Preparation of sample solutions
A total of 4.0 g WO sample was extracted with 40 mL methanol using Soxhlet extractor for 12 h until the complete extraction. Afterwards, the solution was filtered and evaporated. The crude remnants were uniformly mixed with 5 mL methanol for further analysis.
Analysis of liquid chromatography-mass spectrometry (LC–MS)
LC–MS was performed using Waters Acquity UPLC system (Waters, Milford, MA, USA) coupled with TOF mass spectrometer (Bruker, Bremen, Germany). The results were acquired using Hystar software (Bruker). Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) served as the stationary phase, 0.1% formic acid in pure water (A) and 0.1% formic acid in acetonitrile (B) with a flow rate of 0.4 mL/min were utilized as the mobile phase. The conditions were as follows: 0–1 min, 5% B; 1–4 min, 20% B; 4–6 min, 25% B; 6–10 min, 40% B; 10–12 min, 60% B; 12–15 min, 100% B; 15–16 min, 40% B; 16–20 min, 5% B. The column temperature was maintained at 40 °C, and the injection volume of each sample was 2 μL.
The mass spectrometer was operated in the negative mode, the scanning range of m/z from 100 to 3000. The mass conditions were as follows: gas (N2) flow rate was 8 L/min and the temperature was 180 °C; the capillary voltage was 4500 V.
Animal model and fracture surgery
Fifty-Four New Zealand white male rabbits (provided by Animal Center of Guangxi University of Chinese Medicine, Guangxi, China), which were 10 months old with body weight ranging from 2.0 to 2.5 kg, were involved in this study. All rabbits were kept singly and fed with a standard laboratory diet and tap water under climate-controlled conditions (25 °C; 55% humidity; 12 h of light alternating with 12 h of darkness). A small patch of skin dermis was excised from the back and the right forelimb of each rabbit. After the injection of anesthetic (30 mg/mL, sodium pentobarbital sodium solution, Sigma-Aldrich, MO, USA) into the vein of the rabbit ear, all the following procedures were performed in disinfection area with rabbits in supine position. A 1.5 cm longitudinal incision was made to expose the middle part of radius. The periosteum was cut and peeled off, and then the radial shaft was exposed. A fracture model was established by bone sheared with peripheral vessel, muscle and cartilage tissue preserved, keeping the ulna integrate. The research was approved by the ethical committee of Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine. The Minimum Standards of Reporting Checklist contained details of the experimental design, statistics and resources used in this study (Additional file 1).
Grouping and treatment
The rabbits were categorized into three groups (n = 18 each group). Rabbits received normal saline only were in control group, those treated with Yunnan Baiyao (YB, Yunnan Baiyao group Co., Ltd., Yunnan, China) were regarded as YB group, and WO group consisted of rabbits treated with WO (Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine, Guangxi, China). Five milligram/kilogram of YB and WO were externally applied on the fracture three times a day with bandage being put on and dressings were changed every 3 days. The fracture parts of control group were bandaged without any drug applied.
Preparation and treatment of specimens
The rabbits were killed at different time point (3, 7, 14, 21, 28, 35 days after operation) by being injected 10–15 mL air into the syringes. After the skin of the fracture was cut immediately, the surrounding muscles, tendons and other soft tissues were stripped. Hyperplastic soft tissue, fibrous and bones callus were halved. One part was fixed in 10% neutral formalin solution and prepared for the following HE stain and IHC analysis; other part was wrapped by tin foil and stored at − 80 °C for western blot. The sacrifice of rabbits and the preservation of the specimens were completed within 10 min to prevent protein degradation and cell autolysis rupture.
Radiography analysis
The procedure of X-ray and micro-CT observation was similar to the previous studies [17, 18]. Briefly, fracture morphology was detected by digital X-ray equipment (Siemens, Chicago, USA). Three-dimensional (3D) images were rendered. A laboratory micro-CT scanner (Scanco Medical AG, Brüttisellen, Switzerland) and the VGStudio MAX software (Dürr, Bietigheim-Bissingen, Germany) were used for the micro-CT examination. The proportion about total void area of all area in transverse section image was calculated, and the fraction of mineralized callus was quantified.
