1.1 Research subjects
1.1.1 Selection of research subjects
10 patients with osteoporosis and osteoporotic fractures who were hospitalized in the Department of Geriatrics of General Hospital of Tianjin Medical University from January 2017 to December 2017 were randomly recruited into the osteoporosis group. 5 health people without osteoporosis were randomly recruited for the control group. The general data of normal and osteoporosis group was shown in Table1. All participants in the experiments were matched for age and gender. This study was approved by the medical ethics committee of Tianjin Medical University General Hospital on 2015 according to the Declaration of Helsinki, and patients provided informed consent before the experiments.
The inclusion criteria for the osteoporosis group included (1) patients who were diagnosed with osteoporosis by dual-energy x-ray absorptiometry or with osteoporotic fractures by imaging and (2) non-violent fractures.
The exclusion criteria were as follows: (1) patients with fractures caused by violence or trauma and (2) patients with other bone diseases, such as osteomalacia, renal osteodystrophy or other metabolic bone diseases or bone tumors.
1.1.2 Analysis of the demographics data of the selected subjects
The sex, age, height, weight and blood pressure of each selected patient were collected to calculate the mean age, mean body mass index (BMI), mean systolic blood pressure (SBP) and mean diastolic blood pressure (DBP). Fasting blood samples were obtained to test the serum levels of total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), serum creatinine (CREA), blood Urea (UREA), blood uric acid (UA), fasting blood glucose (FPG), blood calcium (Ca), blood phosphorus (P), blood potassium (K), blood sodium (Na), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), 25-hydroxyvitamin D, osteocalcin (OC), parathyroid hormone (PTH), procollagen type I N-terminal peptide (PINP) and C-terminal telopeptide of type I collagen (CTX).
1.2.3 Measurement of bone density and diagnosis of osteoporosis
The medical staff of the Bone Densitometry Section of the Department of Geriatrics at the General Hospital of Tianjin Medical University used dual-energy X-ray absorptiometry (Lunar DPX Prodigy, GE Healthcare, USA) to measure the bone density of the 2nd to the 5th lumbar vertebrae (L2-L4) and the hip (left femoral neck, trochanter and Ward’s triangle) of the patients in each group. The total bone density (g/cm2) was calculated to obtain T-Scores. The diagnostic criteria for osteoporosis were based on T-Scores ≤ –2.5 in any of the following sites: lumbar vertebrae, femoral neck or total hip.
1.2 Fecal DNA extraction and testing
Stool specimens were collected from the patients in the osteoporosis and healthy control groups using a sterile specimen container. Approximately 1 gram of feces was placed in the sterile stool specimen container, and the container was immediately sealed and stored in liquid nitrogen. The samples were sent to the laboratory within 1 hour for storage in a –80 ºC freezer. A cador Pathogen 96 QIAcube HT Kit (QIAGEN, USA) was used to extract bacterial genomic DNA from the stool samples. The concentrations of the genomic DNA samples (in 2 µl) were measured with an ultraviolet-visible spectrophotometer. Pure DNA should exhibit an obvious absorption peak at an optical density of 260 nm (OD260) with an OD260/OD280 ratio of approximately 1.8.
Construction of a high-throughput sequencing library and Illumina-based sequencing using a MiSeq instrument were performed by GENEWIZ (Suzhou, China). The sequencing library was constructed using the MetaVxTM library construction kit (GENEWIZ, Inc., South Plainfield, NJ, USA). Custom polymerase chain reaction (PCR) primers were used to amplify two highly variable regions of the bacterial 16S rDNA gene (the V3 and V4 regions) using 30–50 ng of DNA as the template. The V3 and V4 regions were amplified using a forward primer with the sequence “CCTACGGRRBGCASCAGKVRVGAAT” and a reverse primer with the sequence “GGACTACNVGGGTWTCTAATCC”. Library quality was evaluated using and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., USA), and the library concentrations were determined using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). After the DNA library was mixed, double-end sequencing (PE, 2×300 bp) was performed using an Illumina MiSeq (Illumina, San Diego, CA, USA) instrument according to manufacturer’s instructions, and the sequence data were analyzed with MiSeq Control Software (MCS) provided by MiSeq.
1.3 Cell culture
Mice osteoblasts MC3T3-E1 were purchased from the Tianjin institute of orthopaedics and tranmatology and cultured in α-MEM with 10% fetal bovine serum (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA,USA) at 37˚C and 5% CO2.Mice osteoclasts RAW 264.7 were obtained from the Tianjin Medical University and cultured in DMEM (High Glucose) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA,USA), penicillin (100 U/ml) and streptomycin (100mg/ml) at 37˚C and 5% CO2.
