Bacteria
Various Lactobacillus species were isolated from commercial yogurts, including Suki, Oikos, and Yoplait yogurt purchased from the supermarket (Table 1). Lactobacillus rhamnosus was purchased from ATCC (LGG, ATCC# 53103, Manassas, VA). All Lactobacilli were grown in the De Man, Rogosa, and Sharpe (MRS) broth (Hardy Diagnostics, Santa Maria, CA) at 37°C and incubated in a BactronEZ SHEL LAB anaerobic chamber (Sheldon Manufacturing, Inc. Cornelius, OR). MRS agar plates were made with 55g Lactobacilli MRS broth of dehydrated culture media and 15 g agar (Fisher Scientific) in one liter of water. The lysogeny broth (LB) medium and agar plates for Escherichia coli (E. coli) growth were purchased from Criterion and prepared according to the manufacturer's instructions.
Mice
BALB/c mice (6-8 weeks of age) were purchased from Jackson Laboratory. Mice were housed and fed in a specific pathogen-free animal house. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Antibodies
The following rabbit anti-mouse antibodies used for Western blotting were purchased from Abcam: Claudin 5 (ab15106), Tjp2 (ab224314), Itgb5 (ab184312), GAPDH (ab181602), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (ab270144).
Lactobacillus purification from yogurt
Yogurt was diluted with 10 volumes of phosphate-buffered saline (PBS) and centrifuged at 700 × g for 5 min at 4°C. The supernatant was washed once with an equal volume of cold PBS and centrifuged at 7,000 x g for 15 min at 4°C. Pellets were then resuspended in an equal volume of medium (MRS broth and simulated gastric juice) at 37°C and incubated for 6 h with constant stirring.
Agar plate culture
After serial dilution in a maximum-recovery diluent, the bacteria were cultured on MRS agar and LB agar plates in an anaerobic chamber and a regular incubator at 37°C. The growth was monitored after 24~48 h and photographs were taken with the BioDoc-ItTM Imaging System (UVP).
Bacterial growth monitoring
Samples were taken at 0, 0.5, 1, 2, 4, and 6 h, to measure the optical density (OD) at 600 nm using a spectrophotometer (BioTek) before and during incubation.
Biofilm formation assay
The overnight culture was diluted 1:100 in the fresh MRS medium containing 0.5% glucose to provide carbon and energy source and grown in a 96-well plate for 24 h at 37°C in the BactronEZ SHEL LAB anaerobic chamber. The quantitative assays were performed in 3 replicate wells for each treatment. After incubation, the culture was removed and washed twice with water to remove unattached cells and media components, significantly lowering the background staining. After15 minutes of staining with 150 µl of 0.1% crystal violet (Sigma) diluted to 2.3% with H2O, cells were washed twice with H2O, and the plates were dried for several hours. The crystal violet stain was solubilized in 150 µl of 30% acetic acid, and optical density was read in a spectrophotometer (BioTek) at 550 nm.
Induction of Colitis by DSS and Docetaxel
Colitis was induced by feeding BALB/c mice 2.5% dextran sulfate sodium salt (DSS, MP Biomedicals, Santa Ana, CA) in drinking water until the end of the experiment. To generate colitis model by a chemotherapeutic agent, injectable solution of docetaxel(Sanofi) was purchased from the pharmacy in our hospital and was administered by intraperitoneal injection at 6mg/kg q4d for 28 days 13-15. 5 X 1010 of LGG were administered to the mice (n=5 mice per group) by oral gavage with/without fasting 2 h daily for 7 days prior to the DSS or docetaxel administration. The PBS control colitis group served as the primary comparison with the LGG intervention group. Bodyweight, stool consistency, and fecal blood were monitored daily. The mice were euthanized on day 28 following 7 days of interventional feeding. Immediately after euthanasia by carbon dioxide inhalation, the abdominal skin was sprayed with 70% ethanol, and blood was taken by cardiac puncture. Next, the colons were quickly flushed with cold PBS (10 mM, pH 7.4) to remove feces and blood and the distal segments (1.0 cm) were fixed in 10% buffered formalin solution for histological examination.
In vivo intestinal permeability assay
Fluorescein-5-isothiocyanate (FITC)-conjugated dextran (MW 4000; Sigma-Aldrich, St. Louis, MO) was administered at a concentration of 60 mg/100 g of body weight by oral gavage to study intestinal permeability in vivo. After 5 h, serum was collected retro-orbitally, and fluorescence intensity was determined with a fluorescence spectrophotometer (BioTek) at emission and excitation wavelengths of 485 nm and 528 nm, respectively. FITC concentration was measured from standard curves generated by serial dilution of FITC-dextran 4000, as described in the previously study16.
