Astellas Foundation for Research on Metabolic Disorders (Grant number: 4024). Donated Fund Laboratory of Diabetes therapeutics is an endowment department, supported with an unrestricted grant from Ono Pharmaceutical Co., Ltd., and received personal fees from AstraZeneca plc, Astellas Pharma Inc., Daiichi Sankyo Co., Ltd., Kyowa Hakko Kirin Company Ltd., Kowa Pharmaceutical Co., Ltd., MSD K.K., Mitsubishi Tanabe Pharma Corp., Novo Nordisk Pharma Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd., outside the submitted work. \nNaoko Nakanishi received grant support from Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 19K23999) (JSPS KAKENHI Grant Number 20K16158) and The Japan Food Chemical Research Foundation, and received personal fees from Kowa Pharmaceutical Co., Ltd., and Novo Nordisk Pharma Ltd.\nRyoichi Sasano has no competing interest.\nTakehiro Ogata has no competing interest.\nMasahide Hamaguchi has received grants from Asahi Kasei Pharma, Nippon Boehringer Ingelheim Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novo Nordisk Pharma Ltd., and Eli Lilly Japan K.K., outside the submitted work.\nMichiaki Fukui has received grants from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Co, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Hakko Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd. Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Terumo Co., Teijin Pharma Ltd., Nippon Chemiphar Co., Ltd., and Johnson \u0026 Johnson K.K. Medical Co., Abbott Japan Co., Ltd., and received personal fees from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharma Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., Mochida Pharma Co., Ltd., Abbott Japan Co., Ltd., Medtronic Japan Co., Ltd., Arkley Inc., Teijin Pharma Ltd. and Nipro Cor., outside the submitted work.","formattedTitle":"Gut dysbiosis induced by a high-salt diet aggravates atherosclerosis by increasing the absorption of saturated fatty acids in ApoE-deficient mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerosis is an inflammatory disease caused by the deposition of oxidized low-density lipoprotein (LDL) in the vascular intima, endothelial damage, inflammatory cytokine expression, macrophage migration, intimal thickening, and plaque formation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Atherosclerosis is associated with the development of cardiovascular diseases (CVDs), which are the leading cause of death worldwide, accounting for approximately 32% of all deaths in 2019 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, excessive salt intake is a major cause of hypertension which is highly associated with atherosclerosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Excessive salt intake is also associated with disturbances in the composition of gut microbiota (dysbiosis) which disrupt the intestinal epithelial barrier and induce inflammatory cytokines. In a human study, fecal salt concentration was significantly associated with the presence of obesity, as well as depletion of \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, particularly \u003cem\u003eBifidobacterium longum\u003c/em\u003e and \u003cem\u003eBifidobacterium adolescentis\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, a previous study comparing the gut microbiota of hypertensive patients and healthy controls have shown higher abundance of the genus \u003cem\u003eBarnesiella\u003c/em\u003e in hypertensive patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, animal studies have reported that excessive salt intake alters the composition of the intestinal microbiota in mice, exacerbating colitis and decreasing the relative abundance of lactobacilli and levels of butyrate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Chronic inflammation caused by inflammatory cytokines leads to the development of atherosclerosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In fact, the risk of myocardial infarction, stroke, and heart failure is 2- to 4-fold higher in patients with inflammatory bowel disease (IBD). For example, data from Danish and other European cohort studies have shown an association between ischemic heart disease and IBD [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These previous reports indicate that chronic intestinal inflammation is closely related to atherosclerosis; however, how dysbiosis and intestinal inflammation by excessive salt intake is associated with atherosclerosis remains unclear.\u003c/p\u003e \u003cp\u003eLDL cholesterol is a risk factor for atherosclerosis and CVDs. The consumption of food rich in saturated fatty acid increases serum LDL cholesterol levels, which promotes inflammatory triggers, abnormal lipid metabolism, and obesity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The apolipoprotein E (ApoE) reduces plasma cholesterol levels and has anti-inflammatory effects. ApoE also inhibits the progression of atherosclerosis, and ApoE-deficient mice are prone to lipid abnormalities and atherosclerosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, excessive intake of saturated fatty acids has been reported to be associated with the development of atherosclerosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Saturated fatty acids are rapidly taken up by cultured macrophages, potentially leading to a greater accumulation of arterial lipids [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInnate lymphocytes (ILCs) are immune cells that lack antigen receptors and play a role in innate immunity. ILCs are associated with several lifestyle-related diseases, as well as infectious and allergic diseases, and ILC2 have been reported to protect against atherosclerosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Moreover, we have previously shown that dysbiosis induces inflammation in the intestinal tract, which promotes the gene expression of saturated fatty acid transporters in the epithelium of the small intestinal, leading to increased absorption of saturated fatty acids and exacerbation of fatty liver disease [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on these reports and the results of our previous study, we hypothesized that dysbiosis caused by a high-salt diet may exacerbate atherosclerosis by altering the expression of saturated fatty acid transporters in the intestinal tract. Therefore, the