Enterococcus faecalis ameliorates hyperuricemia and maintains the epithelium barrier in a hyperuricemia mouse model

doi:10.21203/rs.3.rs-2261921/v1

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Enterococcus faecalis ameliorates hyperuricemia and maintains the epithelium barrier in a hyperuricemia mouse model

https://doi.org/10.21203/rs.3.rs-2261921/v1

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Background

Hyperuricemia (HUA) is a major risk factor of gout and many other metabolic syndromes and shows a rapidly increasing incidence worldwide. Previous found that the gut microbiota and intestinal tract play important roles in the pathogenesis of HUA. However, most studies only revealed the association of gut microbiota and HUA without exploring the mechanistic causality, it remains unclear which gut microbiota play crucial roles in HUA.

Results

In the present study, we aimed to investigate key members of the gut microbiota in HUA and validate their effects on uric acid (UA) regulation. To this end, we deleted parts of the gut microbiota with antibiotics (NC-Ab group) and found that the abundance of Enterococcususwas significantly increased and the serum UA was significantly higher in antibiotic-fed mice (NC-Ab group) than no-antibiotic-fed mice (NC group). Subsequently, E. faecalis, the most important enterococcal species, was isolated and applied to HUA model mice for 2 weeks. We found E. faecalissupplementation could effectively alleviate HUA. Additional mechanistic investigation discovered that E. faecalis treatment could decrease the generation of UA and increase the excretion of UA through inhibition of xanthine oxidase (XO) activity and upregulating the expression of urate transporters (Abcg2, Glut9). Additionally, oral gavage with E. faecalis resulted in improvements in inflammation, restoration of impaired gut barrier, and amelioration of hepatic and renal function.

Conclusions

Our study demonstrated that E. faecalis plays a key role in regulating the UA balance and maintaining the intestinal barrier. E. faecalis could decrease UA level and protect gut barrier through inhibiting XO activity and increasing intestinal excretion. These findings may hold promise for developing intervention strategies for HUA.

Hyperuricemia

gut microbiota

Enterococcus faecalis

epithelium barrier

urate transporter

xanthine oxidase

SCFAs

uric acid

Hyperuricemia (HUA), a metabolic disease characterized by high levels of uric acid (UA) in the serum, is caused by the dysregulation of UA generation and excretion [1]. HUA is a major risk factor of gout and many other metabolic syndromes [2] [3] [4], such as cardiovascular disease and type 2 diabetes, and shows a rapidly increasing incidence worldwide. In China, the prevalence of HUA in 2008 in was approximately 13%[2]. Approximately 4/5 of UA (the end product of purine metabolism) is catabolized from nucleic acids in the cells while the remaining 1/5 is produced from purine-rich foods [5]. Almost 2/3 of UA is excreted by the kidney and 1/3 by the gut [6]. When the kidney is impaired, UA is mainly eliminated by intestinal excretion pathway as a compensatory mechanism [7]. Urate-lowering therapy, such as compounds of inhibiting UA synthesis (Allopurinol) and promoting excretion (benzbromarone), is widely used to control HUA [8]. However, owing to the potential adverse effects of these clinical drugs, it is necessary to discover alternative strategies for HUA treatment.

The gut microbiota are defined as a large number of bacteria that colonize the gut. The gut microbiota are known to have a nonnegligible effect on human health [9]. and gut microbiota dysbiosis is associated with tumors [10], immune [11] [12], metabolic disease[13] and nervous system diseases [14]. Transplanted fecal microbiota has also been recognized as an effective therapeutic method in many diseases [15] [16]. During purine metabolism, 1/3 of UA is excreted in the intestine and metabolized by gut microbiota such as Escherichia coli [17] and Clostridium [18] [19]. A high UA concentration in the blood will impact the intestinal environment and induce variation in the gut microbiota. Alteration of the gut microbiota also affects host metabolism and function. Indeed, the gut microbiota have been shown to differ between patients with HUA/gout and healthy individuals. Specific genera, such as Phascolarctobacterium, Bacteroides, and Ruminococcus_gnavus_group genera, are associated with high levels of UA, while a decreased abundance of Bifidobacterium, Prevotella, Butyricicoccus, Oscillobacter, and Ruminococcaceae_UCG_010 is negatively correlated with HUA[20] [21]. The gut microbiota participate in components of UA metabolism, and Clostridium is known to degrade UA directly. E. coli [17] has the ability to secrete xanthine dehydrogenase, which [22] is a core enzyme involved in converting hypoxanthine and xanthine to UA. Several transporters have been found to promote UA secretion in the intestine [23], although whether they are regulated by the gut microbiota remains unclear. These previous findings suggest that the gut microbiota and intestinal tract play important roles in the pathogenesis of HUA. However, as most studies have identified the association of gut microbiota and HUA without exploring the mechanistic causality, it remains unclear which gut microbiota play crucial roles in HUA. Koch’s postulates [24] are a set of scientific methods that are used to identify causative agents in infectious diseases. Similar to Koch’s method, it is necessary to reproduce HUA in appropriate models with pure cultures of potentially important members of the gut microbiota to establish their causative role in HUA.

In this study, we aimed to investigate key members of the gut microbiota in HUA and validate their effects on UA regulation. To this end, we deleted parts of the gut microbiota with antibiotics (NC-Ab group) and found that the abundance of Enterococcusus was significantly increased and the serum UA was significantly higher in antibiotic-fed mice (NC-Ab group) than no-antibiotic-fed mice (NC group). Subsequently, E. faecalis, the most important enterococcal species[25],, was isolated and applied to HUA model mice. The level of serum UA was obviously low in E. faecalis-fed HUA mice. Additionally, oral gavage with E. faecalis resulted in improvements in inflammation, inhibition of XO activity, restoration of impaired gut barrier, and amelioration of hepatic and renal function. Our study demonstrated that E. faecalis plays a key role in regulating the UA balance and maintaining the intestinal barrier. These findings may hold promise for developing intervention strategies for HUA.

