Fecal microbiota transplantation regulates blood pressure by altering gut microbiota composition and intestinal mucosal barrier function in spontaneously hypertensive rats

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In this study, we used fecal microbiota transplantation to determine the impact of microbiota composition on blood pressure in spontaneous hypertensive rats (SHRs), using normotensive Wistar Kyoto (WKY) rats as controls. Methods: SHRs were randomly divided into 2 groups ( n = 10/group), SHR and SHR-T (SHR plus fecal transplantation) and WKY into WKY and WKY-T (WKY plus fecal transplantation). SHR-T received fecal transplantation from WKY while WKY-T received fecal transplantation from SHR. Blood pressure was measured from the tail artery in conscious rats. 16S rDNA gene amplicon sequencing was used to analyze bacterial composition. Circulating levels of diamine oxidase, D-lactate, FITC-Dextrans, and lipopolysaccharide were determined. Hematoxylin and eosin (H&E) staining was used to observe structural changes in the intestinal mucosa. Immunofluorescence, Western blot, and RT-PCR were utilized to determine changes in the expression of tight junction proteins. Results: Following cross fecal transplantation, blood pressure decreased in SHR and increased in WKY. Significant differences in gut microbial composition were found between hypertensive and normotensive rats, specifically regarding the relative abundance of lactic and butyric acid-producing bacteria. Changes in gut microbiota composition also impacted the intestinal mucosal barrier integrity. Moreover, fecal transplantation affected the expression of tight junction proteins that may impact intestinal mucosal permeability and structural integrity. Conclusion: Blood pressure may be associated with butyric acid-producing intestinal microbiota and its function in regulating the integrity of intestinal mucosal barrier. fecal microbiota transplantation gut microbiota hypertension intestinal mucosal barrier spontaneously hypertensive rats Figures Figure 1 Figure 2 Figure 3 INTRODUCTION In recent years, fecal microbiota transplantation has received widespread attention as a major method of intervention in intestinal microecology, allowing increased understanding of the role of intestinal microbiota in the occurrence and development of diseases. Fecal microbiota transplantation is a technique where functional microorganisms are separated from the feces of healthy individuals and transplanted into the recipient's digestive tract. This procedure rebuilds the patient's intestinal microecological balance, thereby treating diseases [ 1 ]. Treatment using fecal bacteria transplantation has gradually expanded from application to digestive system diseases [ 2 – 4 ], such as Clostridium difficile infection, inflammatory bowel disease, and irritable bowel syndrome, to central nervous system diseases such as Alzheimer’s disease [ 5 ]. Increasing evidence has shown that gut microbiota imbalance is closely related to cardiovascular disease [ 6 , 7 ]. A large number of studies suggest that hypertension is related to gut microbiota imbalance [ 6 , 8 , 9 ]. In animal experiments fecal microbiota transplantation has been shown to change blood pressure sensitivity in salt-sensitive rats [ 10 ]. Durgan et al. [ 11 ] transplanted the feces of high-fat-fed obstructive sleep apnea-induced hypertensive rats to normally fed obstructive sleep apnea-induced hypertensive rats, which led to an increase in blood pressure in the normally fed rats. Transplanting the feces from stroke-prone spontaneous hypertensive rats (SHRs) into normotensive Wistar Kyoto (WKY) rats treated with antibiotics led to increased Systolic blood pressure in the WKY rats, whereas systolic blood pressure decreased in stroke-prone SHRs transplanted with feces from WKY rats [ 12 ]. These results suggest that changes in gut microbiota composition can affect blood pressure. When fecal bacteria from a human hypertensive patient was transplanted to sterile mice, their blood pressure increased, indicating that hypertension can be transferred through the microbiota. Although many studies show that hypertension is accompanied by gut microbiota imbalance and intestinal mucosal barrier damage and dysfunction [ 6 , 13 , 14 ], the underlying mechanism remains unclear. In order to investigate the role of gut microbiota in the pathogenesis of hypertension, we hypothesized that intestinal fecal microbiota cross-transplantation between SHRs and WKY rats would change fecal microbiota composition in recipient rats and thereby alter blood pressure. Intestinal barrier dysfunction has been implicated in hypertension [ 15 , 16 ], and butyric acid has been shown to improve intestinal barrier function [ 17 ]. In the current study, we found that the composition of butyric acid-producing bacterial species changed after fecal microbiota transplantation. As the intestinal mucosa is composed of intestinal epithelial cells and tight junctions, the integrity of which is regulated by the expression of tight junction proteins, including ZO-1, occludin, and claudin [ 18 , 19 ], we further hypothesized that changes in gut microbiota composition after fecal microbiota transplantation would change the expression of tight junction proteins. MATERIALS AND METHODS Animals Male 14-week-old SHRs ( n = 20) and WKY rats ( n = 20) rats were used (350 ± 20 g) (Vital River Laboratory Animal Technology, China). SHRs were randomly divided into 2 groups (10/group), SHR and SHR-T (SHR plus fecal transplantation). WKY rats were randomly divided into 2 groups (10/group), WKY and WKY-T (WKY plus fecal transplantation). SHR-T and WKY-T underwent cross-transplantation of fecal bacteria. Before transplantation, tail artery pressure was measured in each group in an awake state. One rat in the WKY-T group died during the transplantation process. Animals were kept in a laboratory with constant temperature (22 ± 2°C), humidity (55 ± 5), and a 12 light/dark cycle. Ethics This study was approved by the Gansu College of Traditional Chinese Medicine Animal Experimen Ethics Committee [2015-002]. Preparation of fecal bacteria liquid Defecation was stimulated and fecal samples (5 g per group) were collected in a sterile beaker to prevent contamination. The fecal samples were dissolved in sterile saline (5 mL/g of feces in 0.9% normal saline). A fecal suspension was collected by coarsely filtering impurities with sterile gauze and centrifuged at 3,000 rpm for 10 min. Supernatant was discarded and the fecal bacteria precipitate was obtained. Physiological saline (20 mL) was added to fecal precipitate and mixed well. The suspension was used immediately for transplant. Fecal bacteria transplant Fecal bacterial liquid from the SHR group was transplanted to the WKY-T group, while fecal bacteria liquid from the WKY group was transplanted to the SHR-T group. A sterile 5 mL syringe was used to take 2 mL of the fecal bacteria liquid. Gavage was used to inject fecal bacteria liquid into the intestine through the anus (once a week for 6 weeks). Blood pressure measurement After 6 weeks of treatment, tail arterial pressure was measured in the early morning using a BP-98A noninvasive blood pressure system (Softron, Tokyo, Japan). Each rat was measured 5 times and the average BP was used. Fecal sampling and 16S ribosomal DNA (rDNA) gene amplicon sequencing Fecal samples were collected in a sterile tube immediately after euthanasia and stored at − 80°C. Microbial DNA was extracted from fecal samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). The V3–V4 hypervariable region of the 16S rDNA gene was amplified using forward primer 515F (GTG CCA GCMGCC GCG GTA A) and reverse primer 806R (GGA CT CHVGGG TWTCT AAT). PCR amplicons were sequenced on an Illumina HiSeq2500 platform, with paired-end reads of 450–460 bp long. Raw sequence data were filtered, processed, and analyzed according to the QIIME (V1.7.0) quality control process. Sequences with ≥ 97% similarity were assigned to the same operational taxonomic unit (OTU). Merging of paired forward and reverse reads was performed by FLASH (fast length adjustment of short reads, v1.2.11) software. OTU clustering on the merged reads was performed by USEARCH (v7.0.1090) software. Representative sequences of OTUs were selected and clustered with the Greengenes database 13 − 8 version at 99% sequence similarity, and then aligned to obtain species annotation information. Taxonomic annotation and abundance analysis were also performed. ANOVA, ANCOM, Kruskal Wallis were used to identify bacteria that had significantly different relative abundances between groups of interest. Alpha and beta diversity indices were used to evaluate differences between samples. Alpha diversity and beta diversity were analyzed using the Qiime2 Diversity plug-in. Chao1 index, Ace index, Shannon index, and Simpson index were used to analyze alpha diversity. The weighted UniFrac distance was used for the principal coordinate analysis (PCoA) of beta diversity. Redundance analysis (RDA) was used to reveal the potential associations between microbial communities and related environmental factors, and the permutation test was performed using the R language VEGAN bag. Enzyme-linked immunoassay (ELISA) Levels of diamine oxidase (DAO) and D-lactate were determined by ELISA. After six weeks of fecal transplant intervention, 5 mL of blood was collected from the pericardium in anesthetized rats (10% chloral hydrate, 0.3 mL/100 g body weight, i.p.). Blood was centrifuged at 3,500 rpm at 4°C for 15 min. Serum was stored at − 20 to − 80°C. ELISA was performed based on the manufacturer’s instructions (Meimian, Jingsu Fiya Biological Technology, China). Azo matrix colorimetric method was used to measure plasma lipopolysaccharide (LPS) contents (Xiamen Limulus Reagent Experimental Factory). FITC-Dextran content determined by fluorescence spectrophotometry FITC-Dextran (MW: 4,000, Sigma, St. Louis, MO, USA) was administered intragastrically (5 mg/100 g body weight) after fasting for 