2.1 Materials and Reagents
Epimedin A (EA, Cas: 110623-72-8, IE0160, purity ≥ 97.0%) and Alendronate sodium (ALN, Cas: 121268-17-5, A6780, purity ≥ 97.0%) were purchased from Solarbio (Beijing, China). Antibodies specific for anti-NFATc1 (ab25916), receptor activator of nuclear factor κB ligand (RANKL, ab239607), RANK (ab305233), anti-NF-κB (ab32536) and anti-p-NF-κB p65 (ab76302) were purchased from Abcam (Cambridge, MA, USA). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), including anti-TRAF6 (#67591), anti- MAPK P-P38 (#4511), anti-MAPK p38 (#8690), P-AKT (#4060), AKT (#4685), GAPDH (#5174), and Anti-rabbit IgG (H + L) (#14708). All antibodies used at the recommended concentrations was in accordance with their peotocols, perviously.
2.2 Animals
A total of 60 female Wistar rats, weighing 240 ± 20 g, 10–12 weeks old, were provided by the SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All rats were accommodated in a temperature-controlled environment with a 12-hour light-dark cycle. All animal experimental procedures complied with the guidelines for the Animal Care and Experiment Committee of Changzhou Hospital of Traditional Chinese Medicine
2.3 Osteoporosis rat model and treatment
After a one-week adaptation period, a portion of the rats underwent sham surgery (n = 12), while the remaining rats underwent surgical ovariectomy (n = 60). Following anesthesia, achieved via intraperitoneal injection of 3% sodium barbiturate (40 mg/kg), the surgical site was shaved, and a lateral incision was made from the mid-axillary line of the quarter rib area to the spinal area. Bilateral ovaries were then excised entirely to induce the osteoporosis model. The sham-operated group underwent the same procedure but without bilateral ovary removal. Subsequently, wound closure was performed for each group of rats, followed by the application of erythromycin ointment to prevent infection. Vaginal smear examinations commenced from the 2nd postoperative day, with continuous daily observations over a week. Rats displaying consistent estrous cycle changes were considered to have undergone successful ovarian removal. The ovariectomized (OVX) rats were randomly allocated into five groups: 1) Model group; 2) EA-L group (5 mg/kg/d Epimedin A); 3) EA-M group (10 mg/kg/d Epimedin A); 4) EA-H group (20 mg/kg/d Epimedin A); and 5) ALN group (2.5 mg/kg/d alendronate). Epimedin A and ALN were dissolved in normal saline and orally administered to rats daily for a duration of 90 days.
2.4 Bone loss assay
At the end of experiments, all rats underwent euthanasia. The left thighbone was subjected to high-resolution micro-CT (Skyscan 1176, Aartselaar, Belgium), and made a three-dimensional finite element model of the rats’ thighbone with MIMICS software. For each sample, standard bone parameters, including bone mineral density (BMD), bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp), were examined and analyzed as previously outlined 12.
2.5 Morphometric analysis of bone tissues
The right thighbone of rat was fixed in 4% paraformaldehyde, decalcified by 10% EDTA, paraffin-embedded and cut into 5 µm sections. Then, these sections underwent for morphometric analysis of bone tissues with hematoxylin and eosin and TRAP staining. The quantitative parameters as follows: osteoclast surface per bone surface (OcS/BS), the ratio of bone volume/tissue volume (BV/TV), and number of osteoclasts per bone perimeter (N.Oc/BS) were analysed using Bioquant Osteo software (Nashville, USA).
2.6 Construction of osteoclast model
The RAW264.7 cells purchased from the American Type Culture Collection (Rockville, MD) were cultured in DMEM medium, containing 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. RAW264.7 cells were cultured in 50 ng/ml RANKL and 10 ng/ml M-CSF, the medium was changed every 3 days, and the factor RANKL was supplemented to induce osteoclast differentiation. 6 days after induction, TRAP staining was performed to observe the morphology of multinucleated osteoclasts after induction.
2.7 Cell viability assay
The cytotoxicity of EA on osteoclastogenesis was detected using CCK-8 assay kit (Dojindo Molecular Technology, Kumamoto, Japan). RANKL-induced RAW264.7 cells were inoculated at a density of 2.5×103 cells/well and co-cultured with EA in different concentrations (0.1 umol/L, 0.2 umol/L and 0.4 umol/L). Two days later, RAW264.7 cells were incubated in a fresh medium containing 10 µL CCK-8 solution for 2 h, and the optical density (OD) was measured at a wavelength of 450 nm using microplate an enzymoleter.