Animals

Twenty-four 7-week-old male urate oxidase gene knockout heterozygous (+/−) C57BL/6J mice (Uox (+/−) mice) were obtained from the Institute of Metabolic Diseases of Qingdao University (Qingdao, China). Animals were kept in a climate- and light-controlled chamber at 23 ± 1°C under a 12/12-h dark/light cycle and given sterile water and standard chow. All of the experimental animals were approved by the Animal Research Ethics Committee of the Affiliated Hospital of Qingdao University (Approval No. AHQU-MAL20221012).

Antibiotic Treatment

The experimental design has been shown in Fig. 3. Seven-week-old male Uox (+/−) mice were randomly assigned to the normal group (NC) and antibiotics-fed normal group (NC-Ab). The NC group was fed with autoclaved double distilled water, and the Ab-NC group was supplemented with water with antibiotic mixture (ampicillin 250 mg/kg + neomycin 250 mg/kg + metronidazole 50 mg/kg). The experiment lasted 5 weeks, during which time the antibiotic mixtures were administered daily. Throughout the experiment, orbital blood and fresh feces samples were collected once a week to measure the biochemical indicators and analyze the intestinal microbiota.

Hyperuricemia mouse model and bacteria E. faecalis colonization

Seven-week-old male Uox (+/−) mice were randomly assigned to the normal group (control), hyperuricemia group (HUA), and hyperuricemia treated with E. faecalis group (E. faecalis-fed-HUA). The E. faecalis-fed-HUA group was fed with autoclaved double distilled water containing antibiotic cocktail (ampicillin 1 g/l, vancomycin 0.5 g/l, neomycin 1 g/l, and metronidazole 1 g/l) for 1 week, before recovering with autoclaved double distilled water without antibiotics for 3 days. The control and HUA groups were fed with autoclaved double distilled water simultaneously. On the 10th day of the experiment, the HUA group was fed yest-rich forage and 250 mg.kg^− 1.d^− 1 potassium oxonate (PO, 97%; Sigma-Aldrich, St Louis, MO, USA) via peritoneal injection at 9:00 am. The E. faecalis-fed-HUA group was treated as the HUA group to establish an HUA mouse model. Meanwhile, each mouse was administered E. faecalis (2 × 10⁹ UFC/ mice) in 0.15 mL phosphate-buffered saline (PBS) (pH = 7.4) by gavage for 20 days. The control group received only standard chow, and oral and intraperitoneal treatments with vehicle (PBS, 0.5% carboxymethyl cellulose, CMC; Macklin, Shanghai, China). The HUA group was administered PBS by gavage. Orbital blood and fresh feces samples were collected once a week to measure the biochemical indicators and analyze the intestinal microbiota, respectively.

The body weight of each mouse was measured once every 2 days. After 30 days, fresh feces were obtained and frozen at − 80°C for further microbiota analysis. After fasting for 12 h, mice were sacrificed via CO₂ inhalation, and blood samples were collected and centrifuged at 3500 rpm for 10 min to obtain serum. The liver, kidney, small intestine, and colon were carefully separated and weighed. The serum and all tissues were snap-frozen and kept at − 80°C until the next experiment.

Analysis Of Biochemical Parameters

From the beginning of the experiments, the serum UA, ALT, AST, Glut, UREA, and CREA levels of each mouse were measured weekly by an automatic biochemical analyzer (Toshiba, Tokyo, Japan). The blood of the mice was drawn from the intra-orbital retrobulbar plexus. The activity of hepatic XO (xanthine oxidase) was determined using ELISA kits (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Intestinal tissues were homogenized using a mortar and pestle with PBS (pH 7.4). The supernatant of the homogenates was obtained for further measurement of inflammatory cytokines following centrifugation for 20 min at 3000 rpm. The levels of inflammatory cytokines, including IL-1β (interleukin-1β), TNF-α (tumor necrosis factor-alpha), and IL-6 (interleukin-6), in the intestinal tissue and serum were detected using ELISA kits (ABclonal Biotechnology Co., Ltd., Wuhan, China). All of the experiments were performed according to the manufacturers’ instructions.

1.1 Quantitative reverse transcription polymerase chain reaction (qRT‑PCR)

Total RNA in the small intestine, liver, and kidney tissues was extracted by TRIzol reagent (Tiangen Biotech Co., Ltd., Beijing, China). An EasyScript Plus cDNA synthesis kit (Takara Bio, Shiga, Japan) was used to synthesize cDNA according to the manufacturer’s protocol. qRT–PCR assay was conducted in a reaction system of 25 µL by applying TB Green Premix Ex Taq reagent (Takara Bio, Shiga, Japan) with a fluorescent PCR instrument (CFX96; Bio-Rad Laboratories Inc., Hercules, CA, USA). The amplification reaction was progressed as follows: at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and at 60°C 30 s. β-actin or Gapdh, as a house-keeping gene, was used as a reference. Relative gene expression levels were determined using the 2^−ΔΔCt method. The primer sequences for the target genes are listed in Table S1.

Wstern Blotting

Proteins were extracted from the intestinal tissue samples using RIPA (radioimmunoprecipitation assay) buffer (Beyotime Institute of Biotechnology, Shanghai, China). Then, the concentrations of proteins were detected by the BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Moreover, 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to separate 30 µg of protein from each sample. Afterwards, the proteins were transferred onto 0.45 µm PVDF (polyvinylidene fluoride) membranes (Millipore, Billerica, MA, USA). Following blocking by skim milk (5% w/v), the membranes were incubated with primary antibodies, including Claudin-5(1:1000; 49564, Cell Signaling Technology, USA), occludin (1:1000; 91131S; Cell Signaling Technology, USA), E-Cadherin (1:1000;3195s; Cell Signaling Technology, USA) and GAPDH (1:1000; 8884; Cell Signaling Technology, USA), overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies. An ECL (enhanced chemiluminescence) reagent was added to PVDF membranes to visualize the protein bands. ImageJ software (provided by National Institutes of Health, Bethesda, MD, USA) was used to calculate the intensity of each protein band. The level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was set as the internal control.