12 h. Four hours later, animals were anesthetized to collect 4 mL of blood from the hepatic portal vein. Blood was centrifuged at 3,000 rpm for 15 min at 4°C. Serum was stored at − 20°C and later used for determining FITC-Dextran (excitation wavelength 485, emission wavelength 528) using a multifunctional microplate reader (Bio Tek Synergy HT, USA). Intestinal mucosa morphological changes determined by H&E staining Intestines (1–2 cm) were collected and fixed in 4% paraformaldehyde. Thin 5 µm paraffin sections were cut and dewaxed by xylenes and ethanol. Sections were stained with hematoxylin for 10 min and rinsed with running water to remove excess staining. Sections were then differentiated with 7% hydrochloric acid and ethanol for a few seconds. Sections were then submerged in 95% ethanol for 30 s, alcohol eosin stain for 30 s, 90% ethanol for 30 s, 100% ethanol for 30 s, and carbolic xylene for 30 s. Sections were cleared in xylenes for 30 s and mounted. Morphological changes in the structure of small intestinal villi were observed. Mouse colons were collected in a sterile bottle, snap frozen in liquid nitrogen, and stored at − 80°C for immunofluorescence, real-time RT-PCR, and Western blotting. Intestinal mucosal tight junction proteins (ZO-1, occludin, and claudin) were determined by immunofluorescence, real-time RT-PCR, and Western blotting. Tight junction proteins determined by immunofluorescence Frozen sections (10 µm) were fixed in 4% paraformaldehyde for 20 min, washed thrice with PBS for 5 min, and blocked with a blocking solution (Thermo Fisher, Waltham, MA, USA) at room temperature for 40 min. ZO-1 and occludin primary antibodies (rabbit polyclonal, Abcam, Cambridge, UK) were added and incubated overnight at 4°C. After washing with PBS thrice for 5 min, IgG-H&L secondary antibody (Abcam, goat anti-rabbit polyclonal) was added and incubated at room temperature for 1 h. The expression of ZO-1 and occludin was observed by laser confocal scanning microscopy. Tight junction protein mRNA expression determined by real-time RT-PCR Frozen colon tissues (50 mg) were ground using an automatic tissue homogenizer (MagNA Lyser, Roche) to extract RNA using Trizol (500 µL). RNA purity and concentration were detected by a nucleic acid microquantifier (Pultton DNA/Proteins Analyzer P100). Reverse transcription was performed according to the instructions from PrimeScript ™ reverse transcription kit. Primer sequences were based on the rat mRNA sequence on the NCBI website and designed with Primer 5.0 software. β-actin primer: forward 5'-GGAGATTACTGCCCTGGCTCCTA-3', reverse 5'-GACTCATCGTACTCCTGCTTGCTG-3; Zo-1 primer: forward 5'-CCATCTTTGGACCGATTGCTG-3', reverse 5'-TAATGCCCGAGCTCCGATG-3'; occludin primer: forward 5'-GTCTTGGGAGCCTTGACATCTTG-3', reverse 5'-GCATTGGTCGAACGTGCATC-3'; claudin-1 primer: forward 5'-CATGAAGTGCATGAGGTGCTTAGAA-3', reverse 5'-TGGCCACTAATGTCGCCAGA-3'. Real-time quantitative fluorescent PCR was performed based on the instructions of the Go Taq® qPCR Master Mix (Promega) real-time quantitative PCR kit. Data obtained were quantitatively analyzed by the 2 − ΔΔCt method. Tight junction protein expression determined by western blotting Colon tissues (50 mg) were collected and homogenized in RIPA lysis solution (500 µL). The homogenized tissues were centrifuged at 12,000 rpm for 15 min at 4°C. Supernatant was collected and protein concentration was determined by the Bicinchoninic Acid Kit (PC0020, Solarbio). Protein loading buffer was added to the sample, heated at 100°C for 5 min, and stored at − 80°C. Protein (50 µg) was loaded on gels with different gel concentrations (ZO-1: 8%, occludin: 12%, claudin-1: 12%) and ran at 100 V for 1.5 h. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (REF IPVH00010, pore size 0.45 µm; Millipore) under 180 mA for 2 h. The membranes were blocked in 5% skimmed milk at room temperature for 2 h, followed by washing with TPBS with shaking 3 times, for 10 min each. Membranes were then incubated with rabbit polyclonal antibodies against ZO-1 (1:500), occludin (1:500), claudin-1 (1:500), and GAPDH (1:1500) overnight at 4°C. After the membranes were washed with TPBS with shaking 3 times, for 10 min each, they were incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1:4000) with shaking at room temperature for 2 h. The membranes were washed with TPBS 3 times, for 10 min each, and washed once with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 5 min. Protein bands were visualized by chemiluminescence and analyzed by Image pro-plus 6.0 software. Results are expressed as normalized intensity to GAPDH. Statistics Data were processed using SPSS 21.0 and presented as mean ± standard deviation. One-way ANOVA or Student’s t- test was used to assess statistical significance, where appropriate. Kruskal Wallis method was used to compare the difference in alpha diversity index among the groups. Significant different were considered at P < 0.05. RESULTS Cross transplantation of fecal bacteria increases blood pressure in WKY rats After transplanting SHR fecal bacteria to WKY rats, systolic, diastolic, and mean blood pressures of the WKY-T group were significantly higher than that of the WKY group. Conversely, blood pressure was significantly decreased in the SHR-T group relative to the SHR group (Fig. 1 ). Fecal microbiota transplantation changes gut microbial composition Chao1, ACE, and Shannon indices in the SHR group tended to be higher (even though not statistically significant) than that in the WKY group, while Simpson’s index was not significantly different between groups. After SHR transplantation with WKY feces, alpha diversity did not decline. However, after WKY transplantation with SHR feces, Chao1 and ACE indices increased while the Shannon index decreased. These results show that fecal bacteria transplantation affected intestinal microbiota alpha diversity. The intestinal microbiota in SHRs increased the number of species of intestinal microbiota in WKY rats (Fig. 2 A). Weighted UniFrac beta diversity analysis showed that gut microbiota composition varied among rats in the SHR group, with that of the WKY group being more similar within that group. After the SHR group was transplanted with feces from the WKY group, gut microbiota composition tended to be more similar at the genus level, indicating that transplantation of WKY fecal bacteria, to a certain extent, promoted the restoration of intestinal microbiota in SHRs. After the WKY group was transplanted with feces from the SHR group, gut microbiota composition did not change substantially at the genus level, indicating that the transplantation of SHR fecal bacteria had no effect on the gut microbiota composition of WKY rats (Fig. 2 B). We identified nine main phyla using 16S rRNA gene amplicon sequencing, namely Firmicutes, Bacteroidetes, Proteobacteria, TM7, Actinobacteria, Tenericutes, Cyanobacteria, Verrucomicrobia, and Elusimicrobia. Firmicutes and Bacteroides had the highest relative abundance. The ratio of Firmicutes to Bacteroides (F/B) was 2.58 in the WKY group and 3.61 in the SHR group. After transplantation, the F/B ratio decreased to 3.37 in the SHR-T group and increased to 2.79 in the WKY-T group. After SHRs were transplanted with WKY feces, the relative abundance of Firmicutes, Verrucomicrobia, and Cyanobacteria decreased, and that of Bacteroides increased. After transplanting SHR feces to WKY rats, however, the relative abundance of Firmicutes and Verrucomicrobia increased, and that of Bacteroides decreased (Fig. 2 C and 2 D). Systolic blood pressure is closely related to changes in microbiota composition Redundancy analysis showed that systolic ( P = 0.0005), mean ( P = 0.0015), and diastolic ( P = 0.0065) pressures were all related to variations in microbiota composition. Among them, systolic blood pressure appeared to be the most closely related to differences in gut microbiota composition (Fig. 2 E). Fecal microbiota transplantation alters the relative abundance of intestinal lactic acid-producing and butyric acid-producing bacteria Based on abundance analysis of 16S rRNA gene amplicon sequencing data, the SHR group had a higher relative abundance of lactic acid-producing Lactobacillus and Turicibacter ( P < 0.01) and reduced relative abundance of butyric acid-producing Clostridium ( P < 0.05) compared with the WKY group. However, following fecal microbiota transplantation from WKY rats, the relative abundance of Lactobacillus was decreased ( P < 0.01), and that of Clostridium ( P < 0.05) and Turicibacter ( P < 0.01) increased in SHRs. Conversely, WKY rats transplanted with SHR feces had increased relative abundance of Lactobacillus ( P < 0.05) and decreased relative abundance of Clostridium and Turicibacter ( P < 0.05). These results suggest that fecal transplantation led to a change in the relative abundance of lactic acid- and butyric acid-producing bacteria, which may promote the conversion of lactic acid to butyric acid (Fig. 2 F). Fecal microbiota transplantation affects intestinal mucosal barrier integrity As discussed earlier, butyric acid promotes intestinal barrier function [ 17 ]. As the relative abundance of lactic acid-producing bacteria changed after fecal microbiota transplantation, we sought to determine the impact of fecal microbiota transplantation on intestinal barrier function. Serum FITC-Dextran and LPS contents ( P < 0.05), as well as serum DAO ( P < 0.01) and D-lactic acid ( P < 0.05), were increased in the WKY-T group relative to WKY groups. Conversely, in the SHR-T group, serum FITC-Dextran, LPS, DAO, and D-lactic acid contents were decreased relative to the SHR group ( P < 0.05). These results indicate that transplantation of fecal bacteria from SHRs to WKY rats lead to increased intestinal mucosal permeability, while transplantation of fecal bacteria from WKY rats to SHRs improves intestinal mucosal barrier function (Fig. 3 A and 3 B). Fecal microbiota transplantation affects colonic mucosal structure Colonic mucosa of rats in the WKY group had intact structure, with neatly