From June to September 2021, the patients who were admitted to our hospital for bone mineral density(BMD) tests were collected. Exclusion criteria: (1)Diabetes, Cushing syndrome, Thyroid or parathyroid dysfunction, osteomalacia, rheumatoid arthritis, multiple myeloma, osteoarthropathy, osteogenesis imperfection, and other disorders that disrupt bone or calcium metabolism are all examples of disorders that disrupt bone or calcium metabolism; (2)Have taken estrogen, steroid hormones, calcitonin, parathyroid hormone, bisphosphate, fluoride, vitamin D, anticonvulsant, diuretic, or other bone-related medicines in the last 6 months; After removing the influencing factors that have a significant impact on the results of a bone mineral density test, 58 cases were selected for retrospective analysis and divided into osteoporosis group (OS, n = 31) and non osteoporosis group (NOS, n = 27) according to the diagnostic criteria of dual energy X-ray bone mineral density (DXA) of Chinese osteoporosis (2018). The ethics review committee of The Second Affiliated Hospital of Zhejiang Chinese Medical University[2019-KL-007-01] has accepted this research design. This study is exempt from requiring written consent because it is the gathering or investigation of previously archived data, records, or diagnostic specimens, and these are open-access resources.
2.2. BMD measures
The lumbar spine and right hip BMD were assessed by DXA scanner(General Electric Company). The lumbar spine measurement range includes two to four vertebral bodies, the femoral neck, and the femoral trochanter. All lumbar imaging pictures were reviewed independently by two competent radiologists to determine the presence of a vertebral fracture in each case. If there is a disagreement, it must be resolved through conversation.
2.3. Complement C3 level measures
All individuals had 5ml of fasting venous blood collected, which was kept at room temperature for 0.5 hours before centrifuging at 3000 G at the speed of relative centrifugal force for 10 minutes, serum separation, and computer detection within 2 hours. According to the manufacturer's instructions, ELISA was used to measure the protein expression levels of Complement C3.
2.4. Cell culture
Raji and MG63 cells (ATCC, Shanghai, China) were grown at 37 ℃ in 5% CO2 in DMEM (Gibco) supplied with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The 293A cell line (ATCC, Shanghai, China) was used to construct a lentivirus containing shC3.
2.5. Construction of recombinant lentivirus
The following sequences were used in C3 siRNA:
Both the forward and reverse sequences were inserted into PLKO.1 (Addgen, Shanghai). Lentiviruses were made by utilizing Lipofectamine 2000(Invitrogen) to triple transfect 90 percent confluent 293A cells with pLKO.1-shC3, psPAX2, and pMD2G (Addgen. Shanghai). After transfection, the medium was replaced with full media for 4–6 hours, and the viral solution was collected after 72 hours. For lentivirus transfection, Raji cells were planted in 24-well plates and divided into three groups, each with 3 wells: blank control, empty vector, and recombinant lentivirus interference.The recombinant lentivirus interference group was infected with C3-shRNA [shC3 (forward)/(reverse)], the empty vector group was infected with control shRNA (shCon), and the blank control group was added PBS in equal amount to other groups for 48 h in serum-free medium.Subsequently, the medium was replaced with regular DMEM containing 10% fetal bovine serum, and the cells were cultivated for 96 hours at room temperature in 5% CO2. The C3 primer in the vector was sent to Shanghai Meiji Biological Co, Ltd. for sequencing. The sequencing results were compared with the original sequence, and there were no base deletions, mutations, and shifts, indicating that the vector had been successfully constructed.
2.6. Co-culture of Raji and MG63 cells in vitro
MG63 cells in the logarithmic growth phase were digested with trypsin (Solarbio, Beijing, T1300-100) and prepared as cell suspension after counting under the microscope. Three groups of cells were separated from the cell suspension, with 300 µl for each group in a 24-well culture plate, inoculated in 3 wells per group, 1×104cells / well, and co-cultured with transfected Raji cells at 37 ℃.