present study aimed to determine the mechanism by which excessive salt intake promotes atherosclerosis and chronic inflammation by evaluating innate immune changes and dysbiosis using a mouse model of atherosclerotic disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e All animal experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (M2021-56, 2021\u0026thinsp;\u0026minus;\u0026thinsp;107). Male B6.129P2-Apoetm1Unc/J (ApoE KO) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and bred in a specific pathogen-free room at this university. Gene disruption was performed by deleting parts of exon 3 and intron 3 of the mouse ApoE locus and inserting a neomycin resistance gene cassette from an E. coli transposon (expressed under a polyomavirus enhancer sequence, an artificial translation start site sequence, and a thymidine kinase gene promoter from herpes simplex virus) by homologous recombination [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The mice were 16-week-old at the beginning of the experimental procedures. They were fed for eight weeks either a high-fat high-sucrose diet (HFHSD; 20% protein, 40% carbohydrate, and 40% fat, coconut oil, 0.3% gm NaCl; D12327, Research Diets, Inc., New Brunswick, NJ, USA) or HFHSD\u0026thinsp;+\u0026thinsp;excessive salt (4% gm NaCl). Paired feeding was performed by supplying an equal amount of feed. The mice were maintained in an environmentally controlled room (temperature, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u0026deg;C; humidity, 40\u0026ndash;60%; and a 12-h light/ dark cycle, from 7 a.m. to 7 p.m). Cumulative oral intake was measured for 8 weeks. The manually weighed fresh food was placed in a trough in each cage once every three days at 9 a.m. and the amount of food was then measured after 24 h. The remnants of the chow were discarded. At 24 weeks of age, mice were fasted overnight and were then sacrificed by the administration of a combination of anesthetics: 4.0 mg/kg, midazolam, 0.3 mg/kg of medetomidine, and 5.0 mg/kg of butorphanol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. At the time of sacrifice, blood was collected by puncturing the left ventricle of the mouse. To remove circulating blood from the mice, the mice were slowly perfused with 10 ml of PBS from the left ventricle, then the epididymal adipose tissue, jejunum, and finally the aorta were dissected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBlood Pressure Measurement\u003c/h2\u003e \u003cp\u003eWe assessed systolic (SBP) and diastolic (DBP) blood pressures under anesthesia at 24 weeks of age using a mouse tail-cuff blood pressure monitor (BP-98AL V3.02; Softron Co. Ltd., Tokyo, Japan), according to the manufacturer\u0026rsquo;s instructions. The mean blood pressure was calculated using the following formula (mean\u0026thinsp;=\u0026thinsp;diastolic pressure\u0026thinsp;+\u0026thinsp;1/3 of pulse pressure).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiochemistry\u003c/h2\u003e \u003cp\u003eBlood samples were collected from fasted mice, and serum samples were collected after centrifugation at 14,000 rpm for 10 min at 4\u0026deg;C. The levels of triglycerides (TG), total cholesterol (T-Chol), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Dehghan et al., 2017), and non-esterified fatty acids (NEFAs) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] were measured using the enzymatic method. Biochemical examinations were performed using a FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eArteriosclerotic Area Measurement\u003c/h2\u003e \u003cp\u003eThe atherosclerotic foci of the aortic annulus were used to measure the atherosclerotic area. Aortas were refluxed in phosphate-buffered hygienic saline, as previously reported, embedded in a frozen tissue embedding agent, fixed with dry ice, and frozen. Frozen sections were obtained using a cryostat until the aortic valve was visible and then sectioned serially at a thickness of 6 \u0026micro;m [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Ten sections per pull were examined.\u003c/p\u003e \u003cp\u003eTissues were fixed in 60% isopropanol for 15 s and stained with oil red-O (Wako Pure Chemicals) for 30 min at room temperature. After staining, images were taken with BZ-X710 (Keyence Co., Osaka, Japan), and the arterial stiffness area was measured using ImageJ (NIH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of Mononuclear Cells from Aortas in Mice\u003c/h2\u003e \u003cp\u003eUsing a dissecting microscope equipped with a cold light source (2.5\u0026times; magnification), fatty tissue adjacent to the adventitia was carefully dissected and removed, leaving the outer aortic membrane intact. Lymph nodes near the aorta were carefully resected.\u003c/p\u003e \u003cp\u003eThe entire aorta was harvested, and the plaque was detached from the intima. The aortic segment was placed in a 60 mm dish in ice-cold fluorescent activated cell sorting (FACS) buffer and stored until enzymatic digestion.\u003c/p\u003e \u003cp\u003eThe aortas were transferred from a 60 mm dish to a 1.5 mL Eppendorf tube and 0.5 mL of an enzyme cocktail was added. The enzyme cocktail contained the following: 400 U/mL collagenase type I, 120 U/mL collagenase type XI, 60 U/mL hyaluronidase, 60 U/mL DNase1 (C0130, C765, H3506, and 11284932001, respectively; Sigma-Aldrich, St. Louis, MO, USA); 20 mM HEPES (15630106; Gibco\u0026trade;, Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco's phosphate-buffered saline containing calcium (DPBS, Thermo Fisher Scientific). The aortic tissue was cut into small pieces using scissors. The mixture was then transferred to a 50 mL Falcon tube and another 2 mL of the enzyme cocktail was added. The tubes containing the aortic tissue pieces were transferred to a water bath at 37\u0026deg;C for 50 min with slow shaking. After 50 min, the digestion solution was poured into a 100 \u0026micro;m cell strainer placed at the top of a new 50 mL Falcon tube. The remaining aortic tissue was crushed with a syringe plunger, and the cell strainer was rinsed with 5 mL FACS buffer. The filtrates were collected and centrifuged at 300 \u0026times; g for 10 min at 4\u0026deg;C. The supernatant was carefully removed after centrifugation, and the cell pellet was suspended in 400 \u0026micro;L of FACS buffer [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTissue Preparation and Flow Cytometry\u003c/h2\u003e \u003cp\u003eStained cells were analyzed using FACS Canto II, and the data were analyzed using FlowJo version 10 software (Ashland, OR, USA). For gating of innate lymphoid cells, the following antibodies, purchased from eBioscience (Thermo Fisher Scientific), were used: Biotin-CD3e (100304; clone: 145-2C11; 1/200), Biotin- CD45R/B220 (103204; clone: RA3\u0026ndash;6B2; 1/200), Biotin-Gr-1 (108404; clone: RB6-8C5; 1/200), Biotin-CD11c (117304; clone: N418; 1/200), Biotin-CD11b (101204; clone: M1/70; 1/200), Biotin-Ter119 (116204; clone: TER-119; 1/200Biotin-FceRIa (134304; clone: MAR-1; 1/200), FITC-Streptavidin (405202; 1/500), PE-Cy7-CD127 (135014; clone: A7R34; 1/100), Pacific Blue-CD45 (103116; clone: 30-F11; 1/100), PE -GATA-3 (clone: TWAJ, 1/50), APC -RORγ (clone: AFKJS-9, 1/50), and Fixable Viability Dye eFluor 780 (1/400) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, the following antibodies (also purchased from eBioscience) for gating of M1 and M2 macrophages were used: APC- CD45.2 (17045482; clone: 104, 1/50), PE-F4/80 (12480182; clone: BM8, 1/50), APC-Cy7-CD11b (47011282; clone: M1/70, 1/50), FITC-CD206 (MA516870; clone: MR5D3, 1/50), and PE-Cy7-CD11c (25011482; clone: N418, 1/50)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of Free Fatty Acids in the Aorta, Sera, and Feces\u003c/h2\u003e \u003cp\u003eFatty acid composition in the aorta, sera, and feces of ApoE KO mice was measured using gas chromatography-mass spectrometry (GC-MS) with an Agilent 7890B/7000D instrument (Agilent Technologies, Santa Clara, CA, USA). Fifteen milligrams of aorta and feces and 25 \u0026micro;L of sera were methylated using a fatty acid methylation kit (Nacalai Tesque Inc., Kyoto, Japan). If the sample weight was less than 15 mg, the concentration was calculated by dividing by the weight. The final product was loaded onto a Varian capillary column (DB-FATWAX UI, Agilent Technologies). The CP-Sil 88 for the FAME capillary column was used for fatty acid separation (100 m \u0026times; an inner diameter of 0.25 mm \u0026times; membrane thickness of 0.20 \u0026micro;m; Agilent Technologies). The column temperature was maintained at 100\u0026deg;C for 4 min and then gradually increased by 3\u0026deg;C/min to 240\u0026deg;C, and held for 7 min. The sample was injected in the split mode with a split ratio of 5:1. Each fatty acid methyl ester was detected in the selected ion monitoring mode. All results were normalized to the peak height of the C17:0 internal standard[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time Polymerase Chain Reaction (q-RT-PCR) of the Aorta and Jejunum\u003c/h2\u003e \u003cp\u003eGene expression was analyzed using q-RT-PCR. Each aorta and jejunum sample was homogenized in ice-cold QIAzol Lysis reagent (Qiagen, Hilden, Germany), and total RNA was isolated using the RNeasy MinElute Cleanup Kit (Qiagen), according to the manufacturer\u0026rsquo;s instructions. Total RNA (0.5 \u0026micro;g) was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) for first-strand cDNA synthesis, using an oligonucleotide dT primer and random hexamer primers according to the manufacturer\u0026rsquo;s recommendations. The reverse transcription reaction was performed for 120 min at 37\u0026deg;C, and the enzyme was then inactivated by incubation at 85\u0026deg;C for 5 min. RT-PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems), according to the manufacturer\u0026rsquo;s instructions. The following PCR conditions were used: one cycle of 2 min at 50\u0026deg;C and 20 s at 95\u0026deg;C, followed by 40 cycles of 1 s at 95\u0026deg;C and 20 s at 60\u0026deg;C.\u003c/p\u003e \u003cp\u003eTotal RNA extracted from the aorta and jejunum was diluted to 5 ng/\u0026micro;L for all samples in DNase-RNase-free water after concentration measurement using Thermo Scientific\u0026trade; NanoDrop Lite (Thermo Fisher Scientific). The relative expression levels of each target gene in the aorta (\u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIfng\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e, \u003cem\u003eIl33\u003c/em\u003e, and \u003cem\u003eFasn\u003c/em\u003e), the jejunum (\u003cem\u003eCd36\u003c/em\u003e, \u003cem\u003eIl22\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e), and epididymal white adipose tissue (eWAT) (\u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIfng\u003c/em\u003e, and \u003cem\u003eTnfa\u003c/em\u003e) were normalized to the \u003cem\u003eGapdh\u003c/em\u003e threshold cycle (CT) value and quantified using the comparative threshold cycle 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔ\u003csup\u003eCT\u003c/sup\u003e method as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We focused on the increase or decrease in fatty acid absorption. Since fatty acid absorption occurs mainly in the small intestine, we evaluated \u003cem\u003eCd36\u003c/em\u003e and \u003cem\u003eIl22\u003c/em\u003e gene expressions in the jejunum instead of the colon in this study. Signals from HFHS-fed ApoE KO mice were assigned a relative value of 1.0. Six mice from each group were examined, and RT-PCR was performed in triplicates for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA Sequencing\u003c/h2\u003e \u003cp\u003eThree 24-week-olds of middle weight in the group were selected, and microbial DNA was extracted from frozen fecal samples using the QIAamp DNA Feces Mini Kit (Qiagen), following the manufacturer\u0026rsquo;s instructions. The V3-V4 region of the 16S rRNA gene was amplified from the DNA using a bacterial universal primer set (341F and 806R). PCR was performed with 20 ng of genomic DNA as a template in a 30 \u0026micro;L reaction mixture using EF-Taq (SolGent, Daejeon, South Korea) for the following cycles: activation of Taq polymerase at 95\u0026deg;C for 2 min, followed by 35 cycles at 95\u0026deg;C, 55\u0026deg;C, and 72\u0026deg;C for 1 min each, finishing with 10 min at 72\u0026deg;C. Amplification products were purified using a multiscreen filter plate (MilliporeSigma, Burlington, MA, USA). 