arranged glands. However, in the SHR group, glands were irregular, the number of goblet cells was reduced, the structure of the lamina propria was loose, and edema was visible, indicating that the intestinal mucosal barrier had been compromised. Compared with the SHR group, the glands in the mucosal barrier in the SHR-T group were neatly arranged and interstitial edema was reduced, indicating recovery of intestinal mucosal barrier structure following fecal transplantation from SHR rats. The colonic morphology in the WKY-T group was similar to that in the SHR group, with disorganized glands and interstitial edema, indicating that fecal transplantation from SHRs had a negative impact on the intestinal mucosal barrier of the WKY-T group (Fig. 3 C). Fecal microbiota transplantation affects tight junction protein expression The mRNA expression of ZO-1, claudin-1, and occludin in colon tissue from the WKY-T group was lower than that of the WKY group ( P < 0.05). In contrast, ZO-1, claudin-1 and occludin mRNA expression was higher in the SHR-T group than that of SHR group ( P < 0.05) (Fig. 3 D). Using immunofluorescence, we showed that ZO-1, occludin, and claudin-1 proteins were mainly distributed on the intestinal epithelial cell membrane. In the SHR group, ZO-1, occludin, claudin-1 protein staining was scattered along the cell membrane, with weakened fluorescence intensity, and a significantly reduced positive staining area compared to the WKY group. In the SHR-T group, although staining was also scattered along the cell membrane with weakened fluorescence intensity compared with the WKY group, there was more staining than in the SHR group. In the WKY-T group, although ZO-1, occludin, and claudin-1 staining was enhanced and the distribution was more continuous relative to the SHR group, the staining was still decreased relative to the WKY group (Fig. 3 E). Similar to mRNA expression, protein levels of ZO-1, claudin-1, and occludin were significantly reduced in SHR compared with the WKY group ( P < 0.01). Compared with the SHR group, protein levels of ZO-1, claudin-1, and occludin were increased in the SHR-T group ( P < 0.05). Compared with the WKY group, the expression of ZO-1, claudin-1, and occludin were significantly reduced in the WKY-T group ( P < 0.05) (Fig. 3 F and 3 G). These results suggest that fecal bacteria transplantation may promote the recovery of the structure and function of the intestinal mucosal barrier in SHRs by up-regulating the expression of tight junction-related proteins. DISCUSSION Our study demonstrates important changes to gut microbiota composition and the structure of the intestinal mucosal barrier in SHRs and WKY rats following cross-transplantation and demonstrates the effect of microbiota composition on BP. Importantly BP decreased in hypertensive rats after fecal microbiota transplantation from normotensive ats, and vice versa. Gut microbial diversity changed significantly after fecal transplantation. Moreover, fecal microbiota transplantation led to changes in the relative abundance of lactic acid- and butyric acid-producing bacteria. Importantly, changes in the composition of butyric acid producing bacteria may be associated with intestinal mucosal permeability and structural integrity, as reflected in changes in the expression of tight junction proteins. These results support the intimate relationship between the gut microbiota and intestinal mucosal barrier function, which in turn could impact blood pressure. In studies related to intestinal microecology, common microecological interventions include drugs, probiotic supplementation, and fecal microbiota transplantation. Fecal microbiota transplantation is important for the study of intestinal microecology as it allows direct alteration of host microecology. A previous study showed that fecal transplantation from a hypertensive donor increased the BP of sterile recipient mice, which demonstrates a causal effect of the gut microbiota on BP [9]. Based on this result, we sought to investigate the mechanistic basis thereof, and found that BP changes are associated with microbiota-induced changes in intestinal mucosal barrier. Gut microbiota composition is closely related to and may regulate the expression of tight junction proteins in the intestinal mucosal barrier. One of the main functions of tight junctions is to regulate intestinal permeability [20]. Tight junctions provide a physical barrier that prevents intramembrane diffusion of lipids and proteins [21]. Gut microbiota dysbiosis is accompanied by decreased expression of the intestinal mucosal tight junction proteins claudin-1, occludin, and ZO-1 [22], which is similar to our findings in SHRs. However, a detailed mechanism has yet to be reported. In the current study, we found an increase in the relative abundance of butyric acid-producing bacteria when transplanting SHR with feces from WKY rats. There are a large number of butyric acid-producing bacteria in the human intestine, including Clostridium , Eubacterium , and Butyrivibrio . When butyric acid-producing bacteria and therefore butyric acid levels are reduced, intestinal mucosal barrier function may be compromised [23]. Butyric acid may affect barrier function by altering the expression of tight junction proteins on the surface of the intestinal mucosa [24,25]. Butyrate therefore promotes mucosal barrier integrity by up-regulating the expression of tight junction proteins in the intestinal epithelium, thereby preventing harmful products such as lipopolysaccharide from entering the blood [26]. As harmful foreign substances cause inflammation, butyric acid can effectively reduce inflammation [27,28]. Previous studies show that circulating pro-inflammatory mediators interleukin-6 and TNF-alpha are related to essential hypertension [29]. Preventing an inflammatory response, and therefore circulating proinflammatory mediators, could provide a plausible link between butyric acid and decreased BP. This idea is supported by the fact that hypertensive patients have significantly reduced butyrate-producing gut bacteria as well as plasma butyrate [7]. Another function of butyric acid is as an energy source for host intestinal epithelial cells, especially in the colon and cecum [30]. In addition, intestinal lactic acid-producing bacteria play an important role in maintaining the colonic intestinal microecological balance. Undigested carbohydrates are first degraded to pyruvate and then reduced to lactic acid by butyric acid-producing bacteria. Lactic acid is then further metabolized to butyric acid [31]. Excessive lactic acid-producing bacteria will lead to a large accumulation of lactic acid that affects colonic pH. Lowering intestinal pH may be one mechanism by which lactic acid promotes the growth of probiotics and inhibits specific pathogen colonization [32]. As the main energy source of intestinal epithelial cells, butyric acid could further improve the integrity of the intestinal mucosal barrier by modulating intestinal cell proliferation, differentiation, and apoptosis, promoting the secretion of antimicrobial peptides, and reducing the structural damage in intestinal mucosa [33]. We found changes in serum DAO, D-lactic acid, FITC-Dextran permeability, and serum LPS after intestinal microbiota transplantation. DAO is an intracellular enzyme expressed in intestinal mucosal epithelial cells that is released to the blood when the intestinal mucosal epithelium is injured. An increase in serum DAO therefore indirectly reflects the degree of intestinal mucosal epithelial cell damage [34]. In contrast, D-lactic acid is a metabolite of bacterial fermentation. D-lactic acid in the blood comes from the microbiota in the gastrointestinal tract that enters the circulation through the intestinal mucosa. Therefore, its detection in peripheral blood reflects the degree of intestinal mucosal damage and permeability [35]. We showed that Chao1 and ACE indices were higher in SHRs than WKY rats, indicating higher species richness in SHR. This may be due to a relative increase in the growth of some normally rare bacteria with a concomitant reduction in the abundance of dominant bacteria, which could lead to an increase in diversity in SHR. In addition, from the Shannon index value, the microbial evenness of the intestinal microbiota in the SHR was higher. Following fecal transplantation to SHRs, alpha diversity did not decline to the level in WKY rats. However, WKY rats transplanted with feces from SHRs had increased intestinal microbial species richness, indicated by Chao1 and ACE. Fecal transplantation therefore appears to affect the species richness of intestinal microbiota to a larger extent in SHR-transplanted WKY rats. Transplantation of SHR fecal bacteria increased the species richness of intestinal microbiota in WKY rats. Several metabolic-related diseases are accompanied by a decrease in the proportion of Bacteroides and an increase in the proportion of Firmicutes [36]. The relative abundance of Bacteroides in DOCA salt hypertensive mice are low [37], which is consistent with our results. The F/B ratio of is an important indicator of the imbalance of intestinal microbiota. Studies have shown that compared with healthy people, the relative abundance and diversity of intestinal microbiota in hypertensive patients are reduced, and F/B is increased [9]. Similarly, we show that the F/B ratio is significantly higher in SHRs compared with WKY rats. Fecal transplantation of SHR with WKY feces, however, decreases the F/B ratio, and vice versa. Lim et al. [38] found that compared with patients with metabolic syndrome, healthy people have a higher diversity and richness of Tenericutes in the intestinal microbiota, which is consistent with our results. A possible explanation is that in hypertension patients, the reproduction of unknown rare or unclassified bacteria is accelerated while the abundance of dominant bacteria is relatively reduced, leading to changes in intestinal microbiota structure and disrupts the stability of intestinal