Bacterial Culture and Preparation of Conditioned Media
Streptococcus thermophilus (S. thermophilus, CICC 24202) and Lactobacillus pentosus (L. pentosus, CICC 6222) were purchased from the China Center of Industrial Culture Collection. S. thermophilus were anaerobically grown at 37C in MRS broth (Fluka, Heidelberg, Germany) containing 0.05% L-cysteine (Roth). L. pentosus cultured in a microaerophilic environment.
The overnight culture of S. thermophilus and L. pentosus were centrifuged at 4000g for 10 minutes and the bacterial pallet separated from the media. The media was discarded and bacterial pellets were resuspended in PBS washed and fixed with 5% formaldehyde for 3 hours at 4oC, washed thrice with sterile PBS before incubating with cell culture media. Conditioned media (CM), bacteria (5 × 107 CFU/ml) from freshly grown cultures were transferred to Dulbecco’s modified Eagle’s medium (DMEM) and cultivated for 3 hours according to the requirements of bacterial species. Bacteria were separated from CM via centrifugation (4300 g, 15 min, RT). pH Adjusted to 7.6, filter-sterilized (0.22 mm) and stored at -80 (supplementary fig S1) (45).
Cell Culture
C28/I2 Human Chondrocyte Cell Line is widely used as a model cell line for studying normal and pathological cartilage repair mechanisms related to chondrocyte biology and physiology were purchased from Sigma-Aldrich (cat # SCC043). DMEM (GIBCO Invitrogen, Carlsbad, CA), 10% FCS (FCS Superior; Biochrom, Berlin, Germany), and 1% antibiotic antimycotic (GIBCO Invitrogen, Carlsbad, CA) in Cell tissue culture flasks (Greiner Bio-One, Frickenhausen, Germany) (5% CO2, 37oC). Cells were grown in CM S.t & CM L.p, both media collectively and 0.1µl Gama Aminobutyric acid (GABA, Sigma# A5835). The results were compared with the no treatment group after 2 days of treatment.
Cells were stimulated with GABA inhibitor for 8 minutes then cultured in CM S.t & CM L.p, both media collectively and 0.1µl GABA for 2 days. The protein and mRNA were extracted to analyze the effect of GABABr.
Animal study
We purchased young (6-8 weeks) C57BL mice from Chongqing Medical University. All mice were kept under sterile conditions with five or less than 5 mice per cage. Mice had free access to food and water. The mice used for all experiments were assigned randomly to control or treatment groups. For treatments, normal C57BL mice were administered PBS, S. thermophilus, L. pentosus, both or GABA (γ aminobutyric acid) via oral gavage for 2 months until the sacrifices. Gavage treatment was initiated 2 weeks before ACLT.
OA was Surgically induced by an Anterior cruciate ligament transection (ACLT) injury to C57BL mice at 9-10 weeks of age. Animals were anesthetized using a mixture of 10% chloral hydrate 400ul/100g administered by a single intraperitoneal injection, and hind limbs were shaved and prepared for surgery, aseptically. For the ACLT surgery, the ACL was transected with k-wire after opening the joint cavity, under a surgical microscope. After rinsing with saline to remove tissue debris, the skin incision was closed with 2-3 sutures (46). After 2 months of the gavage treatment, mice were euthanized and samples were collected for mRNA, protein quantification, and histological assessment of tissues. Whole blood was drained via eye rupture (supplementary fig-S2).
Protocol for oral administration
Oral feeding was initiated 14 days before ACLT and continued up to 6 weeks. In feeding protocols, individual or combination bacteria was suspended in 1 ml PBS and the turbid suspension was administered orally using gavage needles thrice a week. Mice were into five groups according to the difference in feeding regime (Fig-2 a, b, supplementary Fig.S2).
Culture S. thermophiles and L. pentosus at 37°C for 24 h. Cells were harvested by centrifugation (4000 rpm for 10 min), washed, and re-suspended in phosphate buffer saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, pH 7.4), for oral administration to mice. A Viable Count for Culture and desired concentration of bacteria was obtained Using OD Values (600 nm). Od was adjusted and bacteria were stored in Aliquots. Aliquots of these suspensions were frozen at -80oC and stored until used. One fresh aliquot was thawed for every new gavage treatment to reduce the changes in bacterial viability.