Histomorphological Analysis

The kidneys, small intestine, liver, and colon were harvested immediately at the end of the experiment and fixed in a 4% paraformaldehyde solution, embedded in paraffin, cut into 5-µm-thick sections, stained with hematoxylin–eosin (H&E), and observed under a microscope to evaluate the morphological characteristics. A representative field of view was selected and viewed at 200× magnification.

Acian Blue Staining

Whole colon tissues were randomly selected (n = 6 per group), cut distally at 0.5 cm, and fixed with Carnoy’s fixative (dry methanol: chloroform: glacial acetic acid at a ratio of 6:3:1). The colons were fixed overnight, washed in methanol 2 × 30 min, ethanol 2 × 15 min, ethanol/xylene (1:1) 15 min, and xylene 2 × 15 min, followed by embedding in paraffin. Colon tissues were sectioned at a 5-µm thickness, deparaffinized, and stained using an Alcian blue staining kit (Leagene, Beijing, China) according to the manufacturer’s instructions.

Immunohistochemistry

The intestinal tissue was harvested immediately at the end of the experiment. the intestinal tissues from mice were fixed and embedded in paraffin as outlined previously, and 4‑µm thick sections were cut from the paraffin blocks. The sections were dewaxed in xylene (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; oriGene Technologies, Inc.) and subjected to antigen retrieval using 10 mmol/l citrate buffer (pH 6.0; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; oriGene Technologies, Inc.) at 120°C for 3 min. Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide for 10 min at room temperature. Then, the sections were incubated at 4°C overnight using rabbit anti-Muc-2 1:2000; 5406, Cell Signaling Technology, USA), Anti-lysozyme (1:500; GB11345; Servicebio, Wuhan, China) and rabbit anti-occludin (1:1000; 91131S; Cell Signaling Technology, USA Co., Ltd.); as a negative control, sections were incubated with PBS alone. Subsequently, the sections were incubated with a goat-anti-rabbit immunoglobulin G secondary antibody (1:500; cat. no. PV9001; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.; oriGene Technologies, Inc.) at room temperature for 1 h and stained with diaminobenzidine (oriGene Technologies, Inc.) at room temperature for 5 min. The sections were counterstained with hematoxylin at room temperature for 5 min and analysis was conducted using an optical microscope (magnification, ×400). The immunoreactive positive signals appeared brown.

Detection Of Short-chain Fatty Acids In The Feces

SCFA analysis was performed as described previously. Briefly, the feces samples were weighed after freeze-drying, immersed in a saturated sodium chloride solution, and homogenized. The mixture was acidified with a solution of sulfuric acid, and SCFAs were extracted by treatment with diethyl ether. The homogenates were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatants were dispensed into tubes containing anhydrous sodium sulfate to remove water. These tubes were centrifuged under the same conditions and the supernatants were analyzed on a gas chromatography–mass spectrometry (GC–MS) device (Thermo Fisher Scientific, Waltham, MA, USA).

16s Ribosomal Rna Gene Sequencing And Data Analysis

Feces were snap frozen with liquid nitrogen and stored at − 80°C. Total genomic DNA was extracted from the samples using the QIAamp Fast DNA Stool Mini Kit (Qiagen, USA). The DNA concentration and purity were monitored on 1% agarose gel, and 16S rRNA genes were amplified using a specific primer with barcode. All PCR reactions were conducted using TransStart FastPfu DNA Polymerase (TransGen, Beijing, China). The following universal bacterial 16S rRNA gene amplicon PCR primers were used: forward primer, 5′-CCTACGGGNGGCWG CAG-3′ and reverse primer, 5′-GACTACHVGGG TATCTAATCC-3′. PCR products were mixed in equidensity ratios. Then, the mixture of PCR products was purified with a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, USA). Sequencing libraries were generated using the NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations, and index codes were added. The library was sequenced on an Illumina MiSeq platform. Sequence alignment, operational taxonomic units (OTUs) picking against the SILVA reference collection, clustering, phylogenetic and taxonomic profiling, and beta diversity analysis were performed using the Quantitative Insights into Microbial Ecology (QIIME) opensource software package. Differential genera bacteria were identified using LEfSe analysis and STAMP. The bacterial metagenome content was predicted from 16S rDNA-based microbial compositions, and specific gene abundance was determined using the PICRUSt2 algorithm. A total of 4905 inferred genes were categorized into 41 KEGG functional pathways. The expression of purine metabolism-related genes was identified.

Isolation and culture of E. faecalis

Fresh feces were collected from five mice in NC-Ab group. Fecal matters were mixed and tenfold serially diluted with sterile anaerobic phosphate-buffered saline (PBS) (pH 7.2; containing l-cysteine at 0.1%). The 10 − 4 dilutions were spread onto Colombian blood plates (Qingdao Hope Bio-Technology Co., Ltd., China;) and incubated at 37°C under anaerobic conditions for 24 h. Based on the colony morphology of Enterococcus and alpha or gamma hemolytic rings, colonies were selected. All the isolates were analyzed under microscope after gram staining. Gram-positive cells were blue purple oval cells. Under microscope they were seen singly or in paired or short chains. With Brooke mass spectrometer MALDI-TOF (IVD MALDI Bityper 2.3, Burker Scientific Technology Co., Ltd., China), the bacteria were identified as E. faecalis. All of the experiments were performed according to the manufacturers’ instructions.