microecology. Further research is needed to verify this possibility. In conclusion, we show that fecal transplantation from hypertensive rats decreases the proportion of butyric acid-producing bacteria in normotensive recipient rats, which is accompanied by an increase in blood pressure. Given the documented role of butyric acid in maintaining mucosal barrier integrity, and that loss of mucosal barrier integrity is associated with hypertension, our results suggest that butyric acid-producing bacteria could improve blood pressure regulation by promoting mucosal barrier integrity. These findings warrant further investigation with monocolonization experiments. Declarations Author Contribution X.X. contributed to study design, conducted research, data analysis, paper writing; H.J. contributed to study design, conducted research, data analysis, paper writing; X.L.helped to design the study, data analysis and gave some advice; C.Y. helped to design the study, revised the manuscript, conducted research, data analysis and gave some advice; Q.Z. contributed to observation and analysis of pathological experiment results; X.Y. helped in collecting laboratory test results in the database and gave some advice; Z.L. helped in Optimizing experimental scheme; S.L. helped in collecting the sample; F.J. helped in collecting the sample.All authors reviewed the manuscript. Acknowledgement Thanks to LetPub provides professional SCI paper editing services Data Availability Statement The data underlying this article will be shared on reasonable request to the author. References Kelly CR, Kahn S, Kashyap P, Laine L, Rubin D, Atreja A , et al . 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Li C, Xiao P, Lin D, Zhong H-J, Zhang R, Zhao Z-G , et al . Risk factors for intestinal barrier impairment in patients with essential hypertension. Front Med (Lausanne) 2021; 7:543698. Nguyen TD, Prykhodko O, Hållenius FF, Nyman M. Monobutyrin reduces liver cholesterol and improves intestinal barrier function in rats fed high-fat diets. Nutrients 2019; 11:308. Ivanov AI. Structure and regulation of intestinal epithelial tight junctions. Current concepts and unanswered questions. In: Cheng CY (editor). Biology and Regulation of Blood-Tissue Barriers Advances in Experimental Medicine and Biology, vol 763 . New York, NY: Springer; 2013. pp. 132–148. Förster C. Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol 2008; 130:55–70. Bednarczyk J, Lukasiuk K. Tight junctions in neurological diseases. Acta Neurobiol Exp (Wars) 2011; 71:393–408. Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Bio 2001; 2:285–293. Matter K, Balda MS. SnapShot: epithelial tight junctions. Cell 2014; 157:992–992.e1. Barcenilla A, Pryde SE, Martin JC, Duncan SH, Stewart CS, Henderson C , et al . Phylogenetic relationships of butyrate-producing bacteria from the human gut. Appl Environ Microbiol 2000; 66:1654–1661. Feng W, Wu Y, Chen G, Fu S, Li B, Huang B , et al . Sodium butyrate attenuates diarrhea in weaned piglets and promotes tight junction protein expression in colon in a GPR109A-dependent manner. Cell Physiol Biochem 2018; 47:1617–1629. Zheng L, Kelly CJ, Battista KD, Schaefer R, Lanis JM, Alexeev EE , et al . Microbial-derived butyrate promotes epithelial barrier function through IL-10 receptor-dependent repression of claudin-2. J Immunol 2017; 199:2976–2984. Yan H, Ajuwon KM. Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway. PLoS One 2017; 12:e0179586. Van Immerseel F, Ducatelle R, De Vos M, Boon N, Van De Wiele T, Verbeke K , et al . Butyric acid-producing anaerobic bacteria as a novel probiotic treatment approach for inflammatory bowel disease. J Med Microbiol 2010; 59:141–143. Mishiro T, Kusunoki R, Otani A, Ansary MMU, Tongu M, Harashima N , et al . Butyric acid attenuates intestinal inflammation in murine DSS-induced colitis model via milk fat globule-EGF factor 8. Lab Invest 2013; 93:834–843. Bautista LE, Vera LM, Arenas IA, Gamarra G. Independent association between inflammatory markers (C-reactive protein, interleukin-6, and TNF-α) and essential hypertension. J Hum Hypertens 2005; 19:149–154. Vinolo MAR, Rodrigues HG, Hatanaka E, Sato FT, Sampaio SC, Curi R. Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils. J Nutr Biochem 2011; 22:849–855. Zhao HB, Ren YL. Research progress in colon microbiota metabolites. Feed Res 2019; 42:93–97. Silk DBA, Davis A, Vulevic J, Tzortzis G, Gibson GR. Clinical trial: the effects of a trans-galactooligosaccharide prebiotic on faecal microbiota and symptoms in irritable bowel syndrome. Aliment Pharmacol Ther 2009; 29:508–518. Chen R, Li P, Hu Y, Xu Y, Zhang L, Yuan J , et al . Effect of intestinal microbiota on the metabolic process of traditional Chinese herbal medicine. Chin J Microecol 2018; 30:990–993. Tang XH. Research on the effect of supplemented and flavored Chengqi decoction on DAO and PCT expression of sepsis in the rat model. J Guiyang Coll Tradit Chin Med 2017; 39:24–27. Wang XY, Cheng AG. Research progression on the gut barrier function and its detecting method. J North China Coal Med Coll 2019; 11:653–654. Xiao S, Fei N, Pang X, Shen J, Wang L, Zhang B , et al . A gut microbiota-targeted dietary intervention for amelioration of chronic inflammation underlying metabolic syndrome. FEMS Microbiol Ecol 2014; 87:357–367. Marques FZ, Nelson E, Chu P-Y, Horlock D, Fiedler A, Ziemann M , et al . High-fiber diet and acetate supplementation change the gut microbiota and prevent the development of hypertension and heart failure in hypertensive mice. Circulation 2017; 135:964–977. Lim MY, You HJ, Yoon HS, Kwon B, Lee JY, Lee S , et al . The effect of heritability and host genetics on the gut microbiota and metabolic syndrome. Gut 2017; 66:1031–1038. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Aug, 2024 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted Editorial decision: Revision requested 20 Jun, 2024 Reviews received at journal 08 Jun, 2024 Reviews received at journal 20 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers agreed at journal 15 May, 2024 Reviewers invited by journal 15 May, 2024 Submission checks completed at journal 14 May, 2024 Editor assigned by journal 14 May, 2024 First submitted to journal 12 May, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4408181","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304740093,"identity":"7c63c311-20dc-4c00-bef6-738b16c16c3b","order_by":0,"name":"Xinghua XU","email":"","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"XU","suffix":""},{"id":304740097,"identity":"0042bbd1-7642-48e1-a324-bb757fc199e8","order_by":1,"name":"Hua JIN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACPiBmBmE2Bh4gVSEhx09ICxuqljMWxpINRGoBAqAWxraKxA0EtbCfPfy5oOIwO5907zGJn/MkGDcwMD98dAOfFp68BOMZZw4zs8mcS5Ps3SbBbM7AZmycg9dhOQbJvG1ALRI5ZhK82yTYLBt42KTxauF/Y3AYpkXy7xwJHoMDhLRI5Bg2w7RI8zZISBCh5Y0xM8+ZdJAWY2uZYxIGks0E/MLPn2P8mafCOll+Ro7hzTc1dfX97M0PH+PTAgPJCCYzEcpBwI5IdaNgFIyCUTASAQBVwzta2SlW6AAAAABJRU5ErkJggg==","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Hua","middleName":"","lastName":"JIN","suffix":""},{"id":304740098,"identity":"fa4a84c4-7d28-46e9-b5b3-220421051d60","order_by":2,"name":"Xiaoling LI","email":"","orcid":"","institution":"Lanzhou University of Second Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"LI","suffix":""},{"id":304740099,"identity":"aac79838-c8a1-44f1-8180-8be0f079b212","order_by":3,"name":"Chunlu YAN","email":"","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chunlu","middleName":"","lastName":"YAN","suffix":""},{"id":304740100,"identity":"560fa07d-2241-4e84-9ff7-fc7ab4251d72","order_by":4,"name":"Qiuju ZHANG","email":"","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qiuju","middleName":"","lastName":"ZHANG","suffix":""},{"id":304740101,"identity":"e45bfd3a-3118-46c9-9884-f61c2a6a1a91","order_by":5,"name":"Xiaoying YU","email":"","orcid":"","institution":"Gansu Provincial Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"YU","suffix":""},{"id":304740102,"identity":"d3a0a96b-b040-40e5-a117-9043d48a3c10","order_by":6,"name":"Zhijun LIU","email":"","orcid":"","institution":"Affiliated Hospital of Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhijun","middleName":"","lastName":"LIU","suffix":""},{"id":304740103,"identity":"8993e5c5-18cf-485b-8927-acb36a941ba1","order_by":7,"name":"Shuangfang LIU","email":"","orcid":"","institution":"Gansu University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuangfang","middleName":"","lastName":"LIU","suffix":""},{"id":304740104,"identity":"49ae0938-00e3-4145-b3eb-69c6ed22f45e","order_by":8,"name":"Feifei ZHU","email":"","orcid":"","institution":"Tianshui Municipal Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"ZHU","suffix":""}],"badges":[],"createdAt":"2024-05-12 10:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4408181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4408181/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12602-024-10344-x","type":"published","date":"2024-08-22T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57007864,"identity":"c00af19a-1ba8-48cb-9924-dcfdb93d619c","added_by":"auto","created_at":"2024-05-23 10:28:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118367,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of fecal bacteria transplantation on blood pressure in rats. Changes in systolic, diastolic, and mean blood pressures in SHRs and WKY rats after 6 weeks of fecal bacterial transplantation. Data are expressed as the mean ± standard deviation (SD) (\u003cem\u003en\u003c/em\u003e = 9–10); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, vs. the WKY-T group, #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the SHR-T group. WKY, Wistar Kyoto; SHR, spontaneous hypertensive rat; WKY-T, WKY transplanted with SHR fecal bacteria; SHR-T, SHR transplanted with WKY fecal bacteria.