Antler preparation
Mature deer antlers were obtained from Beijing Tong Ren Tang Chinese Medicine Co., Ltd., (Beijing, China). A total of 200 g antler was dried, milled and sliced, and then extracted 4 times by dissolving in 1 l distilled water and boiling for 5 h. Following filtration with 3 levels of sterile gauze (TZSB; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the liquid containing the antler extract was concentrated at 20% by boiling for 5 h and stored at 4°C for 3 days.
Animals
A total of 30 male New Zealand rabbits, 4 months old and weighing 2.6–3.2 kg, were obtained from the Experimental Animal Center of the Affiliated Hospital of Inner Mongolia Medical University (Hohot, China). Rabbits were housed in an animal chamber maintained at 22±2°C with a relative humidity of 50±5% and a 12/12 h light/dark cycle. Access to food and water was ad libitum. All animal experiments in the present study were approved by the Animal Care and Use Committee of the Affiliated Hospital of Inner Mongolia Medical University (Hohhot, China) and followed the ethical guidelines set by the European Community guidelines (38).
Establishment of SAMFH rabbit model
SANFH rabbit models were established following a previously described method (39,40). The 30 healthy New Zealand rabbits were randomly divided into 5 groups (n=6): A control, ANFH, ANFH + antler (250 mg/kg), ANFH + antler (500 mg/kg) and ANFH + antler (1,000 mg/kg) group. All rabbits received an injection of 10 ml/kg horse serum (100%; Thermo Fisher Scientific, Inc., Waltham, MA, USA) via the ear vein for 2 weeks. Subsequently, 6 ml/kg/day horse serum was injected for 2 days.
Rabbits in the experimental groups received an intraperitoneal injection of 20 mg/kg methylprednisolone (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) twice a week, over a 2-week period. Rabbits in the control group were intraperitoneally injected with the same volume of saline. Subsequently, 200,000 U penicillin was injected into the buttock of each rabbit. After 2 weeks, 1 rabbit in each group was randomly selected to confirm whether the SANFH model was successfully established by performing a femoral head histopathological examination. The femoral head was separated and fixed with 2% glutaraldehyde at 4°C overnight (Beijing Solarbio Science & Technology Co., Ltd.), decalcified with 5% nitric acid (Beijing Solarbio Science & Technology Co., Ltd.), dehydrated with a graded series of ethanol (70, 80, 90, 95 and 100%) embedded in paraffin and cut into 5 µm slices using a microtome (HM 355S; Thermo Fisher Scientific, Inc.). Sections were subsequently dewaxed with xylene, rehydrated with a graded series of ethanol (100, 95, 90, 80 and 70%) and stained with hematoxylin at 25°C for 10 min and eosin at 25°C for 1 min. Stained sections were observed under a light microscope (magnification, ×200).
Following confirmation of successful establishment of the SANFH model, the antler extract was dissolved in distilled water to form final concentrations of 250 mg/kg, 500 mg/kg and 1,000 mg/kg. The different concentrations of antler extracts were administered to rabbits in the ANFH + antler (250 mg/kg), ANFH + antler (500 mg/kg) and ANFH + antler (1,000 mg/kg) groups, respectively, every day for 60 days by intraperitoneal injection. Rabbits in the control and ANFH groups were intraperitoneally injected with the same volume of saline.
CT and triglyceride determination
Following treatments, all rabbits were anesthetized with a combination of xylazine (6 mg/kg) and ketamine (40 mg/kg; both Seebio Biotech Co., Ltd., Shanghai, China) according to previous report and sacrificed by cervical dislocation (41). Blood was collected from the ear vein in EDTA tubes and centrifuged at 4°C and 5,000 × g to separate serum. CT and triglyceride concentrations in the blood samples of all rabbits were measured using an automatic biochemistry analyzer (Siemens AG, Munich, Germany).
Histopathological examination
Femoral heads, including the metaphyses and thighbones, of all the rabbits were collected and cut into sections. Sections were fixed in 2% glutaraldehyde (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 25°C for 24 h. Sections were then embedded in paraffin and cut into slices 5 µm thick. Following deparaffinization and rehydration with graded ethanol, sections were stained with hematoxylin and eosin (Thermo Fisher Scientific, Inc.). Histopathological changes of the femoral heads were observed under a light microscope (Olympus Corporation, Tokyo, Japan).