2.8 In vitro experimental grouping
RAW264.7 cells pretreated with RANKL/M-CSF were divided into EA treated group and EA free group (control group). RAW264.7 cells cultured with RANKL/M-CSF were added with 0.1 µM (EA-L), 0.2 µM (EA-M), and 0.4 µM Epimedin A (EA-H) for 5 days at 37˚C, respectively. Then, cells in each group were performed for cell viability assay and the next experiments.
2.9 Construction and infection of lentivirus
TRAF6 interference sequence (TRAF6-siRNA, 22034) were as synthesized by Geneneral Biol (Anhui) Co., Ltd (Hefei, China) and inserted into the hU6-MCS-CMV-Puromycin vector. Then, their plasmids were transfected into 293T cells. RAW264.7 cells were infected with lentivirus-mediated TRAF6-siRNA or its negative control lentivirus (Control-siRNA) for 48 h, and then changed complete optimal medium without lentiviral. At 72 h after infection, the RNA interference efficiency of RAW264.7 cells was determined by western blot. After RAW264.7 cells were infected with lentivirus (TRAF6-siRNA or Control-siRNA), they were also treated with RANKL/M-CSF to induce differentiation in the absence or presence of Epimedin A.
2.10 TRAP staining
RAW264.7 cells were cultured in 96-well plates with varying concentrations of Epimedin A in the presence of RANKL/M-CSF. After differentiation into osteoclasts, the cells were fixed in 4% paraformaldehyde for 10 minutes, washed with cold PBS, and stained with a tartrate-resistant acid phosphatase kit (P0332, Beyotime) following the manufacturer’s instructions. TRAP-positive cells, indicating mature osteoclasts, were counted under a microscope.
2.11 Immunofluorescence assay
Each group’s RAW264.7 cells were grown on glass coverslips in order to measure the relative expression of the intracellular protein NFATc1. In summary, 4.0% paraformaldehyde in PBS was used to fix the osteoclasts for 30 minutes. Following three PBS washes, cells were permeabilized for five minutes with 0.25% Triton X-100 (v/v), and then they were blocked for one hour in PBS containing 3% bovine serum albumin. TRITc rhodamine-conjugated phalloidin was used to stain the F-actin rings for 30 minutes, while DAPI solution was used to stain the nuclei for 30 seconds at room temperature. Lastly, following RAW264.After seven cells were cleaned with PBS, fluorescence pictures were captured and viewed using an Olympus BX51 fluorescent microscope.
2.12 Quantitative real-time PCR
Each group's grown cells underwent three rounds of washing in cold PBS before being lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). After isolating total RNA in accordance with the manufacturer's instructions, cDNA was produced by reverse transcription. Next, real-time PCR was used to quantify the differential expression of the particular genes (normalized by GAPDH level); this was done using an ABI-7500 system (Applied Biosystems, CA, USA). 2 µl of cDNA, 20 µl SYBR Mixture (Takara, Liaoning, China), 16 µl ddH20, 1 µl forward primers (10 µM), and 1 µl reverse primers (10 µM) made up the reaction mixture. The following were the cycling conditions: 95 ℃ for ten minutes, then 35 cycles of 15 seconds at 95 ℃ and one minute at 60 ℃ for 1 min, and a final step at 4 ℃ for 10 min.
2.13 Western blot
Cell cultures were supplemented with 200 µL of RIPA lysis solution containing 2 µL of PMSF (Thermo, USA). Following centrifugation and ultrasonography of the cell solution on ice, the supernatant was obtained, and a BCA assay kit (Solarbio, Beijing, China) was used to measure the protein contents in it. The protein samples were then electrophoresed one after the other for two hours at 80V. Proteins were then moved to nitrocellulose membranes for 1.5 hours at a constant flow rate of 200 mA. Primary and matching secondary antibodies were used for western blotting. As an internal control, the relative protein levels were standardized to tubulin. Software called Image J was used to quantify the density of protein bands.
2.14 Statistical analysis
Each set of data was presented as mean ± standard deviation (SD) and was typical of at least three separate studies. A one-way ANOVA or the Student's t-test were used to assess the significance of the quantitative results.