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4408181/v1/a4b3f7e4bf653cb1f794134c.jpg"},{"id":57007867,"identity":"014e86cf-0cf1-45b6-b182-185630891f05","added_by":"auto","created_at":"2024-05-23 10:28:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2322372,"visible":true,"origin":"","legend":"\u003cp\u003eFecal microbiota transplantation changes gut microbial composition in rats. A. ACE, Chao1, Shannon, and Simpson indices to evaluate gut microbial diversity. \u003cem\u003en\u003c/em\u003e = 6–7. B. Weighted UniFrac beta diversity analysis. The closer the distance between the samples in the figure, the greater the similarity in microbiota composition between samples. C. Gut microbiota family-level composition. D. Fermicutes/Bacteroides ratios by group. E. Redundancy analysis demonstrates the relationship between microbiota composition and blood pressure. F. Relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, and \u003cem\u003eTuricibacter\u003c/em\u003e by group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4408181/v1/ff54c66b80058c8cd73b529e.png"},{"id":57007866,"identity":"65be6fed-aa50-483e-9d0a-b6b1c4667fb7","added_by":"auto","created_at":"2024-05-23 10:28:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7461068,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of fecal microbiota transplantation on rat intestinal mucosal barrier. A. The concentration of serum FITC-Dextrans content and serum LPS (\u003cem\u003en\u003c/em\u003e = 3). B. The concentration of D-lactate and DAO (\u003cem\u003en\u003c/em\u003e= 9–10), *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the WKY group; #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. the SHR group. C. Intestinal mucosal morphology and structural changes. D. immunofluorescence images to show ZO-1, claudin-1, occludin distribution and expression. The arrows indicate the ZO-1, claudin-1, and occludin distribution and expression in intestinal epithelium. E. Real-time RT-PCR for tight junction protein mRNA expression (\u003cem\u003en\u003c/em\u003e = 3). F and G. Western blotting of tight junction protein expression. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4408181/v1/5dfa8762400fb1cae9a98107.png"},{"id":63300995,"identity":"434afe47-4ce0-4cca-8f47-21ff5e5468b7","added_by":"auto","created_at":"2024-08-26 16:18:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13680761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4408181/v1/37a849eb-ec4b-4edd-8bcc-ab76c2ba15aa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fecal microbiota transplantation regulates blood pressure by altering gut microbiota composition and intestinal mucosal barrier function in spontaneously hypertensive rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn recent years, fecal microbiota transplantation has received widespread attention as a major method of intervention in intestinal microecology, allowing increased understanding of the role of intestinal microbiota in the occurrence and development of diseases. Fecal microbiota transplantation is a technique where functional microorganisms are separated from the feces of healthy individuals and transplanted into the recipient's digestive tract. This procedure rebuilds the patient's intestinal microecological balance, thereby treating diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Treatment using fecal bacteria transplantation has gradually expanded from application to digestive system diseases [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], such as \u003cem\u003eClostridium difficile\u003c/em\u003e infection, inflammatory bowel disease, and irritable bowel syndrome, to central nervous system diseases such as Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIncreasing evidence has shown that gut microbiota imbalance is closely related to cardiovascular disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A large number of studies suggest that hypertension is related to gut microbiota imbalance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In animal experiments fecal microbiota transplantation has been shown to change blood pressure sensitivity in salt-sensitive rats [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Durgan \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] transplanted the feces of high-fat-fed obstructive sleep apnea-induced hypertensive rats to normally fed obstructive sleep apnea-induced hypertensive rats, which led to an increase in blood pressure in the normally fed rats. Transplanting the feces from stroke-prone spontaneous hypertensive rats (SHRs) into normotensive Wistar Kyoto (WKY) rats treated with antibiotics led to increased Systolic blood pressure in the WKY rats, whereas systolic blood pressure decreased in stroke-prone SHRs transplanted with feces from WKY rats [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These results suggest that changes in gut microbiota composition can affect blood pressure. When fecal bacteria from a human hypertensive patient was transplanted to sterile mice, their blood pressure increased, indicating that hypertension can be transferred through the microbiota. Although many studies show that hypertension is accompanied by gut microbiota imbalance and intestinal mucosal barrier damage and dysfunction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the underlying mechanism remains unclear. In order to investigate the role of gut microbiota in the pathogenesis of hypertension, we hypothesized that intestinal fecal microbiota cross-transplantation between SHRs and WKY rats would change fecal microbiota composition in recipient rats and thereby alter blood pressure.\u003c/p\u003e \u003cp\u003eIntestinal barrier dysfunction has been implicated in hypertension [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and butyric acid has been shown to improve intestinal barrier function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the current study, we found that the composition of butyric acid-producing bacterial species changed after fecal microbiota transplantation. As the intestinal mucosa is composed of intestinal epithelial cells and tight junctions, the integrity of which is regulated by the expression of tight junction proteins, including ZO-1, occludin, and claudin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we further hypothesized that changes in gut microbiota composition after fecal microbiota transplantation would change the expression of tight junction proteins.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale 14-week-old SHRs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) and WKY rats (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) rats were used (350\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) (Vital River Laboratory Animal Technology, China). SHRs were randomly divided into 2 groups (10/group), SHR and SHR-T (SHR plus fecal transplantation). WKY rats were randomly divided into 2 groups (10/group), WKY and WKY-T (WKY plus fecal transplantation). SHR-T and WKY-T underwent cross-transplantation of fecal bacteria. Before transplantation, tail artery pressure was measured in each group in an awake state. One rat in the WKY-T group died during the transplantation process. Animals were kept in a laboratory with constant temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;5), and a 12 light/dark cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e This study was approved by the Gansu College of Traditional Chinese Medicine Animal Experimen Ethics Committee [2015-002].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of fecal bacteria liquid\u003c/h2\u003e \u003cp\u003eDefecation was stimulated and fecal samples (5 g per group) were collected in a sterile beaker to prevent contamination. The fecal samples were dissolved in sterile saline (5 mL/g of feces in 0.9% normal saline). A fecal suspension was collected by coarsely filtering impurities with sterile gauze and centrifuged at 3,000 rpm for 10 min. Supernatant was discarded and the fecal bacteria precipitate was obtained. Physiological saline (20 mL) was added to fecal precipitate and mixed well. The suspension was used immediately for transplant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFecal bacteria transplant\u003c/h2\u003e \u003cp\u003eFecal bacterial liquid from the SHR group was transplanted to the WKY-T group, while fecal bacteria liquid from the WKY group was transplanted to the SHR-T group. A sterile 5 mL syringe was used to take 2 mL of the fecal bacteria liquid. Gavage was used to inject fecal bacteria liquid into the intestine through the anus (once a week for 6 weeks).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBlood pressure measurement\u003c/h2\u003e \u003cp\u003eAfter 6 weeks of treatment, tail arterial pressure was measured in the early morning using a BP-98A noninvasive blood pressure system (Softron, Tokyo, Japan). Each rat was measured 5 times and the average BP was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFecal sampling and 16S ribosomal DNA (rDNA) gene amplicon sequencing\u003c/h2\u003e \u003cp\u003eFecal samples were collected in a sterile tube immediately after euthanasia and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Microbial DNA was extracted from fecal samples using the E.Z.N.A.\u0026reg; soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.). The V3\u0026ndash;V4 hypervariable region of the 16S rDNA gene was amplified using forward primer 515F (GTG CCA GCMGCC GCG GTA A) and reverse primer 806R (GGA CT CHVGGG TWTCT AAT). PCR amplicons were sequenced on an Illumina HiSeq2500 platform, with paired-end reads of 450\u0026ndash;460 bp long. Raw sequence data were filtered, processed, and analyzed according to the QIIME (V1.7.0) quality control process. Sequences with \u0026ge;\u0026thinsp;97% similarity were assigned to the same operational taxonomic unit (OTU). Merging of paired forward and reverse reads was performed by FLASH (fast length adjustment of short reads, v1.2.11) software. OTU clustering on the merged reads was performed by USEARCH (v7.0.1090) software. Representative sequences of OTUs were selected and clustered with the Greengenes database 13\u0026thinsp;\u0026minus;\u0026thinsp;8 version at 99% sequence similarity, and then aligned to obtain species annotation information. Taxonomic annotation and abundance analysis were also performed. ANOVA, ANCOM, Kruskal Wallis were used to identify bacteria that had significantly different relative abundances between groups of interest. Alpha and beta diversity indices were used to evaluate differences between samples. Alpha diversity and beta diversity were analyzed using the Qiime2 Diversity plug-in. Chao1 index, Ace index, Shannon index, and Simpson index were used to analyze alpha diversity. The weighted UniFrac distance was used for the principal coordinate analysis (PCoA) of beta diversity. Redundance analysis (RDA) was used to reveal the potential associations between microbial communities and related environmental factors, and the permutation test was performed using the R language VEGAN bag.