16S rRNA gene sequencing of E. faecalis and phylogenetic analysis

A TIANamp Bacteria DNA Kit (TIANGEN Biotechnology, China) was used to obtain high-quality genomic DNA of E. faecalis strains. The 16S rDNA was amplified as described by Sibley et al. The sequence of potential isolates for species identification was prepared using the BLAST engine (NCBI) and the nucleotide sequences were submitted to GenBank for reference. The morphology of E. faecalis was analyzed by scanning electron microscopy (SEM S-3400 N II Hitachi, Japan).

NCM460 cells co-culture with E. faecalis.

For the measurement of UA level in vitro, the NCM460 cells were grown in 6-well tissue culture plates for 24 h. The cultures were fed by aspirating the culture medium at 48-h intervals and replacing it with fresh DMEM. Twelve hours before adding the bacteria, the medium was aspirated and replaced with antibiotic-free DMEM; 2 h before adding the bacterial cells, the medium was again removed and replaced by fresh antibiotic-free DMEM. Overnight cultures of E. faecalis and its derivatives were washed twice and resuspended in PBS, and the optical density at 600 nm (OD600) of the suspension was determined. The viable-cell count of the suspension was estimated from a previously prepared standard curve that related optical density to viable-cell numbers, from which it was determined that a cell suspension with an OD600 of 1.0 contained 2 × 10⁸ CFU/ml. NCM460 cell cultures in 6-well plates were previously determined to contain 2 × 10⁶ viable cells per well on average; the amount of bacterial suspension added was adjusted to achieve a multiplicity of infection (MOI) of 10:1. The plates were incubated at 37°C for 4 h in the presence of 5% CO₂. Finally, the supernatant was centrifuged at 12,000× g for 10 min, and the UA level in the supernatant was detected using a UA kit (Jiancheng, Nanjing, China). This kit provides a fluorescence-based method for detecting UA. In the assay, uricase transforms UA to allantoin, hydrogen peroxide (H2O2), and carbon dioxide. In the presence of horseradish peroxidase, H2O2 reacts with 10- acetyl-3, 7-dihydroxyphenoxazine to produce the highly fluorescent compound resorufin. Resorufin fluorescence was then analyzed at an excitation wavelength of 510 nm, after which the concentration of UA in the supernatant samples were calculated using the equation determined from different doses of standards.

E. faecalis treated with inosine

To measure E. faecalis degradation of inosine, 10⁹ probiotic cells were isolated and incubated in 1 ml of trisodium phosphate solution (0.1 M) containing 1.25 mM inosine for 60 min. Then, 0.9 ml of supernatant was collected and mixed with 0.1 mL of HClO₄(0.1mol/L) to prevent further degradation. UA levels in the supernatant were analyzed using UA kits (Jiancheng, Nanjing, China).

Cell Culture

Caco-2 human intestinal cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (MEM) (Invitrogen) containing 10% fetal bovine serum (FBS; Gibco, Adelaide, Australia). Cells were grown in a humidified incubator containing 5% CO₂ at 37°C. During the experiments, a growth arrest period in serum-free medium was observed overnight before stimulation. Cells were treated with soluble uric acid (0,6,8,10 or 16 mg/dl) for 24h.The solution was filtered through a 0.22-µm pore size filter (Millipore, Shanghai, China) before use.

Statistical analysis

The results are presented as the mean ± SEM (standard error of the mean). All of the statistical analyses were performed using SPSS 19.0 (IBM Inc., Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA) software. ANOVA (one-way analysis of variance) followed by Tukey’s post hoc test was used to compare differences between the groups. P < 0.05 was considered to be statistically significant. The significant differences between the two groups were analyzed using an independent samples T-test.

Determination of key microbiota in HUA

To investigate the key gut microbiota in HUA, an antibiotic cocktail in drinking water was given to mice to deplete components of the gut microbiota (Fig. 1A). The antibiotic-fed mice (NC-Ab) and control mice (NC) groups showed no significant differences in baseline body weight and UA level (Fig. S1). The level of serum UA in mice was significantly elevated after antibiotic feeding for 1 week (P < 0.05) (Fig. 1B), indicating that the serum UA was affected by antibiotic administration. We supposed that the antibiotic killed and altered certain microbiota in the intestine, which could affect the level of UA. We first attempted to prove that the gut microbiota was changed by antibiotic treatment. To this end, 16S rRNA gene sequencing was used to analyze the gut microbiota. Compared to the NC group, the richness and diversity indices were significantly decreased in the NC-Ab group (Chao [P = 0.02], Shannon [P = 0.039]) (Fig. 1C). Unweighted UniFrac-based Principal coordinates analysis (Unweighted UniFrac-based PCoA) analysis showed that the microbiota of the NC-Ab group were easily distinguished from those of the NC group (Fig. 1D). The results showed that 12,605 (63.01%) amplicon sequence variants/operational taxonomic units (ASV/OUT) were found only in the NC-Ab group. And 3,267 (32.23%) ASV/OUT were unique in the NC group. Only 4.67% of ASV/OUT were shared between the two groups (Fig. 1E), indicating significantly different microbiota compositions between NC-Ab and NC mice. The abundance of Enterococcus was significantly higher in the Ab-NC group at the genus level (Fig. 1F). Dominant species analysis supported that Enterococcus was almost the only microbiota at the genus level, and minimal Lactobacillus were observed in the Ab-NC group (Fig. 1H). In contrast, the NC group showed a higher abundance of Lactobacillus and a reduced abundance of Enterococcus (Fig. 1H). We further confirmed this finding using Ggtree package analysis. The data demonstrated that Lactobacillaceae was almost replaced by Enterococcaceae at the family level after antibiotic treatment (Fig. 1G). These results suggest that Enterococcus is a nonnegligible microbiotas in UA regulation. Enterococcus belongs to Enterococcaceae[26], while Enterococcaceae and Lactobacillaceae belong to Lactobacillales at the order level [26], thus, antibiotic treatment changed the gut microbiota from the order level.