1.4 Bacterial culture and supernatant extraction
Cryogenic vials containing the lactobacillus acidophilus (LABS)and Lactobacillus rhamnosus (LGG) solution stored in a liquid nitrogen tank were removed and completely thawed at room temperature. One milliliter of the bacterial solution was pipetted into 100 ml of the prepared deMan, Rogosa and Sharpe (MRS) medium in a sterile laminar flow work bench, and the inoculated culture dishes were incubated at 37 °C. After 72 hours, the bacterial growth was stable and the subsequent experiments were carried out. Culture medium containing LABS andLGG was transferred to a centrifuge tube and centrifuged in a high-speed refrigerated centrifuge at 4000 r/min for 10 min at 4 °C. After centrifugation, the supernatant was filtered through a membrane (0.22 μm) 3 times to obtain bacterial supernatant.
1.5 Osteoblast and osteoclasts proliferation
MC3T3-E1 and RAW 264.7 cells (2.5 ×103 per well) treated with bacterial culture supernatant (mixed at ratios of 1:20, 1:50 and 1:100) or sodium butyrate (0.5 mM) were inoculated in 96-well plates and incubated for 48 h. Then 20 μL of tetrazolium dye (MTT) solution (5 mg/mL) was added to each well, and the plates were incubated at 37 °C in 5% CO2 for 4 hours. The medium was aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to the wells. The plates were then stirred for 10 min. After the crystals were completely dissolved, the OD values were measured at 490 nm using a microplate reader. Phosphate buffer saline (PBS) was as control and added in 96-well plates.
1.6 Determination of ALP activity and OCN
MC3T3-E1 cells were inducted by induction medium with vitamin C (50 mg/L) and β-sodium glycerophosphate (10 mM) or uninduction medium without vitamin C and β-sodium glycerophosphate, and LABS mixed at a ratio of 1:20 and 0.5 mM sodium butyrate medium were collected after induction for 7 days, respectively. PBS was as control and added in plates. The media were centrifuged for 10 min at 1500 r/min, and the supernatants were collected. The alkaline phosphatase (ALP) activity and osteocalcin (OCN) concentration were measured by ELISA assay according to the manufacturer’s instructions.
1.7 Realtime PCR assay
MC3T3-E1 and RAW 264.7 cells are treated in medium containing induction medium which MC3T3-E1 cells are inducted by vitamin C (50 mg/L) and β-sodium glycerophosphate (10 mM), RAW264.7 cells are inducted by 50 μg/L RANKL, and LABS mixed at a ratio of 1:20 or 0.5 mM sodium butyrate medium were collected. PBS was as control and added in plates. The total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manual’s instructions. Then cDNA was obtained by oligo-dT primers or stem-loop reverse transcriptase (RT) primers by RT-PCR kit (Takara Bio Inc.), and mRNA expression of the target genes were detected using real-time PCR assay (Applied Biosystems 7500 Real-Time PCR). The RNA was used as a template for complementary DNA (cDNA) synthesis with the following conditions: denaturation at 95℃ 10min, followed by 40 cycles at 95℃ 15s, 60℃ 60s, 60℃ 15s and a fnal elongation at 95°C for 15 s. RT-qPCR was performed to detect wingless-type MMTV integration site family member 2 (WNT2), β-catenin gene (CTNNB1) and receptor activator for nuclear factor-κ B (RANK), and GAPDH was used as the internal reference gene. The quantified results were calculated using the 2−ΔΔCq method (13). The full details of the primers used in these experiments are shown in Table 2
1.8 Effects of bacterial supernatant on osteoblast and osteoclasts culturing and induction
MC3T3-E1 cells are inducted by vitamin C (50 mg/L) and β-sodium glycerophosphate (10 mM), RAW264.7 cells are inducted by 50 μg/L RANKL. Above induction medium are refreshed every 3 days.
The extracted LABS and LGG bacterial supernatant was mixed with at ratios of 1:20 to generate bacterial supernatant culture medium and 0.5 mM sodium butyrate (represent butanoic acids) in which MC3T3-E1 and RAW 264.7 cells were incubated at 37 °C in 5% CO2. The bacterial supernatant culture medium and sodium butyrate was replaced every 48 hours. PBS was as control and added in plates.
ALP/ Alizarin red staining was performed on the MC3T3-E1 cells after 7 days of incubation in induction medium. Two milliliters of citric acid concentrate was added to 100 ml of deionized water and then mixed with acetone (2:3 ratio) to produce a fixing solution. The prepared fixing solution was added to the culture plates to fix the osteoblasts for 3 min. Substrate was applied dropwise to completely cover the fixed osteoblasts, and the plates were incubated at 37 °C for 15 min in the dark before being washed with distilled water for 5 min. The cells were stained with Alizarin red solution for 7 min and then washed with distilled water for 5 min. The slides were dried at room temperature and sealed. The cells were observed and photographed under a microscope.
Tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 osteoclasts was performed after treating them with the same method for 5 days.
1.9 Statistical Analysis
Data was represented by mean ± SD and analyzed by SPSS 11.0 software. The one way ANOVA analysis tukey’s posthoc was used for general measurement data. R software (Version, 2.15.3) was used to nonmetric multidimensional scaling (NMDS) analysis. P < 0.05 was defined as a significant difference.