16S rRNA sequencing was performed using a MiSeq sequencer (Illumina, San Diego, CA, USA) according to the manufacturer\u0026rsquo;s instructions (Macrogen, Seoul, Korea). QIIME version 1.9.1 was used to filter sequences for quality[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Scores less than 75% and mismatches in the barcode or primers were eliminated from the files. The number of operational taxonomic units (OTUs) was determined using the UCLUST algorithm at 97% similarity[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Taxonomic assignment of 16S rRNA was performed with the Greengenes core-set-aligned with UCLUST and UNITE sequence sets for ITS using BLAST (UNITE, 2017).\u003c/p\u003e \u003cp\u003eThe relative abundance of the phenotypic categories of the taxonomic groups was predicted using METAGENAssist, which is a statistical tool for comparative metagenomics [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Data filtering was based on interquartile range, row normalization by sum, and column normalization based on autoscaling. In addition, the Firmicutes/Bacteroidetes ratio was calculated. Alpha diversity was defined as the diversity within an individual sample using the Shannon index [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], Chao1 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and Gini-Simpson index [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, differences in microbial communities between the two groups were investigated using the phylogeny-based weighted UniFrac distance metric and principal coordinate analysis (PCoA) plots, and non-hierarchical \u003cem\u003eK\u003c/em\u003e-means cluster analysis was performed with the number of clusters to be generated pre-specified as two using the Tinn-R Gui version 1.19.4.7, R version 1.36[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe relative abundance of phyla in the groups was evaluated by unpaired \u003cem\u003et\u003c/em\u003e-test using the JMP version 13.2 software (SAS Institute Inc., Cary, NC, USA). Furthermore, the relative abundance of bacterial genera between groups was evaluated using linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/lefse/\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/lefse/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Using a normalized relative abundance matrix, LEfSe showed taxa with significantly different abundances, and the effect size of the feature was estimated using LDA. A \u003cem\u003ep\u003c/em\u003e-value threshold of 0.05 (Wilcoxon rank-sum test) and an effect size threshold of 2 were used for all biomarkers discussed in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using JMP ver. 13.0 software (SAS, Cary, NC, USA). Differences between the two groups were compared using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and asterisks were indicated in the figures as follows to express statistical significance; *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Statistical significance of body weight and oral intake were analyzed using two-way repeated measures ANOVA followed by Bonferroni's tests. Figures were generated using GraphPad Prism software (version 9.0; San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEffects of HFHSD With High-salt Loading on Body Weight, Serum Lipids\u003c/h2\u003e\n \u003cp\u003eApoE KO mice were either fed with HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl or with HFHSD only, and their body weights, blood pressure, and serum lipid levels were compared. At 21 weeks of age, the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl or group had significantly lower body weights than the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, both groups were pair-fed with similar food intake.\u003c/p\u003e\n \u003cp\u003eThe systolic, diastolic, mean blood pressure and pulse pressure were not different between the HFHSD and HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl groups (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-F).\u003c/p\u003e\n \u003cp\u003eSerum lipid levels, such as TG, T-chol, LDL-chol, HDL-chol, and NEFA, were investigated. The HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group had higher serum TG, T-chol, LDL-chol, and NEFA levels but lower serum HDL-cholesterol levels than the HFHSD group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG-K).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eEffects of HFHSD With High-salt Loading on Atherosclerotic Foci of the Aortic Annulus\u003c/h2\u003e\n \u003cp\u003eNext, the area of arterial atherosclerosis in the aortic annulus was measured (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eL). The atherosclerosis area in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was larger than that in the HFHSD group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eM).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eEffects of HFHSD With High-salt Loading on the weight of visceral fat mass\u003c/h2\u003e\n \u003cp\u003eTo assess visceral fat mass, epididymal fat was adopted in this study and weighed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eN \u003cstrong\u003eand O\u003c/strong\u003e). Absolute and relative weight of epididymal fat weight of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than those of the HFHSD group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eDynamics of Inflammatory and Anti-inflammatory Cells in the Aorta\u003c/h2\u003e\n \u003cp\u003eFlow cytometric analysis was performed to determine the number of cells involved in innate immunity in the aorta in the two groups. The M1/M2 macrophage ratio in the aorta of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was higher than that of the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The percentage of ILC1\u0026thinsp;+\u0026thinsp;CD45\u003csup\u003e+\u003c/sup\u003e cells of the aorta in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was higher than that in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), whereas that of ILC2 in CD45\u003csup\u003e+\u003c/sup\u003e cells was lower in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eSaturated Fatty Acids in Feces, Sera, and Aorta\u003c/h2\u003e\n \u003cp\u003eThe concentrations of saturated fatty acids, such as lauric, myristic, palmitic, and stearic acids, in feces, sera, and aortas were investigated by GC/MS. The concentration of saturated fatty acids in the feces of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than that of the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). On the other hand, the concentrations of saturated fatty acids in the sera and aorta of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group were higher than those in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-L).