Screening and identification of Enterococcus in mice feces

To explore whether Enterococcus affects the UA level, an enterococcus was isolated from NC-Ab mice feces. To the naked eye, colonies on Columbia blood agar were small, grayish, white, moist and raised with alpha or gamma hemolytic rings (Fig. 2c). Cells were analyzed under microscope after gram staining. We could observe that cells were Gram-positive and blue purple. These cells were single, in pairs, in short or long chains (Fig. 2a). With Brooke mass spectrometer MALDI-TOF, the bacteria were identified as E. faecalis (Fig. 2b). Phylogenetic analysis on the basis of 16S rRNA gene sequences also identified the bacterial strain as being E. faecalis (Fig. 2d-e). E. faecalis and E. faecium were the most important enterococcal species [25]. Previous studies have shown that administration of E. faecalis improved constipation in pig and villous atrophy in mice and pigs [27].

E. faecium decreased UA levels both in vitro and vivo

To clarify the effects of E. faecium on UA, we cultured E. faecalis with Colombian liquid medium and found that the UA level was not significantly decreased compared to the control (Fig. S3). We then co-cultured the supernatant of E. faecalis with NCM-460 cells (Fig. 3a), and found that the supernatant level was lower than that of the blank group, although there were not statistically significant (Fig. 3c). Furthermore, when we treated NCM-460 with live E. faecalis, the UA level of the supernatant also decreased (Fig. 3b). These data suggest that E. faecalis could decrease the UA levels in vitro. Considering inosine is an intermediate product during purine metabolism which could be metabolized into xanthine, we speculated that the ability of E. faecalis to degrade inosine in the gut would decrease of UA level. We treated live E. faecalis with inosine(1.25Mm). And E. faecalis treated with PBS as control group. We found that the UA level in inosine treatment group was significantly lower than that of the control (Fig. 3d). These results indicate that the E. faecalis regulated UA level related to inosine metabolism, although the mechanism remains unclear. To verify that E. faecalis could decrease UA in vivo, we used male Uox(+/-) mice to establish an HUA model with high- yeast diet and PO intraperitoneal injection. Uox(+/-) mice was shown to lead to more stable high UA levels than WT C57BL/6J mice. Compared to the control group (NC group), UA levels of HUA mice were significantly elevated, suggesting that the HUA model was successful (Fig. 3f). We then treated HUA mice with E. faecalis for 20 days (E. faecalis-fed HUA group) (Fig. 2e) and observed a reduction in the serum UA level in E. faecalis-fed HUA mice (Fig. 1g). This indicated that E. faecalis treatment could reduce serum UA levels. The above results suggest that the composition of gut microbiota was adjusted with the increase in serum UA and E. faecalis could regulated UA level.

E. faecalis regulated liver function and renal function in HUA mice

The liver and kidney are important organs in UA generation and excretion, involved in hyperuricemia pathogenesis. The levels of AST, ALT, creatine, and urea in serum were measured to explore the effects of E. faecalis on the liver and kidney. As shown in Fig. 4a and b, the concentrations of AST and ALT were non-significantly elevated in HUA mice (P > 0.05), indicating slightly impaired liver function. To further evaluate the hepatic histological structure, we stained hepatic tissues with H&E and observed swollen hepatocytes with irregular arrangement (Fig. 4f). After E. faecalis treatment, the levels of AST and ALT were sharply declined and the histological structure was restored to normal conditions (Fig. 3f). Unlike liver function, the levels of urea and creatinine were remarkably increased in the HUA group compared to the control group, indicating impaired renal function in HUA mice (Fig. 4b and c). The serum urea concentration was obviously decreased after E. faecalis supplementation, whereas the creatinine level did not significantly decrease, suggesting that E. faecalis treatment slightly alleviated renal impairment (Fig. 4b and c). The H&E stained kidneys from HUA mice showed obvious Bowman’s capsule dilatation, cytoplasmic vacuolation and dilatation, irregularly arranged cells, and renal tubular dilatation, which were significantly ameliorated by E. faecalis intervention (Fig. 4e).

E. faecalis attenuated inflammation in HUA mice

Previous studies reported that patients with hyperuricemia suffer from an inflammatory response [28]. To investigate the function of E. faecalis in inflammation in HUA mice, the concentrations of IL-6, TNF-a, and IL-1β in the serum and intestine were detected (Fig. 5a–f). The levels of IL-6 and TNF-a in both the serum and intestine were significantly higher in HUA mice compared to control mice. E. faecalis treatment decreased the serum IL-6 and TNF-a levels, while non-significantly reversing the inflammatory cytokines in the intestine. The IL-1β concentration in the intestine was also elevated in HUA mice, while the serum level was comparable between all groups, which may be due to the rather low level of IL-1β in serum. These results indicated that E. faecalis could regulate systematic inflammation rather than locally intestinal inflammation, which is consistent with a previous study that showed that E. faecalis could increase the number of IgA-producing cells in blood without modifying the percentage of IL-6-producing cells in the intestine [29].

E. faecalis affected the fecal SCFA levels in HUA mice

SCFAs are the products of intestinal microbiota fermentation, which are crucial molecules connecting host and gut microbiota. The concentrations of SCFAs, including acetate, propionate, iso-butyrate, butyrate, isovalerate, valerate, and caproic acid in the feces, were measured, and principal component analysis of SCFAs was used to assess discrepancy between the three groups. As shown in Fig. 6a, partial least squares-discriminate analysis (PLS-DA) showed a distinct distribution of SCFAs in the three groups, indicating that SCFAs were changed by E. faecalis treatment. As shown in Fig. 6b–g, all types of SCFAs were notably reduced in HUA mice compared to control mice (P < 0.05). Isovaleric acid and caproic acid were significantly increased after E. faecalis treatment in HUA mice (P < 0.05), while acetate, propionate, and butyrate were not reversed by E. faecalis administration (P > 0.05). These findings suggest that E. faecalis could partly reverse the reduction in SCFAs in HUA mice.

