2.1 Preparation of cells and probiotics
CT-26 cells were purchased from Shanghai Cell Bank and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. CT-26 cells were maintained in a 5% CO2 humidified chamber at 37℃.
L.reuteri JMR-01（NCBI Accession No. MT362007）was isolated from the fecal of cured mice bearing breast tumor. JMR-01 cultured in deMan, Rogosa and Sharpe (MRS) broth at 37℃ for 18 h; living probiotic JMR-01 (LP) was the precipitate harvested by centrifuging the fermentation solution at 8000 r/min after discarding supernatant, and the precipitate was then resuspending in a volume of phosphate-buffered solution (PBS) equal to supernatant and mixing evenly; living probiotic cell （LP-C）was obtained from the precipitate by centrifuging LP；living probiotic supernatant (LP-S) was obtained from the supernatant by centrifuging LP. Inactived probiotics (IP) was prepared by autoclaving LP at 105℃ for 30 minutes; inactived probiotic cell（IP-C） was obtained from the precipitate by centrifuging IP；inactived probiotic supernatant (IP-S) was obtained from the supernatant by centrifuging IP. Living probiotic combined with inactived probiotics (LP+IP) was the mixture of LP-C and IP; LP+IP cell （LP+IP-C）was obtained from the precipitate by centrifuging LP+IP; LP+IP supernatant (LP+IP-S) was obtained from the supernatant by centrifuging LP+IP.
2.2 Cell viability assay
The anti-proliferation effect of JMR-01 on CT-26 cells was examined by using MTT assay. 100 µL of CT-26 cells were seeded into 96-well plates at a concentration of 106 cells/mL and incubated overnight for stabilization. Next 100 µL LP, IP, LP+IP, IP-S and LP+IP-S were added to each well for 24 h to 48 h in air supplemented with 5% CO2 at 37 ℃. The negative control group was treated with RPMI-1640. After 24 h and 48 h, 10 µl of MTT solution (5 mg/ml) was added to each well and incubated for another 4 h. At the end of experiments, the medium was removed and the blue formazan crystals were solubilized with 200 µl of dimethyl-sulfoxide (DMSO, Solarbio), then the absorbance values were measured at 540 nm.
The optimization experiment was divided into 6 groups, which were LP: IP-B = 1:1, 1:2, 1:1/2; LP: IP =1:1, 1:2, 1:1/2（cells to CFU）. The negative control group was CT-26 cells without adding bacteria. The following experimental steps were abided by MTT assay described above. Inhibition rate = (1 –Atreatment/Acontrol) ×100% (A=absorbance).
2.3 Animal experiment
Male BALB/c mice (6-8-week-old) were obtained from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. They were maintained in polycarbonate cages under controlled environment conditions at 23±2℃ and 60%–75% relative humidity in a 12 h light/dark cycle. All mice had free access to water and standard commercial rat chow. After one week adaptation period, mice were subcutaneously injected with CT-26 cells (106 CFU/mL, 100 µL/per mouse). When the volume of tumor reached 900 mm3, the mice were then randomly divided into 6 groups including normal group, tumor control group, IR, IR+LP, IR+IP and IR+LP+IP group. Except for normal group, IR and tumor control group were conducted gavage with 100 µL PBS buffer, the other groups were semidiurnal gavaged with 100 µL LP, IP, LP+IP (109 CFU/mL) respectively for 7 days before irradiation to realize the colonization of probiotics in mice and the above treatment was continued after irradiation until the end of the experiment. 12C6+ irradiation was conducted when the tumor volumes in each group reached nearly 1400 mm3. Then, mice in all groups except for normal and tumor control group were put into the mouse fixator, and the body except for tumor was covered with aluminum plate. The mice were irradiated at a dose of 4 Gy and a dose rate of 4 Gy/min with 12C6+ ion beam provided by Heavy Ion Research Facility at Lanzhou (HIRFL). Tumor sizes were monitored every other day and volumes were calculated using the following formula: Volume (mm3) = longest diameter × (shortest diameter)2 /2. The tumor inhibition rate was calculated when there was significantly difference between tumor control group and other treatment group (P<0.05). The tumor growth inhibition rates= (Vcontrol - Vtreatment)/ Vcontrol (V= Volume). All animal experiments were approved by the Institute of Modern Physics Animal Ethics Committee.