Histological analysis
Tissues were fixed overnight with buffered 10% formalin solution (SF93–20; Fisher Scientific, Fair Lawn, NJ) at 4°C and dehydrated by immersing in a graded ethanol series, 70%, 80%, 95%, 100% ethanol for 40 min each. Tissues were embedded in paraffin and subsequently cut into ultra-thin slices (5 mm) using a microtome, deparaffinized with xylene (Fisher), and rehydrated by decreasing concentrations of ethanol and PBS. Tissue sections were stained with hematoxylin and eosin (H&E), and slides were scanned with an Aperio ScanScope, as previously described17.
Enzyme-linked immunosorbent assay (ELISA)
The cytokine TNFa, IL-1b, and IL-6 levels in mouse colon mucus were quantified using ELISA kits (eBioscience) according to the manufacturer’s instructions. Briefly, excess binding sites were blocked with 200 µl of 1x ELISA/ELISPoT Diluent (eBioscience) for 1 h at 22°C. The microtiter plates were coated with the anti-mouse TNFa, IL-1b, or IL-6 antibody at 1:200 overnight at 4°C. After washing three times with PBS containing 0.05% Tween 20, the plates were incubated with the detection antibody in blocking buffer for 1 h at 22°C. The plates were washed three times and avidin conjugated with horseradish peroxidase (HRP), and substrate were added. Subsequently, absorbance at 405 nm using a microtiter plate reader (BioTek Synergy HT) was determined.
Labeling of bacteria with PKH26
Bacteria were labeled with PKH26 Fluorescent Cell Linker Kits (Sigma) in accordance with the manufacturer's instructions. After a wash with PBS, bacterial pellets were suspended in 250-500 µl of diluent C with 2-4 µl of PKH26 and subsequently incubated for 30 min at room temperature. After centrifugation for 5 minutes at 13,000x g, labeled LGG nanovectors were resuspended for further experiments.
RNA extraction.
Total RNA was isolated from murine tissues using an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. In brief, 100 mg of the tissue was homogenized using a tissue grinder and was disrupted in QIAzol Lysis Reagent. Tissue. The homogenate was mixed with 140 ml of chloroform, centrifuged, the upper aqueous phase was mixed with 1.5 volumes of ethanol, and was loaded into the RNeasy spin column. The flow-through was discarded, and the column was washed with RWT and RPE, respectively. The flow-through was discarded after centrifugation, and the column was washed with RWT and RPE. Total RNA was eluted with RNase-free water, and the quality and quantity of the isolated RNA were analyzed with Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies).
mRNA expression by Quantitative Real-Time PCR
For analysis of mRNA expression, 1 µg of total RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen) and quantitated using primers (Sangon, Shanghai) with SsoAdvancedTM Universal SYBR Green Supermix (BioRad); GAPDH was used for normalization. The primer sequences are listed in Table 2. qPCR was performed using the Applied Biosystems 7500 System, with each reaction performed in triplicate. Analysis and fold-changes were determined using the comparative threshold cycle (Ct) method. The change in mRNA expression was calculated as the fold-change. Gene expression was normalized to the control expression by calculating the ∆ Ct = (Ct of control − Ct of the gene). Setting the expression value of GAPDH to 1.0, the relative expression values were calculated as 2ΔCt. The change in miRNA or mRNA expression was calculated as fold-change.
Tight junction qPCR array
For analysis of tight junction genes mRNA expression, 2 µg of total RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen), and quantitation was performed using mouse Tight Junctions RT2 ProfilerTM PCR Array (Qiagen, PAMM-143) with SsoAdvancedTM Universal SYBR Green Supermix (BioRad); GAPDH was used for normalization. The data analysis was processed online at https://dataanalysis2.qiagen.com/pcr.
Western blot analysis
Samples were incubated in the SDS loading buffer at 95°C for 5 min and separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting to polyvinylidene difluoride membranes (Bio-Rad). After blocking nonspecific binding sites for 1 h in 5% nonfat dried milk in PBST (0.05% Tween 20 in PBS), the membrane was incubated for 1 h at room temperature or overnight at 4°C with the primary antibody. After three washes in PBST, the membrane was incubated with HRP-conjugated goat anti-mouse antibody for 45 min at room temperature, washed three times in PBST, and the signal was detected using enhanced chemiluminescence (ECL kit from Amersham Biosciences).
Quantification and statistical analysis
Unless otherwise indicated, all statistical analyses were performed with SPSS 16.0 software. Data are presented as mean ± standard deviation (SD). The significance of mean values between the two groups was analyzed using the Student's t-test. The differences between individual groups were analyzed by a one- or two-way ANOVA test. The differences were considered significant when the p-value was less than 0.05 or 0.01.