Elevated Ua Directly Damaged The Intestinal Barrier

Previous studies have shown an imbalance in intestinal homeostasis during HUA[4]. As shown in sections of intestinal tissues by H&E-staining, regional epithelial shedding, fewer and fractured villi, and mucosal and submucosal edema were observed in the intestines of HUA mice (Fig. 7a). Additionally, HUA mice had a visible reduction in mucins compared to control mice by Alcian blue staining (Fig. 7b). Along with mucins, the tight junction protein occludin and the adhesion protein E-cadherin act together as the first line of defense at the mucosal barrier. To explore the possible reasons for the impaired gut barrier in HUA mice in vitro, we treated Caco-2 cells with various concentrations of UA for 24 h. The results showed that the higher the UA concentration, the lower the protein expression of Muc-2, claudin, and E-cadherin (Fig. 7e–f). The results suggest that elevated UA directly damages the intestinal barrier.

E. faecalis maintained intestinal homeostasis in HUA mice

As shown in Fig. 2f, the UA level of HUA mice was decreased by E. faecalis management. We hypothesize that E. faecalis could abolish the damaged intestinal homeostasis caused by higher UA through downregulation of the serum UA level. We then used IHC to detect the protein expression of Muc-2, occludin, and lysozyme (antimicrobial peptides) in the HUA group and E. faecalis-fed HUA. The results showed that all of the proteins were strikingly downregulated in HUA mice, but were distinctly increased after E. faecalis treatment compared to HUA mice (Fig. 8a–f). These results supported the hypothesis that E. faecalis could reverse the impaired intestinal barrier caused by HUA through decreasing the serum UA level in HUA mice.

E. faecalis upregulated urate transporters in HUA mice

The serum UA level was affected by excretion and generation, and 1/3 of UA was secreted by the intestine. First, we detected the UA levels in the feces and intestine. As shown in Fig. 9a and b, the UA level was dramatically lower in HUA mice than that in control mice. Additionally, the UA levels in the intestine and feces were remarkably increased in E. faecalis-fed HUA mice vs. HUA mice. These data implied that treatment of E. faecalis increased the UA excretion through the intestinal lumen, resulting a reduction in serum UA, which directly protected the intestinal barrier from hyperuricemia. To clarify how E. faecalis affects UA excretion in the intestine, we detected several urate transporters that participate in UA secretion in the intestine, including Abcg2, Glut9, and Scl17A4[30]⁻[31]. As shown in Fig. 9c, the relative mRNA level of Abcg2 was distinctly lower in HUA mice than that in the control mice, while E. faecalis supplementation increased Abcg2 expression in the E. faecalis-fed HUA group (P < 0.05), indicating that E. faecalis could upregulate Abcg2 expression to increase UA excretion in the intestinal lumen. In contrast to Abcg2, the expression of Glut9 and Slc17A4 was notably increased in HUA mice, with a reduction in expression after E. faecalis treatment (Fig. 9d and e). These findings suggest a compensatory function of Glut9 and Slc17A4 in UA secretion, which is different from that of Abcg2. Next, Abcg2 protein expression was detected by IHC to confirm the function of E. faecalis in Abcg2. The data showed that Abcg2 expression was lower in HUA mice than it in control mice. After E. faecalis treatment, the level of Abcg2 was significantly increased compared to that of HUA mice, in accordance with the mRNA level (Fig. 9I and J). Kidney is the main route of UA excretion. The mRNA level of Abcg2, Scl17a4 and Glut9 in renal tissues also been tested. The Glut 9 and Abcg2 mRNA levels were lower in the HUA group compared to the control group, but were recovered by E. faecalis treatment (Fig. 9f and g). However, E. faecalis treatment could not upregulate the mRNA level of Slc17A4, which was downregulated by HUA (Fig. 9h). The above data suggest that E. faecalis decreased serum UA through increased UA excretion by upregulating the expression of urate transporters in both the intestine and kidney.

E. faecalis inhibited XO activity in HUA mice

Regarding the generation of UA, XO is one of the core enzymes in UA synthesis that converts hypoxanthine and xanthine to UA (Fig. 10a). As shown in Fig. 10b–d, XO activity was significantly higher in the intestine, kidney, and liver of HUA mice than that in control mice. When treated with E. faecalis, remarkably decreased hepatic XO, renal XO, and intestinal XO activities were found in the E. faecalis-fed-HUA group. (Fig. 10b–d). These findings suggested that XO activity could be inhibited by E. faecalis intervention in HUA mice, reducing UA synthesis.

The dramatic development of next-generation sequencing technologies has facilitated greater understanding of the microbiota residing in the intestine. Microbiota participate in host metabolic diseases, such as obesity, diabetes, fatty liver, and gout[32]. Research has also revealed that the gut microbiota is central to hyperuricemia [33]. The dysbiosis of gut microbiota is known to occur in patients with HUA and gout [34] [35]. Elucidating the main microbiota and using probiotics or deleting pathogenic microbiota to decrease UA levels and restore gut homeostasis may be an efficient strategy to treat hyperuricemia.