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eEnzyme-linked immunoassay (ELISA)\u003c/h2\u003e \u003cp\u003eLevels of diamine oxidase (DAO) and D-lactate were determined by ELISA. After six weeks of fecal transplant intervention, 5 mL of blood was collected from the pericardium in anesthetized rats (10% chloral hydrate, 0.3 mL/100 g body weight, i.p.). Blood was centrifuged at 3,500 rpm at 4\u0026deg;C for 15 min. Serum was stored at \u0026minus;\u0026thinsp;20 to \u0026minus;\u0026thinsp;80\u0026deg;C. ELISA was performed based on the manufacturer\u0026rsquo;s instructions (Meimian, Jingsu Fiya Biological Technology, China). Azo matrix colorimetric method was used to measure plasma lipopolysaccharide (LPS) contents (Xiamen Limulus Reagent Experimental Factory).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFITC-Dextran content determined by fluorescence spectrophotometry\u003c/h2\u003e \u003cp\u003eFITC-Dextran (MW: 4,000, Sigma, St. Louis, MO, USA) was administered intragastrically (5 mg/100 g body weight) after fasting for 12 h. Four hours later, animals were anesthetized to collect 4 mL of blood from the hepatic portal vein. Blood was centrifuged at 3,000 rpm for 15 min at 4\u0026deg;C. Serum was stored at \u0026minus;\u0026thinsp;20\u0026deg;C and later used for determining FITC-Dextran (excitation wavelength 485, emission wavelength 528) using a multifunctional microplate reader (Bio Tek Synergy HT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntestinal mucosa morphological changes determined by H\u0026amp;E staining\u003c/h2\u003e \u003cp\u003eIntestines (1\u0026ndash;2 cm) were collected and fixed in 4% paraformaldehyde. Thin 5 \u0026micro;m paraffin sections were cut and dewaxed by xylenes and ethanol. Sections were stained with hematoxylin for 10 min and rinsed with running water to remove excess staining. Sections were then differentiated with 7% hydrochloric acid and ethanol for a few seconds. Sections were then submerged in 95% ethanol for 30 s, alcohol eosin stain for 30 s, 90% ethanol for 30 s, 100% ethanol for 30 s, and carbolic xylene for 30 s. Sections were cleared in xylenes for 30 s and mounted. Morphological changes in the structure of small intestinal villi were observed.\u003c/p\u003e \u003cp\u003eMouse colons were collected in a sterile bottle, snap frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for immunofluorescence, real-time RT-PCR, and Western blotting. Intestinal mucosal tight junction proteins (ZO-1, occludin, and claudin) were determined by immunofluorescence, real-time RT-PCR, and Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTight junction proteins determined by immunofluorescence\u003c/h2\u003e \u003cp\u003eFrozen sections (10 \u0026micro;m) were fixed in 4% paraformaldehyde for 20 min, washed thrice with PBS for 5 min, and blocked with a blocking solution (Thermo Fisher, Waltham, MA, USA) at room temperature for 40 min. ZO-1 and occludin primary antibodies (rabbit polyclonal, Abcam, Cambridge, UK) were added and incubated overnight at 4\u0026deg;C. After washing with PBS thrice for 5 min, IgG-H\u0026amp;L secondary antibody (Abcam, goat anti-rabbit polyclonal) was added and incubated at room temperature for 1 h. The expression of ZO-1 and occludin was observed by laser confocal scanning microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTight junction protein mRNA expression determined by real-time RT-PCR\u003c/h2\u003e \u003cp\u003eFrozen colon tissues (50 mg) were ground using an automatic tissue homogenizer (MagNA Lyser, Roche) to extract RNA using Trizol (500 \u0026micro;L). RNA purity and concentration were detected by a nucleic acid microquantifier (Pultton DNA/Proteins Analyzer P100). Reverse transcription was performed according to the instructions from PrimeScript\u003csup\u003e\u0026trade;\u003c/sup\u003e reverse transcription kit. Primer sequences were based on the rat mRNA sequence on the NCBI website and designed with Primer 5.0 software. β-actin primer: forward 5'-GGAGATTACTGCCCTGGCTCCTA-3', reverse 5'-GACTCATCGTACTCCTGCTTGCTG-3; Zo-1 primer: forward 5'-CCATCTTTGGACCGATTGCTG-3', reverse 5'-TAATGCCCGAGCTCCGATG-3'; occludin primer: forward 5'-GTCTTGGGAGCCTTGACATCTTG-3', reverse 5'-GCATTGGTCGAACGTGCATC-3'; claudin-1 primer: forward 5'-CATGAAGTGCATGAGGTGCTTAGAA-3', reverse 5'-TGGCCACTAATGTCGCCAGA-3'. Real-time quantitative fluorescent PCR was performed based on the instructions of the Go Taq\u0026reg; qPCR Master Mix (Promega) real-time quantitative PCR kit. Data obtained were quantitatively analyzed by the 2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTight junction protein expression determined by western blotting\u003c/h2\u003e \u003cp\u003eColon tissues (50 mg) were collected and homogenized in RIPA lysis solution (500 \u0026micro;L). The homogenized tissues were centrifuged at 12,000 rpm for 15 min at 4\u0026deg;C. Supernatant was collected and protein concentration was determined by the Bicinchoninic Acid Kit (PC0020, Solarbio). Protein loading buffer was added to the sample, heated at 100\u0026deg;C for 5 min, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Protein (50 \u0026micro;g) was loaded on gels with different gel concentrations (ZO-1: 8%, occludin: 12%, claudin-1: 12%) and ran at 100 V for 1.5 h. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (REF IPVH00010, pore size 0.45 \u0026micro;m; Millipore) under 180 mA for 2 h. The membranes were blocked in 5% skimmed milk at room temperature for 2 h, followed by washing with TPBS with shaking 3 times, for 10 min each. Membranes were then incubated with rabbit polyclonal antibodies against ZO-1 (1:500), occludin (1:500), claudin-1 (1:500), and GAPDH (1:1500) overnight at 4\u0026deg;C. After the membranes were washed with TPBS with shaking 3 times, for 10 min each, they were incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1:4000) with shaking at room temperature for 2 h. The membranes were washed with TPBS 3 times, for 10 min each, and washed once with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 5 min. Protein bands were visualized by chemiluminescence and analyzed by Image pro-plus 6.0 software. Results are expressed as normalized intensity to GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData were processed using SPSS 21.0 and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way ANOVA or Student\u0026rsquo;s \u003cem\u003et-\u003c/em\u003etest was used to assess statistical significance, where appropriate. Kruskal Wallis method was used to compare the difference in alpha diversity index among the groups. Significant different were considered at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCross transplantation of fecal bacteria increases blood pressure in WKY rats\u003c/h2\u003e \u003cp\u003eAfter transplanting SHR fecal bacteria to WKY rats, systolic, diastolic, and mean blood pressures of the WKY-T group were significantly higher than that of the WKY group. Conversely, blood pressure was significantly decreased in the SHR-T group relative to the SHR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiota transplantation changes gut microbial composition\u003c/h2\u003e \u003cp\u003eChao1, ACE, and Shannon indices in the SHR group tended to be higher (even though not statistically significant) than that in the WKY group, while Simpson\u0026rsquo;s index was not significantly different between groups. After SHR transplantation with WKY feces, alpha diversity did not decline. However, after WKY transplantation with SHR feces, Chao1 and ACE indices increased while the Shannon index decreased. These results show that fecal bacteria transplantation affected intestinal microbiota alpha diversity. The intestinal microbiota in SHRs increased the number of species of intestinal microbiota in WKY rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWeighted UniFrac beta diversity analysis showed that gut microbiota composition varied among rats in the SHR group, with that of the WKY group being more similar within that group. After the SHR group was transplanted with feces from the WKY group, gut microbiota composition tended to be more similar at the genus level, indicating that transplantation of WKY fecal bacteria, to a certain extent, promoted the restoration of intestinal microbiota in SHRs. After the WKY group was transplanted with feces from the SHR group, gut microbiota composition did not change substantially at the genus level, indicating that the transplantation of SHR fecal bacteria had no effect on the gut microbiota composition of WKY rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe identified nine main phyla using 16S rRNA gene amplicon sequencing, namely Firmicutes, Bacteroidetes, Proteobacteria, TM7, Actinobacteria, Tenericutes, Cyanobacteria, Verrucomicrobia, and Elusimicrobia. Firmicutes and Bacteroides had the highest relative abundance. The ratio of Firmicutes to Bacteroides (F/B) was 2.58 in the WKY group and 3.61 in the SHR group. After transplantation, the F/B ratio decreased to 3.37 in the SHR-T group and increased to 2.79 in the WKY-T group.