Construction of mouse liver fibrosis model
Fifteen Balb/c male mice (6–7 weeks old) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and randomly grouped as follows: Control (n = 5), CCl4 (n = 5) and CCl4 + resveratrol (n = 5). For the CCl4 group, the mice were intraperitoneally injected with 0.5 µL/g CCl4 (dissolved in olive oil) twice a week for 4 weeks. For the Control group, the mice were intraperitoneally injected with the same amount of olive oil twice a week for 4 weeks. For the CCl4 + resveratrol group, the mice were treated with 30 mg/kg resveratrol daily by gavage, following by intraperitoneally injection of 0.5 µL/g CCl4 twice a week for 4 weeks. For the overlapped day (the day mice received both resveratrol and CCl4 subjection), the interval time between resveratrol gavage and CCl4 induction was approximately 5 h. Four weeks immediately after above treatment, the mice were sacrificed under euthanasia condition and the tissue samples and blood samples were collected. All the animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University and followed the principles outlined in the Declaration of Helsinki.
Sirius red staining
The degree of liver fibrosis in mice was evaluated using Sirius red staining following the previously described method with minor changes (Chen et al. 2017). The liver tissues of mice were prepared into 5 μm sections, and then the Sirius red staining solution (Solarbio, Beijing, China) was applied to stain the above sections referring to the standard procedure of the manufacturer. The Image J software (National Institutes of Health, Bethesda, MD) was conducted to quantify the area of liver fibrosis in mice.
The mouse liver tissues were fixed with 4% paraformaldehyde and prepared into 5 μm sections. The sections were incubated with 3% hydrogen peroxide for 15 min and then continued to incubate with 5% BSA for approximately 20 min. Next, the sections were incubated with anti-α-SMA (#19245, 1:500, Santa Cruz Biotechnology, USA) and anti-Collagen I (#72026, 1:200, Santa Cruz Biotechnology) overnight at 4 °C. The sections were further incubated with the specific secondary antibodies at room temperature for 1 h. Subsequently, the sections were treated with DAB chromogen (Abcam, Cambridge, UK) and re-stained with hematoxylin. All data were assessed using microscope (DS-U3, Nikon, Tokyo, Japan) and ImageJ software (National Institutes of Health, Bethesda, MD). A minimum of 5 representative fields per biological replicate were taken into analysis.
Detection of the serum markers for liver fibrosis
Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), indirect bilirubin (IBil), total bilirubin (TBil), direct bilirubin (DBil) and hydroxyproline (Hyp) levels are common indicators for evaluating liver function (Elsayed Elgarawany et al. 2020; Ambade et al. 2019). After collecting the mouse blood samples, the contents of ALT (ab282882, Abcam, UK), AST (ab263882), ALP (ab285274) and Hyp (A030-3-1, Jiancheng, Nanjing, China) were measured by corresponding ELISA kits followed the instructions of the manufacturer. The levels of IBil, TBil and DBil were detected using a standard clinical automated analyser (SRL, Tokyo, Japan).
After the mouse colon tissues were harvested, the tissues were fixed with 4% paraformaldehyde and prepared into 5 μm sections. Followed by the sections blocked with 5% BSA, the sections were incubated with anti-zonula occludens-1 (ZO-1, #13663, 1:400, Santa Cruz Biotechnology) at 4 °C overnight. Next, the above sections were incubated with the specific secondary antibodies at room temperature for approximately 1 h and were stained with 4,6-diamidino-2-phenylindole (DAPI, Solarbio) for 10 min. For the Caco-2 monolayer cells, the cells were fixed by 4% paraformaldehyde and the non-specific sites were blocked with 10% FBS. The remaining methods were the same as above. Ultimately, the immunofluorescence signals were observed under a confocal microscope (BX51TF, Olympus, Japan).
16S rRNA sequencing and data processing
The genomic DNA from mouse feces was isolated using a PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). Then, the Hot Master PCR mixture (5Prime, Gaithersburg, MD, USA) and specific primers targeting the 16S rRNA region V4 were conducted to amplify the 16S rRNA gene. Ultimately, the amplified product was sent to the biotech company (TSINGKE, Beijing, China) for the sequencing analysis. For the data processing, the diversity and composition of microbial communities were assessed using alpha-diversity and beta-diversity (Sims et al. 2019); and the genus-level differences were identified and the ternary plot analysis, and the relative abundance of species to investigate the gut microbiota of mice with obvious changes after CCl4 induction and resveratrol intervention.