The number of live cells after freezing and thawing was determined by colony-forming unit (CFU) counting on MRS-C agar after 48 h incubation. For both tested strains, more than 90% of cells were alive upon thawing and no significant differences were found during storage time. The number of vial cells was determined several times throughout the gavage period of two months,
The amount of each treatment according to body weight was as follows: (G1) S. thermophilus (2 × 1010 CFU/kg, 500 mg/kg); (G2) L. pentosus (2 × 1010 CFU/kg, 500mg/kg) (G3)Mixture (S. thermophilus + L. pentosus; 2 × 1010 CFU/kg, 500 mg/kg); (4) GABA (50 mg/kg); (5) PBS (phosphate buffer saline). Using body surface area-based formula (47), the dose of GABA for mice (4 mg/20g) was determined based on the amount recommended previously. We also fed 0.4 mg/20g/day of GABA via drinking water to mice to mimic continuous GABA supply identical to postbiotics. Since the optimal dose of S. thermophilus + L. pentosus was not reported, we used an effective dose showing suppressive effect against OA based on previous studies (15, 28).
Real-time Q–PCR
To extract RNA, the intestine and knee joints that underwent surgery and no-surgery controls were immediately transferred to liquid nitrogen on dissection. Tissues were pulverized with help of mortar and pestle. Total RNA was extracted from knee joints using BioTeke (RP1202) RNA Rapid extraction kit and was reverse transcribed to cDNA using SuperScript II reverse transcriptase following the manufacturer’s protocol. RT‐PCR reactions were carried out using the PowerUp SYBR Green Master Mix Kit (Applied Biosystems) following the cycling protocol: 50°C for 30 min, followed by 40 cycles at 95°C for 15 min, 94°C for 15 s, and 60°C for 45 s. The primer sequences used for RT‐PCR are enlisted in supplementary table S1.
Real-time PCR for Aggrecan, Adamts-5 COL2, IL4, Il6, IL10, TNF-α, and SOX9, was performed using the BIO-RAD Real-Time PCR System with SYBR Green RT supper Mix for qPCR (Applied Biosystems by Life Technologies, 4367659). All signals were normalized to that for GABDH. Relative gene expression was calculated by the ∆∆CT method, ∆CT was calculated using the GABDH as a reference gene. ∆∆CT was calculated relative to the non-surgery control samples.
In the in vitro studies total RNA was extracted from cells were compared between treatment and no treatment groups like mentioned above. See Supplementary Table S1 for a list of primers.
Tissue fixation and histologic preparation
Mice were euthanized using Chongqing Medical university-approved method. Both knee joints and intestine were removed and fixed for 72 hours at 4oc in 4% formaldehyde (neutral PH). Knee joints were decalcified in EDTA for 14 days, and all tissues were processed using a microwave processor and were embedded in paraffin. Tissue blocks were sectioned in the midsagittal plane through the medial compartment of the joint. 5-μm thick sections were cut from each paraffinized tissue. Safranin-O/Fast Green (catalog G1371, Solarbio life sciences) and Alcian Blue Hematoxylin/Orange (catalog G1121, Solarbio life science) staining’s were performed to analyze knee joint tissue. Knee tissue sections were stained with Safranin O/Fast Green for OARSI scoring and histomorphometric analysis. Intestine sections were used for Immunofluorescence (IF) staining. Unstained knee sections were employed for Immunohistochemistry (IHC) and IF staining.
Histomorphometric analysis of cartilage
Tibial and femoral cartilage area, total chondrocyte number, and Safranin O+ chondrocytes were quantified by a blinded observer via histomorphometry as previously described. Briefly, using Safranin O-stained sections, the articular cartilage area was measured using the ImageJ software. For each specimen, measurements were taken from one slide at each of the 3 levels (50μm apart) on both the tibial plateau and the femoral condyle in a 200-μm wide area of interest centered on the joint. Total chondrocyte and Safranin O+ chondrocytes were calculated. Three measurements were obtained across the joint for each specimen and the average was calculated.
Mouse OARSI scoring
To estimate the amount of cartilage repair or degeneration, a semiquantitative histopathologic grading system was employed through blinded graded observations by two observers who followed the Osteoarthritis Research Society International (OARSI) scoring system (48, 49). Utilizing this system, Safranin O-stained joint sections were graded based on the following scale: 0, normal cartilage; 0.5, loss of proteoglycan stain without cartilage damage; 1, mild superficial fibrillation without cartilage loss; 2, fibrillation/cliffing extending below the superficial zone coupled with partial loss of surface lamina; 3, vertical clefts/erosion of the cartilage to the calcified zone over 75% of the cartilage surface.