2.7. RNA extraction and real-time PCR (RT-PCR)
Total RNA was extracted with Invitrogen's Trizol reagent, and cDNA was made following the manufacturer's instructions. The primer sequences for the test were as follows:
C3, forward: 5'-CACCAGCAGACCGTAACCATC-3'; reverse: 5'-GCAGCCTTGACTTCCACTTCC-3'
OPG, forward: 5'-ATCAGTTGGTGGGAATGAAG-3'; reverse: 5'-GGGATGACACAGAAGATAGTAG-3'
GAPDH, forward: 5'-GGAGTCTACTGGCGTCTTCAC-3'; reverse: 5'- ATGAGCCCTTCCACGATGC − 3'
The amplification conditions were: initial denaturation at 95 ℃ for 10 min, followed by 40 cycles of denaturation at 95 ℃ for 15 sec and annealing extension at 60 ℃ for 15 sec. SYBR green dye (Invitrogen) was used to measure mRNA levels, and the comparative Ct method was used to calculate relative mRNA levels. GAPDH was used to standardize all reactions. The relative differences between PCR findings were calculated using Comparative Ct.
2.8. Western blotting analysis
The cells were lysed until completely cracked in RIPA lysate containing protease and phosphatase inhibitors (BYL40825, JRDUN, Shanghai, China), then ,centrifuged at 12000 rpm for 10 minutes at 4 ℃. The total protein concentration in the supernatant was determined using a BCA protein analysis kit(Beyotime, Shanghai, China). SDS-PAGE was used to separate the same amount of protein (25g) and transfer it to PVDF membranes (Bio-Rad). The membrane was blocked for 1 hour with 4% skimmed milk before being incubated overnight at 4 ℃ with the primary antibodies C3 (1:2000, Abcam), and GAPDH (1:2000, Abcam). The membrane was incubated in 37 ℃ for 1 hour with HRP-conjugated antibodies (Wuhan Boster Bioengineering Co, Ltd.). and then the enhanced ECL chemiluminescence reagent (Millipore) and GelDoc imaging system (Millipore) were used to quantify immunoreactive bands.
2.9. MG63 cell viability
After the corresponding incubation times (3 h, 6 h, 12 h, 24 h, 48 h, 72 h), Cell Counting Kit-8 (CCK-8) and serum-free essential basic medium were mixed in a 1:10 volume ratio, each test well received 300 ul of mixture, which was then incubated for 1 hour at 37 ℃ in a 5 percent CO2 incubator. Absorbance at 450 nm was used to determine cell viability.
2.10. MG63 cell apoptosis analysis
To explore cell apoptosis,annexin V fluorescein isothiocyanate (FITC)-conjugated and propidium iodide were used to label the MG63 cells.The cells were then examined using a FACSCalibur flow cytometer (BD Biosciences).
2.11. ALP activity assay
Following the manufacturer's instructions, an ALP assay kit(Sigma) was used to detect ALP activity. In each well of a 24-well plate, cells were mixed with the substrate buffer. The reaction was terminated with 0.2 M sodium hydroxide after 15 minutes of incubation at 37 ℃. A microplate spectrophotometer was used to test sample absorbance at 405 nm.Following the manufacturer's directions, the amounts of proteins in cell lysates were measured using a BCA protein assay kit. Enzyme activity was measured in units per gram of protein. At pH 9.8 at 37 ℃, the release of 1 nmol p-nitrophenol per minute was defined as one unit of enzyme activity.
2.12 Statistical analysis
The data were all given as means ± standard deviation (mean ± SD). An independent sample t-test was used to compare the mean of the two groups of measurement data. One-way ANOVA with post hoc Tukey's multiple comparison tests was used to assess differences between data from three groups. SPSS 20.0 was used for the analyses (IBM). Statistical significance was assigned to values with p < 0.05.