Osteoblast culture
A total of 2 male newborn New Zealand rabbits, 10 days old, weight 20.17±1.03 kg, were obtained from the Experimental Animal Center of the Affiliated Hospital of Inner Mongolia Medical University and sacrificed as described above. Rabbits were housed in an animal chamber maintained at 22±2°C with a relative humidity of 50±5% and a 12 h light/dark cycle with access to food and water ad libitum. Primary osteoblasts were obtained from newborn rabbit calvarias. Tissues were digested with trypsin 3 times and incubated with type I collagenase and 0.25% trypsin-EDTA in Hank's buffer solution (all Thermo Fisher Scientific, Inc.). The tissues were then cut into pieces <0.5 mm3 and placed on the inner surface of the dish to release the cells. Cells were cultured with Dulbecco's Modified Eagle medium (DMEM; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C in 5% CO2 for 24 h. The tissues were then washed with 1X PBS to discard unattached cells. The medium was replenished every 2–3 days and cells were passaged until 90% confluence was reached.
Antler serum treatment
Osteoblasts were seeded at 1×106/ml and cultured in a 96-well plate. Cells from rabbits in the 5 groups including the control, ANFH, ANFH + antler (250 mg/kg), ANFH + antler (500 mg/kg) and ANFH + antler (1,000 mg/kg) groups and there were 3 replicates from each group. A total of 40 µl methylprednisolone solution (1 µmol/l) was added to the ANFH, ANFH + antler (250 mg/kg), ANFH + antler (500 mg/kg) and ANFH + antler (1,000 mg/kg) groups, while 40 µg/DMEM was added to the cells in the control group. Following incubation for 24 h, medium was replenished and serum samples collected from the rabbits (1:40) were added to the corresponding group of cells for 24 h.
Western blotting
Total proteins from the femoral head tissue or the cells treated with antler serum were extracted using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) and measured using the BCA method (Thermo Fisher Scientific, Inc.). A total of 75 µg protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% separating gel and 5% stocking gel. Following SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane (Merck KGaA). The membrane was blocked with 5% skimmed milk at 25°C for 2 h. Rabbit anti-11β-HSD1 and rabbit anti-11β-HSD2 primary antibodies (1:1,000, Wuhan Sanyang Biotechnology, Wuhan, China) were incubated with the membrane at 4°C overnight. Membranes were washed with PBS 3 times for 7 min. Membranes were incubated with goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove PA, USA) at 25°C for 1 h. β-actin was used as an internal control. An electrochemiluminescence system (Thermo Fisher Scientific, Inc.) was used for exposure.
Alkaline phosphatase (ALP) detection
Following treatment with antler serum for 72 h, 20 µl 0.05% Triton X-100 was added to each well and incubated at 25°C for 5 min to induce cell lysis. An ALP assay kit (AP0100-1KT; Sigma-Aldrich; Merck KGaA) was used to detect ALP levels in the blood serum and osteoblasts. A total of 980 µl (blank) and 960 µl (test and control) of reaction buffer was pipetted into cuvettes and 20 µl of 0.67 M pNPP solution was added to each cuvette and equilibrated at 37°C. Subsequently, 20 µl of sample was added to each test cuvette, and 20 µl of diluted ALP solution was added to the enzyme control cuvette. Cuvettes were mixed by inversion and the A405 nm was recorded (SpectraMax i3X plant reader; Molecular Devices, LLC, Sunnyvale, CA, USA). Obtained the maximum linear rate (∆A405 nm/minute).
Measurement of cell proliferation
Following treatment with antler serum for 72 h, 10 µl cell counting kit-8 (Thermo Fisher Scientific, Inc.) was added to each well and incubated at 37°C for 20 min. An automatic biochemistry analyzer was used to measure the optical density (OD) value at 450 nm.
Cell cycle analysis
Following treatment with antler serum for 72 h, cells were collected and fixed in pre-cooled 70% ethanol at 4°C overnight. A Cell Cycle and Apoptosis Analysis kit (C1052, Beyotime Institute of Biotechnology) was used to assess the cell cycle according to the manufacturer's protocol. A flow cytometer (Cytomics FC 500; Beckman Coulter, Inc., Brea, CA, USA) was used to detect and determine the proportion of cells in each phase of the cell cycle.
Statistical analysis
All data were analyzed using SPSS 19.0 software (IBM Corp, Armonk, NY, USA) and presented as the mean ± standard deviations. One-way ANOVA followed by Dunnett's test was used for comparison and analysis between groups. P<0.05 was considered to indicate a statistically significant difference.