Antibiotics are classes of chemical compounds, which, at low concentrations, are able to kill or inhibit the growth of microorganisms [36]. Widespread use of antibiotics alters the history of infectious diseases and gut microbiota. Antibiotics impact both harmful and healthy bacteria due to their broad spectrum of action. The changes in gut microbiota following antibiotic treatment have been widely investigated[37]. and the impairment of gut microbiota and GI epithelia is the main conclusion [38]. Besides the side effect on gut microbiota, antibiotics have also been shown to have neuroprotective function in neurodegenerative disease by rebalancing the dysbiosis of gut microbiota [36]. Additionally, antibiotic treatment has been used to deplete almost all gut microbiota prior to bacterial colonization [39]. Many studies have found that modulation of the gut microbiota by antibiotics leads to clinical manifestations [36]. Indeed, Eiji Miyauchi et al.[40] administered mice with an antibiotic cocktail to deplete the gut microbiota, and found that the mice demonstrated attenuation of experimental autoimmune encephalomyelitis (EAE). Similarly, ampicillin treatment alone also limited the development of EAE. The bacterial strain depleted by ampicillin was isolated and used to inoculate EAE mice, which resulted in an increased severity of EAE. The results implied that antibiotic treatment could be used as an efficient method to select key members in millions of gut microbiota by depleting some bacterial strains.

Here, we employed an antibiotic strategy to reduce a component of the gut microbiota to elucidate the key microorganisms in HUA. The result showed that altered gut microbiota resulted in an increased serum UA level. At the order level, Enterococcaceae was significantly increased and almost replaced the abundance of Lactobacillaceae. At the genus level, Enterococcus was richer after antibiotic treatment, consistent with a study of gosling gout⁴². In the gosling gout study, Enterococcus was found to be enriched in gosling with gout [41]. These data indicate that Enterococcus is closely related to HUA, and may play a nonnegligible role in regulation of the UA level. In our study, the application of the most important enterococcal species[25]. E. faecalis, in HUA mice indeed resulted in a reduction in serum UA.

The gastrointestinal tract, the most important interface between the body and environment, plays roles in nutrient absorption and acts as barrier against pathogenic microbes [42]. Therefore, the integrity of the gut barrier is critical for health. The mucus barrier is one of the first lines of protection [42], which has a primary role in intestinal protection against mechanical, chemical, and biological attacks and contributes to the maintenance of intestinal homeostasis. Mucin2 (Muc2) is the best characterized secreted mucin of the GI tract[42]. Tight junctions are essential for the gut barrier and their physiological role is selective paracellular diffusion, which is essential to maintain tissue homoeostasis [43] ⁴⁶. Occludin is a member of the tight junction proteins [43]. Lysozyme[39], another member of gut barrier, is produced by Paneth cell. We selected these three proteins as intestinal barrier markers. The intestinal barrier is known to be impaired in HUA mice. In our study, we found notably decreased expression of Muc-2, occludin, and lysozyme in HUA mice, while E. faecalis treatment increased their expression. The results of in vitro experiments showed that the higher UA concentration, the lower the expression of Muc2, occludin, and E-cadherin, which indicated that a high UA level could directly damage the intestinal barrier. As mentioned above, E. faecalis treatment could decrease the serum UA level. These results support the role of E. faecalis in gut barrier maintenance by reducing the UA level.

As a necessary organ for UA excretion, the intestine could secrete UA into the intestinal lumen through urate transporters. Abcg2[23], a high-capacity urate transporter expressed on the apical membrane in the intestine, was downregulated in HUA mice, and the expression of was distinctly upregulated after E. faecium treatment. In UA generation terms, XO is a core enzyme converting hypoxanthine and xanthine to UA. The HUA mice had higher XO activity in the main UA synthesis organs, including the liver, intestine, and kidney. Moreover, E. faecalis intervention inhibited XO activity and upregulated Abcg2, which resulted in reduced UA production and increased UA excretion, thereby leading to decreases in the serum UA.

Our results showed that the levels of inflammatory cytokines (IL-6, TNF-α) in the serum and intestine were significantly higher in HUA mice than in control mice, indicating that HUA mice had low-grade intestinal and systemic inflammation. E. faecalis treatment could decrease serous IL-6 and TNF-α level. In this research, the intestinal IL-1β level was also much higher in HUA mice than in control mice, while the serous level was inconsistent with intestinal level, possibly because the IL-1β level was too low to detect differences. Pro-inflammatory cytokines and LPS have been reported to upregulate hepatic XO activity[44]. In our study, the HUA mice exhibited higher XO activity in the liver, kidney, and intestine compared to controlled mice, which coordinated with the fluctuation of inflammatory cytokine levels in the serum and intestine. When HUA mice were treated with E. faecalis, lower hepatic and renal XO activity, and less serous inflammatory cytokines were found. These data suggests that the normalization of XO activity may be related to decreased systemic inflammatory responses post-E. faecalis treatment. Interestingly, the intestinal inflammation was not decreased by E. faecalis treatment. But E. faecalis appeared to be able to affect intestinal XO activity. How E. faecalis skipped the intestine to directly regulate systemic inflammatory responses remains unclear and requires further investigation.

SCFAs are vital metabolites for maintaining intestinal homeostasis and are produced by anaerobic fermentation of dietary fibers in the gut [45], including acetate, propionate, butyrate, isobutyric acid, isovaleric acid, valeric acid, and caproic acid. In the current study, the SCFA concentrations were sharply decreased in HUA mice vs. the control group, consistent with the findings of a previous cohort study[46]. E. faecalis treatment increased isovaleric acid and caproic acid concentrations, while the levels of acetate, propionate, and butyrate were not elevated by E. faecalis. It has been reported that acetic acid inhibits XO activity, which acts as an inhibitor of XO [47]. The lack of increase in acetate concentration in E. faecalis-treated HUA mice implicates that other, as yet unclear factors could regulate XO activity. Butyrate has beneficial effects on cellular energy and intestinal homeostasis[48] and has been proven to inhibit inflammation and maintain epithelial barrier integrity [49]. In our study, the butyrate level was declined and gut barrier was impaired in HUA mice. Moreover, the gut barrier was repaired in the E. faecalis-fed HUA group, while the butyrate concentration was not improved consistently, suggesting that the gut barrier is protected by E. faecalis independent of butyrate. However, we found that isovaleric acid, another SCFA, was increased after E. faecalis treatment. Isovaleric acid has been shown to induce colonic smooth muscle relaxation[50], but whether it affects the barrier function requires further research.