\u003c/p\u003e \u003cp\u003eAfter SHRs were transplanted with WKY feces, the relative abundance of Firmicutes, Verrucomicrobia, and Cyanobacteria decreased, and that of Bacteroides increased. After transplanting SHR feces to WKY rats, however, the relative abundance of Firmicutes and Verrucomicrobia increased, and that of Bacteroides decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSystolic blood pressure is closely related to changes in microbiota composition\u003c/h2\u003e \u003cp\u003eRedundancy analysis showed that systolic (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005), mean (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0015), and diastolic (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0065) pressures were all related to variations in microbiota composition. Among them, systolic blood pressure appeared to be the most closely related to differences in gut microbiota composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiota transplantation alters the relative abundance of intestinal lactic acid-producing and butyric acid-producing bacteria\u003c/h2\u003e \u003cp\u003eBased on abundance analysis of 16S rRNA gene amplicon sequencing data, the SHR group had a higher relative abundance of lactic acid-producing \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and reduced relative abundance of butyric acid-producing \u003cem\u003eClostridium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the WKY group. However, following fecal microbiota transplantation from WKY rats, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e was decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and that of \u003cem\u003eClostridium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and \u003cem\u003eTuricibacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) increased in SHRs. Conversely, WKY rats transplanted with SHR feces had increased relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased relative abundance of \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggest that fecal transplantation led to a change in the relative abundance of lactic acid- and butyric acid-producing bacteria, which may promote the conversion of lactic acid to butyric acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiota transplantation affects intestinal mucosal barrier integrity\u003c/h2\u003e \u003cp\u003eAs discussed earlier, butyric acid promotes intestinal barrier function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As the relative abundance of lactic acid-producing bacteria changed after fecal microbiota transplantation, we sought to determine the impact of fecal microbiota transplantation on intestinal barrier function. Serum FITC-Dextran and LPS contents (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as well as serum DAO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and D-lactic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), were increased in the WKY-T group relative to WKY groups. Conversely, in the SHR-T group, serum FITC-Dextran, LPS, DAO, and D-lactic acid contents were decreased relative to the SHR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that transplantation of fecal bacteria from SHRs to WKY rats lead to increased intestinal mucosal permeability, while transplantation of fecal bacteria from WKY rats to SHRs improves intestinal mucosal barrier function (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eFecal microbiota transplantation affects colonic mucosal structure\u003c/h2\u003e \u003cp\u003eColonic mucosa of rats in the WKY group had intact structure, with neatly arranged glands. However, in the SHR group, glands were irregular, the number of goblet cells was reduced, the structure of the lamina propria was loose, and edema was visible, indicating that the intestinal mucosal barrier had been compromised. Compared with the SHR group, the glands in the mucosal barrier in the SHR-T group were neatly arranged and interstitial edema was reduced, indicating recovery of intestinal mucosal barrier structure following fecal transplantation from SHR rats. The colonic morphology in the WKY-T group was similar to that in the SHR group, with disorganized glands and interstitial edema, indicating that fecal transplantation from SHRs had a negative impact on the intestinal mucosal barrier of the WKY-T group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFecal microbiota transplantation affects tight junction protein expression\u003c/h2\u003e \u003cp\u003eThe mRNA expression of ZO-1, claudin-1, and occludin in colon tissue from the WKY-T group was lower than that of the WKY group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, ZO-1, claudin-1 and occludin mRNA expression was higher in the SHR-T group than that of SHR group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eUsing immunofluorescence, we showed that ZO-1, occludin, and claudin-1 proteins were mainly distributed on the intestinal epithelial cell membrane. In the SHR group, ZO-1, occludin, claudin-1 protein staining was scattered along the cell membrane, with weakened fluorescence intensity, and a significantly reduced positive staining area compared to the WKY group. In the SHR-T group, although staining was also scattered along the cell membrane with weakened fluorescence intensity compared with the WKY group, there was more staining than in the SHR group. In the WKY-T group, although ZO-1, occludin, and claudin-1 staining was enhanced and the distribution was more continuous relative to the SHR group, the staining was still decreased relative to the WKY group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eSimilar to mRNA expression, protein levels of ZO-1, claudin-1, and occludin were significantly reduced in SHR compared with the WKY group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with the SHR group, protein levels of ZO-1, claudin-1, and occludin were increased in the SHR-T group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the WKY group, the expression of ZO-1, claudin-1, and occludin were significantly reduced in the WKY-T group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eThese results suggest that fecal bacteria transplantation may promote the recovery of the structure and function of the intestinal mucosal barrier in SHRs by up-regulating the expression of tight junction-related proteins.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study demonstrates important changes to gut microbiota composition and the structure of the intestinal mucosal barrier in SHRs and WKY rats following cross-transplantation and demonstrates the effect of microbiota composition on BP. Importantly BP decreased in hypertensive rats after fecal microbiota transplantation from normotensive ats, and vice versa. Gut microbial diversity changed significantly after fecal transplantation. Moreover, fecal microbiota transplantation led to changes in the relative abundance of lactic acid- and butyric acid-producing bacteria. Importantly, changes in the composition of butyric acid producing bacteria may be associated with intestinal mucosal permeability and structural integrity, as reflected in changes in the expression of tight junction proteins. These results support the intimate relationship between the gut microbiota and intestinal mucosal barrier function, which in turn could impact blood pressure.\u003c/p\u003e\n\u003cp\u003eIn studies related to intestinal microecology, common microecological interventions include drugs, probiotic supplementation, and fecal microbiota transplantation. Fecal microbiota transplantation is important for the study of intestinal microecology as it allows direct alteration of host microecology. A previous study showed that fecal transplantation from a hypertensive donor increased the BP of sterile recipient mice, which demonstrates a causal effect of the gut microbiota on BP [9]. Based on this result, we sought to investigate the mechanistic basis thereof, and found that BP changes are associated with microbiota-induced changes in intestinal mucosal barrier.\u003c/p\u003e\n\u003cp\u003eGut microbiota composition is closely related to and may regulate the expression of tight junction proteins in the intestinal mucosal barrier. One of the main functions of tight junctions is to regulate intestinal permeability [20]. Tight junctions provide a physical barrier that prevents intramembrane diffusion of lipids and proteins [21]. Gut microbiota dysbiosis is accompanied by decreased expression of the intestinal mucosal tight junction proteins claudin-1, occludin, and ZO-1 [22], which is similar to our findings in SHRs. However, a detailed mechanism has yet to be reported. In the current study, we found an increase in the relative abundance of butyric acid-producing bacteria when transplanting SHR with feces from WKY rats. There are a large number of butyric acid-producing bacteria in the human intestine, including \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eEubacterium\u003c/em\u003e, and \u003cem\u003eButyrivibrio\u003c/em\u003e. When butyric acid-producing bacteria and therefore butyric acid levels are reduced, intestinal mucosal barrier function may be compromised [23]. Butyric acid may affect barrier function by altering the expression of tight junction proteins on the surface of the intestinal mucosa [24,25]. Butyrate therefore promotes mucosal barrier integrity by up-regulating the expression of tight junction proteins in the intestinal epithelium, thereby preventing harmful products such as lipopolysaccharide from entering the blood [26]. As harmful foreign substances cause inflammation, butyric acid can effectively reduce inflammation [27,28]. Previous studies show that circulating pro-inflammatory mediators interleukin-6 and TNF-alpha are related to essential hypertension [29]. Preventing an inflammatory response, and therefore circulating proinflammatory mediators, could provide a plausible link between butyric acid and decreased BP. This idea is supported by the fact that hypertensive patients have significantly reduced butyrate-producing gut bacteria as well as plasma butyrate [7].