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eEffects of HFHSD With High-salt Loading on the Expression of Genes Related to Inflammatory Cytokines in the Aorta and With Anti-inflammatory Cytokines and Fatty Acid Transporters in the Jejunum\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eGene expression of inflammatory cytokines and fatty acid transporters was analyzed using q-RT-PCR to determine the effect of HFHSD with high-salt loading on chronic inflammation. The expression levels of genes related to inflammatory cytokines, such as \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIfng\u003c/em\u003e, and \u003cem\u003eTnfa\u003c/em\u003e, in the aorta of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group, were higher than those in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). In addition, the expression level of \u003cem\u003eIl33\u003c/em\u003e, which is one of the factors that activate ILC2, in the aorta of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than that of the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). On the other hand, the expression levels of \u003cem\u003eFasn\u003c/em\u003e, a gene related to fatty acid metabolism, was not different between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). The \u003cem\u003eCd36\u003c/em\u003e expression level in the jejunum of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was higher than that of the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG), whereas the \u003cem\u003eIl22\u003c/em\u003e expression level in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than that in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH). In addition, \u003cem\u003eIl6\u003c/em\u003e gene expression in the jejunum of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was higher than that of the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI). Finally, gene expression in visceral adipose tissue was also evaluated based on the assumption that inflammation in visceral fat may have been exacerbated as the cause of the decrease in visceral fat mass despite the development of atherosclerosis in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group. In eWAT, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIfng\u003c/em\u003e, and \u003cem\u003eTnfa\u003c/em\u003e expressions in the HFHSD\u0026thinsp;+\u0026thinsp;NaCl4% group were higher than those in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ-M).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eEffects of HFHSD With High-salt Loading on the Gut Microbiota\u003c/h2\u003e\n \u003cp\u003eWe performed 16s rRNA sequencing to determine the effect of high-salt load HFHSD on the gut microbiota. The most abundant phylum was Bacteroidetes in both groups (HFHSD: 47.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1%, HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl: 52.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4%). The second and third most abundant phyla in the HFHSD group were Firmicutes and Proteobacteria, respectively, whereas those in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group were Proteobacteria and Firmicutes, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eP\u003c/em\u003e-values analyzed using the \u003cem\u003et\u003c/em\u003e-test are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. Next, the Firmicutes/Bacteroidetes phyla (F/B) ratio, whose increase or decrease has been reported to be associated with various diseases, was calculated [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The F/B ratio in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than that in the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). The Shannon index, Chao1 index, and Gini-Simpson index were used to assess the diversity of the gut microbiota, which revealed that the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group had lower diversity than the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). PCoA plots of unweighted and weighted UniFrac distances were constructed to compare the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG \u003cstrong\u003eand H\u003c/strong\u003e). The clustering results showed that the HFHSD and HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl groups belonged to different clusters.\u003c/p\u003e\n \u003cp\u003eAdditionally, we used the LEfSe algorithm to identify specific taxa that were variably distributed between the two groups. Four taxa (including the class Bacteroidia, order \u003cem\u003eBacteroidales\u003c/em\u003e, and genus \u003cem\u003eBarnesiella\u003c/em\u003e) were over-represented, and five taxa (including the class Alphaproteobacteria, genus \u003cem\u003eStreptococcus\u003c/em\u003e, family \u003cem\u003eLachnospiraceae\u003c/em\u003e, family \u003cem\u003eRuminococcaceae\u003c/em\u003e, and genus \u003cem\u003eAllobaculum\u003c/em\u003e) were under-represented in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group compared to the HFHSD group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study using ApoE KO mice, we evaluated the effects of excessive salt intake on atherosclerosis development and showed that excessive salt intake alters the gut microbiota, increases the gene expression levels of long-chain fatty acid transporters in the small intestine, and the absorption of palmitic acid, a saturated fatty acid, thereby increasing serum saturated fatty acid levels, expanding atherosclerotic foci, and reducing anti-inflammatory innate cells.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that excessive intake of saturated fatty acids is associated with the development of atherosclerosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and that saturated fatty acids are rapidly taken up by cultured macrophages, potentially leading to a greater accumulation of arterial lipids[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In clinical practice, the current cardiovascular guidelines recommend reducing saturated fatty acids and replacing them with unsaturated fatty acids[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, recent epidemiological studies have failed to reveal an increased risk of CVDs associated with diets high in saturated fat [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, several studies have found no association between saturated fat intake and carotid intima-media thickness [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In contrast, high serum saturated fatty acid levels have been shown to increase the risk of coronary artery disease[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Taken together, these studies suggest that increased serum saturated fatty acid levels might be more strongly associated with atherosclerosis than saturated fatty acid intake. In the present study, although there was no difference in caloric intake between the HFHSD and HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl groups after pair feeding, serum lipid levels were significantly higher in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group than in the HFHSD group. Based on these results, we evaluated the expression levels of genes related to inflammation in the small intestine to determine whether inflammation caused by a high-salt diet alters the dynamics of nutrient absorption from the intestinal tract. \u003cem\u003eIl22\u003c/em\u003e mRNA levels in the small intestine of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group were lower than those in the HFHSD group. IL22 is an important cytokine that plays a role in the thickening of the mucin layer of the small intestine and protects against intestinal inflammation by enhancing the expression of antimicrobial proteins in epithelial cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In contrast, \u003cem\u003eCd36\u003c/em\u003e mRNA levels, which are long-chain fatty acid transporters, increased following the administration of excessive salt in this study. In our previous study, we reported that dysbiosis caused by excessive intake of saturated fatty acids, sucrose, or trans-fatty acids causes inflammation in the intestinal tract, which in turn increases \u003cem\u003eCd36\u003c/em\u003e expression levels in the small intestine [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Dysbiosis is difficult to define precisely, but this study used changes in the Firmicutes/Bacteroidetes (F/B) ratio and reduced diversity as markers. Increased F/B ratio has been reported to be associated with obesity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], whereas decreased the ratio has been reported to be associated with type 2 diabetes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and inflammatory bowel disease [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, Morgan, et al. reported that the phylum Proteobacteria increased in IBD patients compared to healthy subjects. In this study, F/B ratio in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group was lower than that in HFHSD group and the abundance of phylum Proteobacteria in the HFHSD\u0026thinsp;+\u0026thinsp;NaCl4% group was higher than that in the HFHSD group. In summary, the excessive salt intake group in this study showed the gut microbiota more similar to IBD than to the gut microbiota that cause obesity and glucose intolerance.\u003c/p\u003e \u003cp\u003eRecently, other groups have reported that Cd36 expression in the small intestine is increased by dysbiosis and intestinal inflammation. Shi, et al. have reported that the supplementation of \u003cem\u003eBacteroides fragilis\u003c/em\u003e, which is reported to be related with obesity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], did not only deteriorated metabolic dysfunction but also increased the expression of Cd36 in small intestine [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the present study, there was no difference in the expression of \u003cem\u003eFasn\u003c/em\u003e, a gene that promotes the conversion of lauric acid to myristic acid and myristic acid to palmitic acid in the aorta between the two groups. In summary, the study suggested that the increase in saturated fatty acids in arteries may be due to increased fatty acid absorption from Cd36, rather than changes in the metabolism of saturated fatty acids in aorta. The present study also suggested that inflammation in the intestine caused by a high-salt diet may have increased the gene expression of long-chain fatty acid transporters in the intestinal tract, resulting in increased saturated fatty acid absorption. The expression of \u003cem\u003eIl6\u003c/em\u003e in the small intestine was also increased by the high salt diet in this study.\u003c/p\u003e \u003cp\u003eAs microorganisms rely on dietary substrates in the gut, the gut microbiota is often proposed as a mediator of the pro-and anti-inflammatory effects of diet. Similar to other nutrients, animal studies have demonstrated that salt-rich foods induce inflammation and autoimmunity through microbial mechanisms, such as the induction of T-helper 17 cells [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Moreover, type 3 innate lymphoid cells have been reported to have a function homologous to that of Th17, are very abundant in the intestinal mucosa, and play a role in intestinal homeostasis by producing interleukin (IL)-22 and IL-17 in response to IL-23 and IL-1β [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the 16s rRNA analyses of gut microbiota, the phylum Bacteroidetes, class Bacteroidia, order \u003cem\u003eBacteroidales\u003c/em\u003e, and genus \u003cem\u003eBarnesiella\u003c/em\u003e were more abundant in HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl mice than in HFHSD mice. \u003cem\u003eBarnesiella\u003c/em\u003e, a genus of the family Porphyromonadaceae and order \u003cem\u003eBacteroidales\u003c/em\u003e, is one of the most abundant genera detected in the mouse intestine. Several studies have revealed that the abundance of the phylum Bacteroidetes, class Bacteroidia, order \u003cem\u003eBacteroidales\u003c/em\u003e, family \u003cem\u003ePorphyromonadaceae\u003c/em\u003e, and genus \u003cem\u003eBarnesiella\u003c/em\u003e in diabetic (\u003cem\u003edb/db\u003c/em\u003e) mice was higher than that in lean mice [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. A previous study comparing the microbiota of patients with hypertension to healthy individuals has revealed that the abundance of the genus \u003cem\u003eBarnesiella\u003c/em\u003e was higher in patients with hypertension [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, the abundance of the genus \u003cem\u003eBarnesiella\u003c/em\u003e increased in IL-22 deficient mice[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The function of the genus \u003cem\u003eBarnesiella\u003c/em\u003e remains unclear; however, several previous studies and the present study suggest that the high-salt diet-induced increase in the genus \u003cem\u003eBarnesiella\u003c/em\u003e is associated with dysbiosis, which might be related to a decrease in IL-22 in the intestinal tract.