Immunohistochemistry (IHC)
Mouse knee joint sections evaluated by IHC were deparaffinized in 2 changes of xylene for 10 minutes each, rehydrated in a series of ethanol (100% ethanol, followed by 95% ethanol, followed by 1 change of 70% ethanol), and rinsed twice in deionized water. An antigen retrieval kit (AR0022; Boster) was prepared for the sections for 1 minute at 37°C. 0.5% percent H2O2 in ddH2Owere used to quench endogenous peroxidases for 10 minutes. The slices were incubated with selected primary antibodies at 4℃ overnight. sections were rinsed in phosphate-buffered saline containing 0.1% tween 20 (PBST, catalog P1397, Millipore Sigma). Next, slides were blocked with 5% normal goat serum followed by overnight incubation at 4°C with rabbit anti-mouse AGCAN (1:200 dilution, catalog DF7561, Affinity), rabbit anti-mouse MMP-13 polyclonal antibody (1:200 dilution, catalog 18165-1-AP, Proteintech), mouse anti-mouse Runx2 polyclonal antibody (1:200 dilution, catalog AF5186 Affininty), slides were incubated with biotinylated anti-rabbit secondary for 30 minutes at room temperature. Slides were once again rinsed in PBS 3 times for 5 minutes each, followed by 2 washes in deionized water for 5 minutes each. Antibody binding to Aggrecan Mmp-13 and Runx2 antigen was detected by a 3-minute incubation with DAB peroxidase substrate (catalog zli90181, OriGene). 3-minute application of DAB peroxidase substrate. Nuclei were then counterstained with Mayer’s hematoxylin solution (catalog AR0005, BOSTER) for 15 seconds.
Immunofluorescence
Intestine and joint samples of mice fixed in 4% paraformaldehyde, were deparaffinized as mentioned above. Permeabilized with PBS containing 0.5% Triton X-100 for 10 min and then blocked with 4% BSA containing 0.25% Triton X-100 for 30 min at room temperature. The intestine tissue slides were incubated with primary antibodies against IL-6 (1:200 dilution, catalog DF6087) and IL-1β (1:200 dilution, catalog AF5103) overnight at 4 °C. The knee joints were incubated with IL-6 (1:200) and COL2 (1:200 dilution, catalog bs-0709R). The cells were rinsed three times with PBS and incubated with goat anti-rabbit IgG (H&L). After washing, the nuclei were counterstained with DAPI for 5 min. Nuclear against IL-6 and IL-1β were imaged by fluorescence microscopy.
Immunoblotting
Proteins were separated by SDS–PAGE and then transferred on membranes. The membranes were blocked for 1 h with 5% BSA and incubated overnight at 4 °C with primary antibodies specific for MMP-13 (1:2000), COLX (1:1000, catalog A6889), IL6(1:500), ß-actin (1:2000, catalog AF7018), and GAPDH (1:2000, catalog AF5009). After washing away unbound primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:5,000; Bio-Rad, 1706515) and anti-rabbit secondary antibodies (1:10,000, catalog SA00001-2) and protein bands were detected using an ECL detection system.
GABA Assay
The frozen samples of small intestinal content were diluted (10%, (w/v)) in PBS and homogenized by vertex. The samples were then centrifuged for 20 minutes at 3000rpm. A commercially available enzyme immunoassay was then used for the quantitative determination of GABA by ELISA (MM-0442M2). Quantification of unknown samples was calculated by comparing their absorbance with a standard curve prepared with known standards and results were standardized to individual sample weights.
The whole blood was drained out via eye rapture and allowed to coagulate naturally. Centrifuged at 3000rpm for 20 minutes, the supernatant was collected and used for ELISA. The fecal water was also used to determine GABA levels. The fecal samples were collected and mixed with distilled water and vertex until slurry was made. Centrifuged like above and the supernatant was used for GABA assay (supplementary figure S3B).
Statistics
All statistical analyses were performed with Prism 7 software from GraphPad (San Diego, CA, USA). Statistical significance was determined to be P < 0.05. To determine the average and standard deviation in the model and characterization methods; all in vivo data expressed as the mean and each data point signifies an individual mouse. All in vitro data expressed as averages ± standard deviation. Statistical significance (P values) was determined by or one-way ANOVA (Tukey’s multiple-comparisons test or Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli).