Reagents and animals

Jin-Tian-Ge (JTG) were prepared from skeletons of several farmed animals, including Cervus elaphus Linnaeus, Cervus nippon Temminck, Capra hircus Linnaeus, and Sus scrofa domesticus Linnaeus by Ginwa Enterprise Group INC, Xi’an, China, and their ingredients includes 18% calcium, 8% phosphorus, peptides and proteins.

The regents used in this study were estradiol valeratse (E2V, Bayer Health Care Co., Ltd., Guangzhou, China); penicillin (Shandong Lukang Pharmaceutical Co., Ltd., Shandong, China); the bio-markers for bone matebolism including commercially enzyme linked immunosorbent assay (ELISA) kit of deoxypyridinoline cosslinks (DPD), procollagen I N-Terminal propeptide (PINP), osteoprotegerin (OPG), osteocalcin (OCN), C-Telopeptide of type I collagen (CTX-I), tartrate resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa B ligand (RANKL), and biochemical kit of calcium (Ca) and alkaline phosphatase (ALP) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); All other reagents were of analytical-grade purity, and purchased from Sinopharm Chemical Reagent Co., Ltd.

Fetal bovine serum (FBS), α-Modified minimal essential medium (α-MEM), phosphate buffered saline (PBS) and penicillin/streptomycin were obtained from Gibco company (USA). Ascorbic acid, β-glycerophosphate, dexamethasone, Acid Phosphatase Kit, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from Peprotech (EC, USA). Antibodies against BMP2 and CtsK were purchased from Abcam (Cambridge, MA, USA). Antibodies against Wnt3a, IkBα, c-Fos, MMP9 and TRAF6 were purchased from Boster Biological Technology (Wuhan, China). Antibodies against Smad4, Smad1/5/9, Runx2, LRP5, β-catenin, P-β-catenin, glycogen synthase kinase-3 β (GSK-3β) and p-GSK-3β, p65, p-p65, NFATc1 and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). The BCA Protein assay kits was from Biyotime (Shanghai, China).

Sixty female Wistar rats ages 10 weeks were purchased from Sippur Will Kay Company and housed at the Experimental Animal Center of Zhejiang Chinese Medicine University (Hangzhou, China, Certificate No. SYXK 2018-0012). The rats were acclimatized on a 12 h light–dark cycle under a temperature of (24 ± 0.5) ℃ and humidity of (47.5 ± 2.5)%. All animals were handled according to National Institute of Health (NIH) guidelines on the ethical use of animals, and received humane care.

Determination of amino acid contents in JTG

The samples of JTG were treated according to hydrolysis method of proteins provided in protocols of Hitachi L-8900 Amino Acid Analyzer. The amino acid in JTG were determined by using Hitachi L-8900 Amino Acid Analyzer (Tokyo, Japan). The chromatographic column is 4.6 mm I.D. × 60 mm L packed with Hitachi custom ion exchange resin (Partical size: 3 µm). The detective wavelength were 570 nm and 440 nm. The elutes and gradient elution procedures were in accordance with the protocols of this instrument.

Animal experimental protocolGrouping and drug administration of rats

Sixty rats were equally randomized into six groups, with ten rats in each group. The rats in Sham group were subjected to cut off some fat tissue close to the ovaries, and the other fifty rats were removed bilateral ovaries as previously described (OVX), and randomly divided into five groups, including OVX model group, positive control group and low, middle and high dose of JTG treatment groups [19]. One week after ovariectomy, the rats in Sham and OVX group were orally administered with distilled water containing 0.5% CMC-Na (1 mL/100 g body weight), the OVX rats in positive control group were orally administered with E2V (0.2 mg/kg body weight, once every day), the OVX rats in JTG treatment groups were orally administered with different doses of JTG (180, 360 and 720 mg/kg body weight, once every day), respectively. The rats were treated orally with vehicle, E2V and JTG for 12 weeks. The body weight of rats was weighed every week to adjust the dosage of E2V and JTG. At the end of the experiment, all rats were sacrificed by cervical dislocation after serum samples collection. The uterus was immediately removed and weighed. This experiment was approved by the Bioethic Committee of the Zhejiang University of Traditional Chinese Medicine (Approval NO. IACUC-20190311-09), and the procedures of the experiment were strictly according to generally accepted international rules and regulations.

Determination of bio-marker levels in serum and urine of rats

Rat blood was collected and allowed to curdle 2 h at room temperature. Serum was collected and frozen at – 80 °C for biochemical markers assay. The levels of bio-markers related with bone metabolism including OCN, OPG, PINP, CTX-I, RANKL, TRACP and ALP were blindly measured with ELISA and biochemical kits following the standard kit procedure.

Rats in each group were placed in metabolic cages before sacrifice, and fasted and free drink water, and the urine were collected for 12 h. Urine was frozen at – 80 °C for biochemical markers assay. The biomarker levels of bone metabolism including DPD and Ca were measured with ELISA and biochemical kits according to the kit protocols.

Micro-CT analysis

The right femur of rats was removed, and then fixed in 4% paraformaldehyde for more than 24 h. The BMD and parameters of bone histomorphometry at distal femoral were blindly analyzed with micro-CT (V1.5.22, Skyscan1172, Belgium). The analysis conditions were as follows: the scanner resolution was 9 μm, with source current of 112 μA, and source voltage of 80 kV. Rotation step was 0.6 degree, exposure time 370 ms, and the frame averaging was 2. Image resolution ratio was 17.81 µm. Three-dimensional reconstruction was conducted with the NRecon analysis workstation. One millimeter below the center of the epiphyseal line was selected as the region of interest (ROI) for the analysis. The parameters of bone histomorphometry were obtained, including bone mineral density (BMD), bone surface to bone volume (BS/BV), relative bone volume (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N).

Three point bending test

The left femur of rats was removed and preserved in – 20 °C. The femurs were thawed to room temperature, and to determine the length, width in long axis and width in short axis using Vernier Caliper. The three point bending test were performed by using Universal Material Testing Machine (Instron 5569). The analysis conditions were as follows: the crosshead speed was 1 mm/min, with span length of 15 mm, and the size of load cell of 1000 N. The sample size was measured by UTM according to the description in literature [24]. The parameters of bone biomechanics including bone material toughness, bending stiffness, maximum load, elastic modulus, maximum strain and maximum stress were obtained by calculating as follows.

Stress (σ, N/mm2) is typically defined as force per unit area, and strain (ε, mm/mm) is typically defined as percentage change in length, or relative deformation [25]. The load can be converted to stress and deformation through engineering formulae, the relationship between stress and strain in bone follows a curve called the stress–strain (σ-ε) curve. The slope of the stress–strain curve within the elastic region is called the elastic modulus (E) [26]. Elastic modulus (E), which is the amount of energy per unit volume necessary to cause damages as well as permanent compositional and structural changes of bone, is calculated from the ratio of stress/strain (σ/ε) [27]. Stress, strain, and elastic modulus can be calculated from the force (F) and displacement (d) of the loaders. For three point loading, the equations are

$$\sigma = \frac}}}}}} , \, \varepsilon = \frac}}}}^ }},\,\,}\,} = \frac = \frac}}}}\frac}^} }}}}},$$

(1)

where c is the distance of force point to the center of mass; L is the length between the two fulcrum; I is cross-sectional moment of inertia around the axis of bending, can be estimated for the cross-section of a long bone by using formulae for a perfectly elliptical cross-section [24, 26, 28, 29]. Maximum load, which is the force that cause a specimen broken at maximum height of the curve, were directly measured from the load–displacement diagram [30]. For bending tests, the intrinsic stiffness of the bone is equal to the elastic modulus (E); and the extrinsic stiffness or bending stiffness is equal to E * I. Furthermore, the bone material toughness, also called as energy absorption, can be derived from the area underneath the stress–strain curve [25, 29]. Maximum stress (σ, N/mm2) and maximum strain (ε, mm/mm) is force per unit area and percentage change in length, respectively, when the bone is broken [25].

Analysis of protein expression in bone tissue of rats

The left femur in hind limbs of rats were collected, and the muscle and excess tissues were removed, the bones marrow were flushed with PBS solution using a syringe. The bone of rats was ground in liquid nitrogen. The proteins of bone tissue were extracted using 200 μL lysate containing phosphatase inhibitors, protease inhibitors, PMSF and IP lysis buffer (v/v 20: 20: 10: 950) in a 1.5 mL eppendorf tube on the ice for 20 min. The extracted mixture was centrifuged (12,000 rpm) at 4 °C for 20 min, and the supernatant containing proteins of bone tissue were transferred into a 1.5 mL Eppendorf tube. The proteins were quantified and denatured for Western-blot analysis.

Osteoblast experimental protocolBMSC culture and identification

BMSCs were isolated from the femur of the 3-week-old Sprague Dawley rat, and cultured in a dish containing α-MEM supplemented with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycinn and 1% l-Glutaminea in humidified atmosphere of 5% CO2 for 10 days. The medium was changed every 3 days. The subculture cells (passage 2–4) were used for subsequent experiments. BMSCs were identified in terms of the morphological character, properties of differentiation into osteoblasts and adipocytes, and expression of CD29 and CD45. This experiment was approved by the Bioethic Committee of Zhejiang University of Traditional Chinese Medicine.

Cell proliferation assay

BMSCs were seeded in a 96-well culture plate at a density of 5 × 104 cells per well and cultured in α-MEM medium with 10% FBS, treated with different concentrations of JTG for 48 h. Cell proliferation was measured by CCK-8 assay. 10 µL CCK-8 solution was added to each well and then incubated for appropriate time. The absorbance was determined at 450 nm with a microplate reader.

ALP staining and activity determination

BMSCs were seeded in a 48-well culture plate at a density of 2.5 × 105 cells per well and cultured in α-MEM medium with 10% FBS, and treated with different concentrations of JTG for 7 days to perform ALP staining and determination of ALP activity. The cells were stained with BCIP/NBT assay kit at 37 °C for 30 min. ALP activity was measured according to the manufacturer’s instructions of the assay kit. Briefly, the cells were washed twice with PBS, and then lysed on ice for 30 min. The cell lysate was collected by centrifuge, and mixed with detection buffer and color substrate, and then incubated for appropriate time. The absorbance was determined at 405 nm with a microplate reader. The alkaline phosphatase activity was calculated according to the definition of enzyme activity provided in the instructions of the assay kit.

Alizarin red staining for analysis of bone mineralized nodule formation

BMSCs were seeded in a 48-well culture plate at a density of 2.5 × 105 cells per well and cultured in osteogenic medium (50 µg/mL ascorbic acid, 10 mM sodium β-glycerophosphate and 10–8 M dexamethasone) and treated with different concentrations of JTG for 21 days. Then, cells were fixed with 4% paraformaldehyde and stained with alizarin red (pH 8.3) at 37 °C for 30 min. Bone nodules were observed under a microscope. To quantify cell mineralization, bone nodules were dissolved with 10% (V/W) cetylpyridinium phosphate chloride, and the absorbance was measured at 570 nm with a microplate reader.

The analysis of expression of protein involved into osteoblast differentiation and activities

BMSCs were seeded in a 6-well culture plate at a density of 1 × 106 cells per well and cultured in α-MEM medium with 10% FBS, and then treated with different concentrations of JTG for 7 days. Subsequently, cells were lysed and the protein content was determined using a BCA protein kit. The western blot analysis was used to determine the expression of proteins.

Osteoclasts experimental protocolOsteoclasts culture

C57BL/6 mice aged 6 weeks were used to isolate bone marrow macrophages (BMMs) from the femur. The extracted bone marrow cells were cultured in α-MEM containing with 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 5 ng/mL M-CSF in a humidified atmosphere of 5% CO2 for 24 h. Non-adherent cells, which were considered as BMMs were collected and cultured in α-MEM medium containing 30 ng/mL M-CSF for 3 days, and then cultured in α-MEM medium containing 30 ng/mL M-CSF and 50 ng/mL RANKL for 48 h to induce cells to differentiate into osteoclasts (OCs).

Viability and TRAP activity assay and TRAP staining of OCs

OCs induced from BMM cells in 96-well plates were treated with various concentrations of JTG for 48 h. The viability of OCs was measured with a MTT kit, and the TRAP activity was determined according to our previous methods. The TRAP activity was represented as nanomoles p-nitrophenol per minute per 100 OCs. For TRAP positive multinucleated cell staining, BMMs-derived OCs were fixed with 4% paraformaldehyde and stained for TRAP activity using an Acid Phosphatase Kit (Sigma-Aldrich, St. Louis, MO, USA). The number and area of OCs containing 3 or more nuclei were counted and photographed using an inverted microscope (Nikon Corporation, Tokyo, Japan).

The staining of F-actin ring of osteoclasts

OCs induced from BMM cells were seeded in confocal culture dishes (diameter, 35 mm), and treated with various concentrations of JTG for 48 h. BMMs-derived OCs were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X-100 for 5 min. Then cells were washed and incubated with fluorescein isothiocyanate (FITC)-phalloidin for 45 min followed by 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich) staining for 10 min. Then, cells were washed and photo-graphed using a laser scanning confocal microscope (Meridian Co., USA).

The analysis of expression of protein associated with osteoclast differentiation and activities

BMMs were plated in 6-well plates at a density of 2 × 106 cells per well and induced by concentrations of 30 ng/mL M-CSF and 50 ng/mL sRANKL for 48 h. The BMMs-derived OCs were treated with various concentrations of JTG for 4 h, and then, were stimulated with 200 ng/mL LPS for 5, 15, 30, 60 min. Finally, the cells were washed with PBS and lysed in RAPI assay buffer for 30 min at 4 °C. The cell protein was collected and their contents were detected by BSA kits. The western blot analysis was used to determine the expression of proteins.

Western-blot analysis

The cell lysates were incubated with a sample buffer of 5 × Laemmli at 100 °C for 5 min. An equal amount of protein was separated by 10% SDS-PAGE and transferred to the PVDF membrane. The membrane was then blocked with 5% BSA for 2 h and incubated with corresponding primary antibodies overnight at 4 °C, and then washed three times with TBST, incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. The protein bands were detected with electro-chemiluminescence (ECL) reagent, and scanned using a chemiluminescence digital imaging system.

Statistical analysis

All data analyses were completed using Graphpad Prism version 5.0 software and IBM SPSS statistic 22.0 software. Data are expressed as the mean ± SD. Data comparison was shown by using one-way analysis of variance (ANOVA). P values lower than 0.05 were considered statistically significant.