As common opportunistic pathogens of humans[25], the use of E. faecalis as probiotics generated serious concern, and has led to an increase in research in this field. In HUA mice, impaired renal function was indicated by a higher serum level of creatinine and urea accompanied by pathological changes such as tubular dilatation and cytoplasmic vacuolation, which were obviously ameliorated by E. faecalis treatment. Similarly, the hepatic function and pathological changes were improved by E. faecalis, suggesting that the effect of E. faecalis on kidney and liver was beneficial rather than pathogenic. As the commensal microbiota of human and animals, E. faecalis predominates the Entercoccus species of the gastrointestinal tract, which is followed by E. faecium, E. durans, and finally, E.Hirae [25] . The use of E. faecalis as a food supplement in several probiotic preparations has been proven to be both effective and safe [25] Additionally, E. faecalis was found to improve the laying rate and affect the cecal microbial community structure in laying hens [51] Furthermore, it improved the growth performance, reduced the diarrhea rate, and increased the proportion of beneficial bacteria in weaned piglets [52]. Besides, a previous study [53] using germ-free, wild-type (WT), and disease-susceptible IL-10(–/–) mice to characterize the impact of E. faecalis, demonstrated that potentiality of E. faecalis to initiate pro-inflammatory responses in the disease-susceptible but not the normal host. Hence E. faecalis could be considered an effective and safe therapy for HUA mice.

In summary, we hypothesized that E. faecalis was the key microbiota involved in regulating the UA level. First, as a commensal microbiota, E. faecalis was proved to be efficacious and relatively safe. Second, E. faecalis could down regulate the UA level. Third, E. faecalis could maintain the epithelial barrier function through decreasing serous UA.

In this study, we screened an E. faecalis strain and demonstrated its ability to relieve hyperuricemia in a mouse model. E. faecalis could restore the mucosal barrier, ameliorate inflammation, improve hepatic and renal function, decrease the serous UA concentration by inhibiting XO activity and increasing intestinal excretion. Our findings implicate E. faecalis as a promising therapeutic candidate for alleviating HUA and associated disorders.

HUA Hyperuricemia

UA uric acid

E. faecalis Enterococcus faecalis

Efa Enterococcus faecalis

ELISA Enzyme-linked-immunosorbent serologic assay

TNF-α Tumor necrosis factor alpha

IL-1β Interleukin-1β

IL-6 Interleukin-6

IL-10 Interleukin-10

XO Xanthine oxidase

WT Wild-type

SCFAs Short-chain fatty acids

PCoA Principal co-ordinates analysis

KEGG Kyoto Encyclopedia of Genes and Genomes

Ethics approval and consent to participate

All animal procedures were conducted according to the guidelines for the care and use of experimental animals established by Qingdao University. The project was approved by the Animal Research Ethics Committee of the Affiliated Hospital of Qingdao University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

The authors’ contributions are as follows: ZT, XL designed the experiments. XL and CH conducted the experiments. XL, CH, JW, CL, LK, WZ and LY collected the samples and performed the analysis of samples. XL, CH, KL, QY, YG, TM and ZT wrote the manuscript, prepared the figures, and was responsible for data compilation and integration. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 81970461).

Availability of data and materials

The datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archive (SRA) repository under accession number PRJNA898992

(available on November 08 2022).

Authors' information

Xin Liu

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China. [email protected]. [email protected]

Chunhua Han

Department of Clinical Laboratory, the Affiliated Hospital of Qingdao

University, 16 Jiangsu Road, 266000 Qingdao, China. [email protected].

Ke Lei, Ph.D

Center of Tumor Immunology and Cytotherapy, Medical Research Center, Affiliated Hospital of Qingdao University, Qingdao, 266003, China. [email protected]

Tao Mao

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China. [email protected].

Qian Yu, Ph.D

Tumor Immunology and Cytotherapy, Medical Research Center, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China. [email protected].

Changgui Li

Institute of Metabolic Diseases, Qingdao University, Qingdao 266003, Shandong, China.

[email protected].

Leyong Ke

Kunming Medical University. Kunming, 650000, Yunnan, China. [email protected].

Jie Wu, Ph.D

Department of pathology. the second affiliated hospital of Xi'an Jiaotong University. Xi'an, 710000, China. [email protected].

Yingjie Guo

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China. [email protected].

Wenqing Zhang

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China. [email protected].

Zibin Tian, M.D and Ph.D

Department of Gastroenterology, The Affiliated Hospital of Qingdao University, Qingdao 266000, Shandong, China. [email protected]

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No competing interests reported.

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Enterococcus faecalis ameliorates hyperuricemia and maintains the epithelium barrier in a hyperuricemia mouse model

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Abstract

Figures

Background

Materials And Methods

Animals

Antibiotic Treatment

Analysis Of Biochemical Parameters

1.1 Quantitative reverse transcription polymerase chain reaction (qRT‑PCR)

Wstern Blotting

Histomorphological Analysis

Acian Blue Staining

Immunohistochemistry

Detection Of Short-chain Fatty Acids In The Feces

16s Ribosomal Rna Gene Sequencing And Data Analysis

Cell Culture

Statistical analysis

Results

Determination of key microbiota in HUA

Elevated Ua Directly Damaged The Intestinal Barrier

Discussion

Conclusion

Abbreviations

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References

Additional Declarations

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