\u003c/p\u003e\n\u003cp\u003eAnother function of butyric acid is as an energy source for host intestinal epithelial cells, especially in the colon and cecum [30]. In addition, intestinal lactic acid-producing bacteria play an important role in maintaining the colonic intestinal microecological balance. Undigested carbohydrates are first degraded to pyruvate and then reduced to lactic acid by butyric acid-producing bacteria. Lactic acid is then further metabolized to butyric acid [31]. Excessive lactic acid-producing bacteria will lead to a large accumulation of lactic acid that affects colonic pH. Lowering intestinal pH may be one mechanism by which lactic acid promotes the growth of probiotics and inhibits specific pathogen colonization [32]. As the main energy source of intestinal epithelial cells, butyric acid could further improve the integrity of the intestinal mucosal barrier by modulating intestinal cell proliferation, differentiation, and apoptosis, promoting the secretion of antimicrobial peptides, and reducing the structural damage in intestinal mucosa [33].\u003c/p\u003e\n\u003cp\u003eWe found changes in serum DAO, D-lactic acid, FITC-Dextran permeability, and serum LPS after intestinal microbiota transplantation. DAO is an intracellular enzyme expressed in intestinal mucosal epithelial cells that is released to the blood when the intestinal mucosal epithelium is injured. An increase in serum DAO therefore indirectly reflects the degree of intestinal mucosal epithelial cell damage [34]. In contrast, D-lactic acid is a metabolite of bacterial fermentation. D-lactic acid in the blood comes from the microbiota in the gastrointestinal tract that enters the circulation through the intestinal mucosa. Therefore, its detection in peripheral blood reflects the degree of intestinal mucosal damage and permeability [35].\u003c/p\u003e\n\u003cp\u003eWe showed that Chao1 and ACE indices were higher in SHRs than WKY rats, indicating higher species richness in SHR. This may be due to a relative increase in the growth of some normally rare bacteria with a concomitant reduction in the abundance of dominant bacteria, which could lead to an increase in diversity in SHR. In addition, from the Shannon index value, the microbial evenness of the intestinal microbiota in the SHR was higher. Following fecal transplantation to SHRs, alpha diversity did not decline to the level in WKY rats. However, WKY rats transplanted with feces from SHRs had increased intestinal microbial species richness, indicated by Chao1 and ACE. Fecal transplantation therefore appears to affect the species richness of intestinal microbiota to a larger extent in SHR-transplanted WKY rats. Transplantation of SHR fecal bacteria increased the species richness of intestinal microbiota in WKY rats.\u003c/p\u003e\n\u003cp\u003eSeveral metabolic-related diseases are accompanied by a decrease in the proportion of Bacteroides and an increase in the proportion of Firmicutes [36]. The relative abundance of Bacteroides in DOCA salt hypertensive mice are low [37], which is consistent with our results. The F/B ratio of is an important indicator of the imbalance of intestinal microbiota. Studies have shown that compared with healthy people, the relative abundance and diversity of intestinal microbiota in hypertensive patients are reduced, and F/B is increased\u0026nbsp;[9]. Similarly, we show that the F/B ratio is significantly higher in SHRs compared with WKY rats. Fecal transplantation of SHR with WKY feces, however, decreases the F/B ratio, and vice versa. Lim\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e [38] found that compared with patients with metabolic syndrome, healthy people have a higher diversity and richness of Tenericutes in the intestinal microbiota, which is consistent with our results. A possible explanation is that in hypertension patients, the reproduction of unknown rare or unclassified bacteria is accelerated while the abundance of dominant bacteria is relatively reduced, leading to changes in intestinal microbiota structure and disrupts the stability of intestinal microecology. Further research is needed to verify this possibility.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we show that fecal transplantation from hypertensive rats decreases the proportion of butyric acid-producing bacteria in normotensive recipient rats, which is accompanied by an increase in blood pressure. Given the documented role of butyric acid in maintaining mucosal barrier integrity, and that loss of mucosal barrier integrity is associated with hypertension, our results suggest that butyric acid-producing bacteria could improve blood pressure regulation by promoting mucosal barrier integrity. These findings warrant further investigation with monocolonization experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.X. contributed to study design, conducted research, data analysis, paper writing; H.J. contributed to study design, conducted research, data analysis, paper writing; X.L.helped to design the study, data analysis and gave some advice; C.Y. helped to design the study, revised the manuscript, conducted research, data analysis and gave some advice; Q.Z. contributed to observation and analysis of pathological experiment results; X.Y. helped in collecting laboratory test results in the database and gave some advice; Z.L. helped in Optimizing experimental scheme; S.L. helped in collecting the sample; F.J. helped in collecting the sample.All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanks to LetPub provides professional SCI paper editing services\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared on reasonable request to the author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKelly CR, Kahn S, Kashyap P, Laine L, Rubin D, Atreja A\u003cem\u003e, et al\u003c/em\u003e. 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The effect of heritability and host genetics on the gut microbiota and metabolic syndrome. \u003cem\u003eGut \u003c/em\u003e2017; 66:1031\u0026ndash;1038.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"fecal microbiota transplantation, gut microbiota, hypertension, intestinal mucosal barrier, spontaneously hypertensive rats","lastPublishedDoi":"10.21203/rs.3.rs-4408181/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4408181/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives:\u003c/strong\u003e Hypertension is accompanied by gut microbiota imbalance, but the role of bacteria in the pathogenesis of hypertension requires further study. In this study, we used fecal microbiota transplantation to determine the impact of microbiota composition on blood pressure in spontaneous hypertensive rats (SHRs), using normotensive Wistar Kyoto (WKY) rats as controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e SHRs were randomly divided into 2 groups (\u003cem\u003en\u003c/em\u003e = 10/group), SHR and SHR-T (SHR plus fecal transplantation) and WKY into WKY and WKY-T (WKY plus fecal transplantation). SHR-T received fecal transplantation from WKY while WKY-T received fecal transplantation from SHR. Blood pressure was measured from the tail artery in conscious rats. 16S rDNA gene amplicon sequencing was used to analyze bacterial composition. Circulating levels of diamine oxidase, D-lactate, FITC-Dextrans, and lipopolysaccharide were determined. Hematoxylin and eosin (H\u0026amp;E) staining was used to observe structural changes in the intestinal mucosa. Immunofluorescence, Western blot, and RT-PCR were utilized to determine changes in the expression of tight junction proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Following cross fecal transplantation, blood pressure decreased in SHR and increased in WKY. Significant differences in gut microbial composition were found between hypertensive and normotensive rats, specifically regarding the relative abundance of lactic and butyric acid-producing bacteria. Changes in gut microbiota composition also impacted the intestinal mucosal barrier integrity. Moreover, fecal transplantation affected the expression of tight junction proteins that may impact intestinal mucosal permeability and structural integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Blood pressure may be associated with butyric acid-producing intestinal microbiota and its function in regulating the integrity of intestinal mucosal barrier.\u003c/p\u003e","manuscriptTitle":"Fecal microbiota transplantation regulates blood pressure by altering gut microbiota composition and intestinal mucosal barrier function in spontaneously hypertensive rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 10:28:18","doi":"10.21203/rs.3.rs-4408181/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-20T17:26:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-08T04:51:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-20T21:21:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147770408420817833505400221837128074259","date":"2024-05-15T22:54:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55538813156091820883952635857877046564","date":"2024-05-15T21:02:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-15T20:49:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-14T08:27:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-14T08:27:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2024-05-12T10:51:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3f066ba1-96d0-41f7-89b9-293d97b91c90","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:14:09+00:00","versionOfRecord":{"articleIdentity":"rs-4408181","link":"https://doi.org/10.1007/s12602-024-10344-x","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2024-08-22 15:57:58","publishedOnDateReadable":"August 22nd, 2024"},"versionCreatedAt":"2024-05-23 10:28:18","video":"","vorDoi":"10.1007/s12602-024-10344-x","vorDoiUrl":"https://doi.org/10.1007/s12602-024-10344-x","workflowStages":[]},"version":"v1","identity":"rs-4408181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4408181","identity":"rs-4408181","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}