\u003c/p\u003e \u003cp\u003eAlthough the present study showed that a high-salt diet can promote the development of atherosclerosis, no obvious increase in blood pressure was detected. One possible reason for this observation is that in other studies of high-salt diet-induced atherosclerosis in ApoE KO mice, the dietary salt concentration was 7\u0026ndash;8% gm and the administration period was 12 weeks[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In contrast, in our previous study, a high-salt diet of 8% gm resulted in extremely low food intake and frequent sudden death of mice during the 12-week treatment period; therefore, we reduced the salt concentration to 4% gm and shortened the treatment period to 8 weeks. Additionally, hypertension has long been known to increase the prevalence of coronary artery disease and the extent and severity of atherosclerosis in both humans [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and animals [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In other words, we examined the effects of increased saturated fatty acid absorption on increased arterial stiffness while excluding the blood pressure component.\u003c/p\u003e \u003cp\u003eIn this study, we investigated innate immune cells in the aorta. Previous studies have reported that ILC2 protects against atherosclerosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In addition, ILC2 transfer to ApoE KO mice reduced the lipid content of the atherosclerotic lesions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, the number of ILC2 cells and the expression level of \u003cem\u003eIl33\u003c/em\u003e in the aorta of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group were lower than those of the HFHSD group. IL-33, which is secreted by ILC2 cells [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], has been reported to play a protective role against the development of atherosclerosis [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The decrease in arterial ILC2 associated with excessive salt intake may exacerbate atherosclerosis by reducing the secretion of IL-33, which has anti-arterial effects.\u003c/p\u003e \u003cp\u003eAs a limitation of this study, we did not reveal significant difference of blood pressure by excessive salt intake. However, a previous study showed that blood pressure did not change with high salt diet in the inactive phase but did in the active phase [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Therefore, re-measuring blood pressure during the active phase could show an increase in blood pressure due to the high-salt diet. In addition, in this study, the body weight of the HFHSD\u0026thinsp;+\u0026thinsp;4% NaCl group was significantly lower than that of the HFHSD group. Furthermore, absolute and relative epididymal fat weights were lower than those of the HFHSD group. Previous studies have shown an inverse correlation between salt intake and body weight gain in mice fed a high-fat diet [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]; DeClercq et al. reported that excess salt intake increased epididymal fat weight in male mice, and Pitynski-Miller et al. reported that excess salt intake in female rats. In summary, the effects of high-salt diets on body weight and visceral fat mass may vary among various backgrounds and require further study. On the other hand, in this study, excessive salt intake induced the inflammation in eWAT. In a previous study, expression of the inflammatory adipocytokines have been reported to increase in a dose-dependent manner upon salt treatment [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Furthermore, the inflammation in adipose tissue induces adipose tissue dysfunction such as decrease in fatty acid intake [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. It has been reported that decreased fatty acid uptake into visceral fat increases ectopic fat accumulation, whereas visceral adipose tissue weight decreases. Since atherosclerosis, a form of ectopic fat accumulation, was advanced in this study, the decrease in body weight in the HFHSD\u0026thinsp;+\u0026thinsp;NaCl4% group may have been due in part to a reduction in visceral fat weight caused by inflammation within visceral fat. Moreover, we did not evaluate the glucose tolerance. Additional data on blood glucose levels could have explained the weak effect of high salt load on the arterial stiffness region. Lastly, we did not have the data of the characterization of the initial fecal sample prior feeding. If we had the data, we could compare the data with the baseline data and more accurately determine the changes in the gut microbiota due to differences in the diet.\u003c/p\u003e \u003cp\u003eIn conclusion, this study revealed the mechanism by which an HFHSD that mimics the modern diet, loaded with excess salt promotes the development of atherosclerosis. We revealed that excessive salt intake causes alterations in the gut microbiota, induces inflammation in the gut, increases the expression of long-chain fatty acid transporters in the small intestine, and increases the influx of saturated fatty acids into the body, thereby worsening atherosclerosis. This study offers insights into the mechanism of aggravation of atherosclerosis which could not be explained by an increase in saturated fatty acid intake alone.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine (M2021-56, 2021-107). All methods were performed in accordance with relevant guidelines and regulations. All methods were reported according to the ARRIVE guidelines for reporting animal experiments (https://arriveguidelines.org/)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTakashi Yoshimura has no competing interest.