Bacteria source and culture
Staphylococcus_lentus, Aerococcus_viridans and Staphylococcus_xylosus were from American Type Culture Collection (ATCC, USA). Staphylococcus_lentus, Aerococcus_viridans and Staphylococcus_xylosus were cultured in the Tryptic Soy Agar (TSA, Thermo Fisher Scientific, Waltham, MA, USA) with 5% defibrinated sheep blood at 37 °C. After that, these bacteria were further cultured in the Tryptic Soy Broth (TSB, Thermo Fisher Scientific) at 37 °C.
In vivo verification of the regulation of Staphylococcus_lentus and Staphylococcus_xylosus on liver fibrosis
To explore the impact of Staphylococcus_lentus, Aerococcus_viridans and Staphylococcus_xylosus on liver fibrosis in vivo, the pure cultures (109 CFU) of gut microbiota were given to the mice in the control, CCl4 or CCl4 + resveratrol group by gavage, and then the liver fibrosis model of mice was induced by intraperitoneal injection of CCl4 (0.5 µL/g). Four weeks later, the mice were sacrificed and the tissue samples and blood samples were isolated. All the animals were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University.
Construction of in vitro model of Staphylococcus biofilm
The cover glass was soaked in the TSB medium. After autoclaving, the bacteria solution (Staphylococcus_xylosus and Staphylococcus_lentus) of the activated TSB liquid medium was inoculated in the TSA medium and cultured at 37 °C overnight. A single colony was selected and inoculated in the TSB medium at 37 °C overnight for nearly 12 h, then diluted to OD600 of 0.4, and further diluted 100-fold. Then, 2 mL of the bacterial liquid was inoculated into the 24-well plates with the cover glass, mixed and sealed with parafilm. The biofilms were formed by continuous incubation in a shaker at 37 °C for nearly 3 days.
The growth characteristics of Staphylococcus_xylosus and Staphylococcus_lentus were conducted as the previously described methods with minor modifications (Rong et al. 2019). After Staphylococcus_xylosus and Staphylococcus_lentus were treated with resveratrol with the minimum inhibitory concentration (MIC, 260 μg/mL), 1/2 MIC, 1/4 MIC, and 1/8 MIC, the cell growth was measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 h using OD600 nm on a microplate reader (Multiskan MK3, Thermo Fisher Scientific).
Besides, after Staphylococcus_xylosus and Staphylococcus_lentus and their biofilms were treated with 1U of penicillin (PNC), the Staphylococcus_xylosus Staphylococcus_lentus biofilms were further treated with 1/8 MIC, and the cell growth was measured at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 h using OD600 nm on a microplate reader (Multiskan MK3, Thermo Fisher Scientific).
Determination of bacterial translocation
Bacterial translocation (BT) is generally considered as the presence of viable organisms in the MLN culture (Teltschik et al. 2012). Specifically, the MLNs were separated aseptically from the ileocecal zone. After grinding the separation, the homogenized MLNs (100 μL) were put in the MacConkey (Thermo Fisher Scientific), Mueller–Hinton (Thermo Fisher Scientific), and whole blood agar (Bio Merieux, Lyon, France) at 37 °C for approximately 2 days. Ultimately, the number of bacteria per gram was quantified using BT (CFU/g).
Detection of lipopolysaccharide-binding protein (LBP) concentration
In accordance with standard reagent manufacturer procedures, we quantified the concentrations of LBP and Albumin in mouse blood samples using a lipopolysaccharide-binding protein (LBP) assay kit (Mlbio, Shanghai, China).
EUB338 fluorescence in situ hybridization analysis
EUB338 fluorescence in situ hybridization (FISH) was carried out following the reported methods (Huang et al. 2020). The colon tissues were fixed with 4% paraformaldehyde and prepared into 5 μm sections. Then the sections were deparaffinized using xylene and rehydrated through an ethanol gradient (95%, 10 min; 90%, 10 min) to water. Subsequently, the sections were incubated with a universal bacterial probe directed against the 16S rRNA gene (EUB338: [Cy3]-GCTGCCTCCCGTAGGAGT-[AmC7 ~ Q + Cy3es]) at 60 °C for nearly 3 h. The NON-EUB 338 probe was a negative control probe to identify the non-specific binding of fluorochromes. The sections were then washed, and the sections were counter-stained with DAPI. All images were obtained under a confocal microscope (BX51TF, Olympus, Japan).
Quantitative real-time PCR (qRT-PCR)
The colon tissues and Caco-2 cells were harvested, and TRIzol reagent (Solarbio, Beijing, China) was applied to isolate the total RNA. The quantity and and quality of RNA were evaluated by a Nanodrop (Thermo Scientific) and limited the A260/A280 ratios from 1.9 to 2.1. Then, RNA was reversely transcribed into cDNA using the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific). The cDNA was subjected to perform qRT-PCR detection on the Bio-Rad CFX96™ Real-Time System with SYBR Green I PCR mix (Thermo Fisher Scientific). GAPDH was used as an internal reference and the relative expression of genes was calculated using the 2−ΔΔCt method. The primers of ZO-1, occludin, ACTA2 and GAPDH were all designed and synthesized by GenePharma (Shanghai, China).
Trans-epithelial electrical resistance (TEER) analysis
TEER is one of the commonly used methods to evaluate cell monolayer integrity (Akbari et al. 2017). Caco-2 cells were cultured in a Transwell chamber and then washed with HBSS containing 5 mM HEPES. After that, HBSS/HEPES were added into the basolateral and apical compartment at 1 mL and 0.2 mL volumes and then incubated at 37 °C and 5% CO2 for nearly 30 min. The TEER was then transferred to a hot plate at 37 °C and was tested by an epithelial voltmeter with chopstick electrodes (Millicellers-2, Billerica, MA, USA). An insert without cells was used as a blank and its mean resistance was subtracted from all samples, and the TEER value was generally expressed as ohm cm2.
Paracellular permeability analysis
Fluorescein isothiocyanate-dextran (FD) and lucifer yellow (LY) were conducted to assess paracellular permeability. Specifically, the Staphylococcus_xylosus and Staphylococcus_lentus cultures were co-cultured in a Transwell chamber with the monolayer of Caco-2 cells. The membrane-impermeable tracers LY and FITC conjugated dextran dissolved in PBS were added to the apical compartment and incubated for nearly 4 h, and the medium was added to the basolateral chamber. After the incubation for 1 h at 37 °C, a total of 100 μL solution was collected from the basolateral sides. The transport buffer volume in the apical was 300 μL, and that in the basal sides was 800 μL. A Synergy HTX multimode plate reader was carried out to test the fluorescent signals of FD (the excitation and emission wavelengths were 485 and 530 nm) or LY (the excitation and emission wavelengths were 428 and 540 nm).
The total proteins were extracted from Caco-2 cells using RIPA lysis buffer (Solarbio), and the concentrations of proteins were quantified by a BCA protein assay kit (Abcam, Cambridge, UK). After that, the proteins with different molecular weights were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred into polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The above membranes were then blocked with 5% skimmed milk at room temperature for approximately 1 h, and incubated with the primary antibodies: anti-Claudin-1 (ab211737, 1:2000, Abcam), anti-Claudin-3 (ab214487, 1:1000, Abcam), anti-Occludin (ab216327, 1:1000, Abcam), anti-JAM-1 (ab269948, 1:1000, Abcam) and anti-β-actin (ab8226, 1 µg/mL, Abcam) at 4 °C overnight. The following day, the membranes were incubated with the secondary antibodies for approximately 1 h at room temperature. The protein bands were visualized and quantified using the Enhanced Chemiluminescence Assay Kit (Abcam) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).
All the data were represented as the mean ± SD of three independent assays. The differences between the two groups were evaluated by Student’s t-test, and the differences among more than two groups were assessed using one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant.
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