Alkaline phosphatase (ALP) assay
At each time point, 4 mL ear margin venous blood stewed in the bio-tube for 30 min was centrifugalized at the speed of 2500 r/min. Then, the upper layer of the serum was removed and ALP activity of the lower layer was detected with ALP assay kit (Sigma-Aldrich, St. Louis, MO, USA). The measurement was performed using Beckman LX 20 Analyzer (Beckman Coulter, Inc., CA, USA).
Hematoxylin–eosin (HE) staining
The bone specimens were fixed in 10% formaldehyde solution for 48 h and decalcified in 15% ethylene diamine tetraacetic acid (EDTA) for 3 weeks. Then, the decalcified samples were embedded in paraffin, cut into 4 mm-thick sections and dehydrated with gradient ethanol. After that, the sections were stained with HE and assessed with the optical microscope.
Immunohistochemistry
The bone specimen sections, which were dewaxed and rehydrated with xylene solution and gradient ethanol, were then hot fixed with 0.01 M Citrate repair solution (pH 6.0) for 2 min in hyperbaric condition. 100 μL anti-VEGF receptor 1 (rabbit, 1: 800, ABcam, US) was added and incubated overnight. Then the sections were incubated with horseradish enzyme labeled goat anti-rabbit IgG (Beijing Zhong Jin Jinqiao company, Biological Technology Co., Ltd., Beijing, China) for 40 min. Afterwards, the sections were treated with 3,3′-diaminobenzidine (DAB) coloring agent for 20 s, hematoxylin re-infection for 1 min and dehydrated. The figures were analyzed with the MIQAS medical image quantitative analysis system (Motic China Group Co., Ltd., Guangdong, China), 5 fields of vision were selected randomly with about 100 cells in each field. In each field, mean optical density, the positive reaction area and total area of cells were measured (cells area was 0.15 mm2). Subsequently, the average rate of positive area was calculated.
Western blot
The harvested bone specimens were cut into pieces, weighed and grinded into powder in liquid nitrogen with radio-immunoprecipitation assay (RIPA) buffer. The total proteins were extracted using protein extraction kit (Millipore, Billerica, MA, USA). Equal weight of proteins were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes and then blocked with 5% skim milk at room temperature for 1 h. Primary antibody anti-rabbit F-box protein 32 (FBXO32) diluted with Tris buffered saline solution (TBST, 1: 2500) was added to the membranes and incubated at 4 °C overnight. The membranes were then incubated with goat monoclonal anti-rabbit, the secondary antibody marked with horseradish peroxidase (HRP, 1: 2000), for 2 h at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. Signal detection was performed using Chemiluminescence reagent kit (Amersham, IL, USA). The antibodies mentioned above were all bought from Beijing Zhong Shan Jinqiao Biological Technology Co., Ltd. (Beijing, China).
Enzyme linked immunosorbent assay (ELISA)
The bone specimens were cut off and rinsed with pre-cooled phosphate buffered solution (PBS) (0.01 M, pH = 7.4) to remove residual blood and weighted. The specific volume of PBS (1:9 w/w) was added to a glass homogenizer and sufficiently grinded on ice. After 5000g homogenate were ultrasonically broken and thawed, it was centrifuged for 10 min and the supernatant was collected. Rabbit VEGF specific enzyme-linked immunoassay kit (TWp027731, TongWei, Shanghai, China) was used to detect the VEGF expression level. Manufacturer’s protocol was strictly followed and the optical density (OD) was measured at 450 nm.
Statistical analysis
All statistical analyses were performed by SPSS 19.0 software (SPSS, IL, USA). The significant differences in numerical data (mean ± SD) were estimated with the analysis of variance (ANOVA). Differences between two groups were analyzed by unpaired t tests. P value less than 0.05 was defined as statistical significance.