\u003c/p\u003e\n\u003cp\u003eTakuro Okamura has no competing interest.\u003c/p\u003e\n\u003cp\u003eHiroki Yuge has no competing interest.\u003c/p\u003e\n\u003cp\u003eYukako Hosomi has no competing interest.\u003c/p\u003e\n\u003cp\u003eEmi Ushigome has received grants from the Japanese Study Group for Physiology and Management of Blood Pressure, the Astellas Foundation for Research on Metabolic Disorders (Grant number: 4024). Donated Fund Laboratory of Diabetes therapeutics is an endowment department, supported with an unrestricted grant from Ono Pharmaceutical Co., Ltd., and received personal fees from AstraZeneca plc, Astellas Pharma Inc., Daiichi Sankyo Co., Ltd., Kyowa Hakko Kirin Company Ltd., Kowa Pharmaceutical Co., Ltd., MSD K.K., Mitsubishi Tanabe Pharma Corp., Novo Nordisk Pharma Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., and Sumitomo Dainippon Pharma Co., Ltd., outside the submitted work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNaoko Nakanishi received grant support from Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 19K23999) (JSPS KAKENHI Grant Number 20K16158) and The Japan Food Chemical Research Foundation, and received personal fees from Kowa Pharmaceutical Co., Ltd., and Novo Nordisk Pharma Ltd.\u003c/p\u003e\n\u003cp\u003eRyoichi Sasano has no competing interest.\u003c/p\u003e\n\u003cp\u003eTakehiro Ogata has no competing interest.\u003c/p\u003e\n\u003cp\u003eMasahide Hamaguchi has received grants from Asahi Kasei Pharma, Nippon Boehringer Ingelheim Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Astellas Pharma Inc., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novo Nordisk Pharma Ltd., and Eli Lilly Japan K.K., outside the submitted work.\u003c/p\u003e\n\u003cp\u003eMichiaki Fukui has received grants from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Co, Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Hakko Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharmaceutical Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd. Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Terumo Co., Teijin Pharma Ltd., Nippon Chemiphar Co., Ltd., and Johnson \u0026amp; Johnson K.K. Medical Co., Abbott Japan Co., Ltd., and received personal fees from Nippon Boehringer Ingelheim Co., Ltd., Kissei Pharma Co., Ltd., Mitsubishi Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Sanofi K.K., Takeda Pharma Co., Ltd., Astellas Pharma Inc., MSD K.K., Kyowa Kirin Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Kowa Pharma Co., Ltd., Novo Nordisk Pharma Ltd., Ono Pharma Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Eli Lilly Japan K.K., Taisho Pharma Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., Mochida Pharma Co., Ltd., Abbott Japan Co., Ltd., Medtronic Japan Co., Ltd., Arkley Inc., Teijin Pharma Ltd. and Nipro Cor., outside the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUshigome Emi received grant support from the Japan Society for the Promotion of Science (grant numbers 18K15897 and 22K08108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.H. originated and designed the study, researched the data, and wrote the manuscript. T.Y., T.O., and M.F. originated and designed the study, researched the data, and reviewed the manuscript. D.Y., Y.H., T.K., E.U., and N.N. researched the data and contributed to the discussion. R.S. and T.O. supported the techniques of research. \u0026nbsp;H.M. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors were involved in the writing of the manuscript and approved the final version of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.com) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDuttaroy AK. Role of Gut Microbiota and Their Metabolites on Atherosclerosis, Hypertension and Human Blood Platelet Function: A Review. 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Int J Mol Sci. 2019;20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2759933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2759933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn this study, we investigated the mechanism by which excessive salt intake aggravates atherosclerosis by evaluating the changes in the gut microbiota, the expression of nutrient transporters in the gut, and the fatty acid composition in atherosclerosis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSixteen-week-old male ApoE-deficient mice were either fed a high-fat, high-sucrose diet (HFHSD) or HFHSD, high-salt diet (HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl) for 8 weeks. The HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group showed progression of atherosclerosis, and gut microbiota analysis revealed that this group had a reduced abundance of \u003cem\u003eAllobaculum\u003c/em\u003e spp., \u003cem\u003eRuminococcaceae\u003c/em\u003e family, \u003cem\u003eLachnospiraceae\u003c/em\u003e family, and \u003cem\u003eAlphaproteobacteria\u003c/em\u003e class compared to the HFHSD group. Furthermore, \u003cem\u003eCd36\u003c/em\u003e gene expression levels were increased in the small intestine of the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group compared to those in the HFHSD group. The concentration of saturated fatty acids in serum and atherosclerotic lesions, was remarkably increased in the HFHSD\u0026thinsp;+\u0026thinsp;4%NaCl group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eDysbiosis induced by excessive salt intake increases the expression of long-chain fatty acid transporters in the intestinal tract, which increases the influx of saturated fatty acids into the body.\u003c/p\u003e","manuscriptTitle":"Gut dysbiosis induced by a high-salt diet aggravates atherosclerosis by increasing the absorption of saturated fatty acids in ApoE-deficient mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-11 22:16:45","doi":"10.21203/rs.3.rs-2759933/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ec4922e-e8f5-4e93-90f6-02877a5d98e8","owner":[],"postedDate":"May 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-02T13:15:42+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-11 22:16:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2759933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2759933","identity":"rs-2759933","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}