2.4 Detection the immunomodulatory function of JMR-01
2.4.1 Lymphocytes proliferation assay
Spleen cells were prepared as reported by Li. Briefly, mice were euthanized by euthanasia. The spleens were minced in RPMI-1640 media and filtered through a fine nylon mesh. Mononuclear cells were isolated after lysis of red blood cells. Subsequently, splenocytes of 105 cells/mL were distributed into 96-well plates. Then 100 μL of JMR-01 LP, IP, LP+IP, LP-S, IP-S, (LP+IP)-S (the ratio with cells was 1:1, 1:10, 1:100), 100 μL peptidoglycan (50 μg/ mL, 100 μg/ mL) (Peptidoglycan was obtained by the following steps: First, JMR-01 was boiled for 20 min in 10% TCA. Suspensions were centrifuged at 10000 r/min for 15 min to collect cell wall precipitate without lipoteichoic acid. The precipitate was dissolved in trypsin-phosphate buffer (1 mg / ml, trypsin; 0.1 mo1/L PBS) and incubated at 37℃ overnight. Then centrifuged at 4000 r/min for 20 min and discarded the insoluble protease precipitate. By centrifuging the supernatant at 15000 r/min for 20 min, the crude extract of peptidoglycan was obtained) were added to 96-well plates and incubated at 37℃ for 48 h. Then 10μL CCK8 was added to each well, and the plates were incubated for 4 h. Splenocytes proliferation was measured by determining the absorbance at 570 nm. Proliferation index was calculated according to the following equation: Proliferation index= Test OD570/Control OD570.
2.4.2 phagocytosis of macrophages assay
Cervical dislocation was performed to sacrifice mice. The peritoneal macrophages were collected by lavage with 5 mL physiological saline solution. Macrophages of 105 cells/mL were distributed into 96-well plates. Then 100 μL of JMR-01 LP, IP, LP+IP, LP-S, IP-S, (LP+IP)-S (the ratio with cells was 1:1, 1:10, 1:100), 100 μL peptidoglycan (50 μg/ mL, 100 μg/ mL) were added to 96-well plates and incubated at 37℃ for 48 h. 100 μL 0.075% neutral red solution was added to each well at 48 h and the macrophages were washed with PBS to remove remanent neutral red after 0.5 h, then 100μL of cell lysis buffer (acetic acid:ethanol=1:1) was added to each well. Phagocytosis was measured by determining the absorbance at 540 nm using an automated microplate reader, and calculated according to the following equation: Phagocytosis index= Test OD540/Control OD540.
2.5 The regulation of gut microbiota by JMR-01
2.5.1 Fecal bacterial counts
To detect the number of intestinal flora, the fecal samples were weighed, homogenized, serially diluted and plated on selective agar according to the implementation manual of technical specifications for inspection and evaluation of health food of the People’s Republic of China (2003). After overnight incubation at 37℃, bacteria colonies were counted. Bifidobacterium BS Medium was used to analyze fecal Bifidobacterium. DeMan, Rogosa, and Sharpe agar (MRS) was used to assess Lactobacillus. In order to explore Enterobacterium, Enterococcus, Bacteroides fragilis, and Clostridium perfringens, fecal sample were plated on Eosin-Methylene Blue Agar, Bile Esculin Azide Agar, BBE agar and Clostridium Perfringens Medium respectively. Fecal bacteria count was expressed as log10 CFU/g feces.
2.5.2 Fecal enzyme assay
To determine fecal enzyme activities, fecal was collected at the beginning of treatment, at 16th day of treatment (tumor volumes of IR+IP group were significantly higher than PBS (P<0.05)), at 30th day of treatment (the tumor volumes of LP, IP and LP+IP were significantly higher than PBS (P<0.05)), at 40th day of treatment (the survival rate was 50% in tumor control group) and at 50th day of treatment (the end of experiment). The fecal samples were frozen immediately after collection and stored at −80°C. Preweighted fecal were homogenized in PBS buffer and then centrifuged at 8000 r/min for 5 min. Its supernatant placed into tubes containing 100 μL 15 mmol/L p-nitrobenzoic acid solution, 500 μL 0.2 mol/L PBS. The incubations were performed at 37℃ for 30 min in the water bath. The reaction was stopped via adding 1mL 20% trichloroacetic acid and read at 240 nm with microplate reader. The amount of decomposition of p-nitrobenzoic acid was calculated from the standard curve of p-aminobenzoic acid concentration, and the enzyme activity was calculated.
2.5.3 Determination of short chain fatty acids (SCFA) in mice fecal
Fecal samples (100 mg) were diluted in 1 mL 0.1% phosphoric acid. Samples were homogenized and centrifuged at 10000 rpm for 10 min at 4℃. Supernatants were passed through 0.45 µm-pore-size nitrocellulose filters. SCFA (acetate, propionate, butyrate) determination was performed by using high-performance liquid chromatography (HPLC) equipped with a C18 Column 300 × 7.8 mm and a UV detector at 210 nm. 20 µl of each sample was injected into the HPLC system and 0.1% Phosphoric acid was used as a mobile phase at a flow rate of 0.5 mL/min. The concentrations of SCFAs were estimated using the linear regression equations generated from acetic, propionic, and butyric acids standard curves. SCFAs concentrations were expressed in mM/g.
2.6 Statistical analysis
The statistical analysis were performed using the GraphPad PRISM 8.0.1 (GraphPad Inc., California, CA, USA), SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and the Origin 9.0 (Origin Lab Corp., Northampton, MA, USA). The number of mice in each group was 14 (normal group=8), cell viability assay were carried out six replicates, and the other experiments were carried out three replicates. The data were presented as means ± standard deviations (SD).