PMOP Rat Model
Forty-five healthy female SD rats weighted 200 - 220 g were housed in SPF animal room at 20~26 °C and the relative humidity was 50~60%, light and dark cycle was 12/12 h. Rats have free access to eat and drink. The study was approved by the animal care committee, and all experimental operations were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
After 1 week of adaptive feeding, SD rats were given 1% pentobarbital sodium (40 mg/kg) anesthetized intraperitoneal injection, the skin was disinfected on the back. The longitudinal incision was made in the median, and the skin, subcutaneous, muscular layers were cut open, then a small incision was made into the abdominal cavity on both sides. The bilateral ovaries were completely removed. After surgery, the rats were injected penicillin (Sigma-Aldrich, USA) into the abdominal cavity to prevent infection. All rats were kept in the same condition, fed in a single cage with standard feed. Five days after the operation, the vaginal epithelial cell smear was observed with a cotton swab, and rats with irregular estrus for 5 consecutive days were regarded as successful modeling. ADSCs cells were isolated as described in a previous study.16
Grouping
A total of 50 rats were randomly selected for bilateral ovarian ablation. Rats were divided into 4 groups randomly (n=9): the model group, the icariin group (250 mg/kg/d, intragastric administration), ADSCs group (ADSCs 2×105 cell/ml, intramedullary injection, 0.5ml each time), icariin combined with ADSCs group (icariin + ADSCs group, icariin 250 mg/kg/d, intragastric administration; ADSCs 2×105cell/ml, intramedullary injection, 0. 5ml each time). The remaining 9 rats as a sham operation group, all operations were the same as the surgery group, except that the ovaries did not remove. Continuous administration for 12 weeks. After the end of the administration, the rats femur were taken, peeled off attached muscles and connective tissues on the femur, the left side femur was fixed on 10% formaldehyde, and the right side femur was frozen at -20 ° C for use.
Haematoxylin and eosin staining and observation
After the femur was fixed for 24 h in each group, the decalcification was performed by 10% EDTA for 30 days. The decalcification solution was replaced every 3 days, the decalcification of bone tissue was checked during fluid exchange. The femoral tissue was placed in 95% ethanol overnight, gradient dehydration to 100% ethanol, 1 h per gradient, then xylene was transparent, embedded in paraffin, and sectioned 6 μm. Sectional dewaxing with xylene, gradient alcohol rehydration, distilled water soaking, hematoxylin (Solarbio, Beijing, China) staining for 20min, washed in running water back to the blue for 10min, eosin (Solarbio, Beijing, China) staining for 10min, gradient alcohol dehydration, xylene transparent, Neutral gum was used for sealing. Pathological changes in femoral tissue were observed by an optical microscope (magnification, x100; Olympus, Japan).
Immunohistochemistry
The femoral tissue were routine sectioned, the baked flakes were dewaxed with xylene and hydrated sequentially with a gradient ethanol solution. The antigen was repaired in citrate buffer for 20 min, 3% hydrogen peroxide solution was inactivated for 30 min, and blocked with 5% BSA for 20 min. The anti- calcitonin receptor (CALCR) polyclonal antibody (1:20, ab11042, Abcam, UK) and anti- cathepsin K (CTSK) polyclonal antibody (1:1000, ab19027, Abcam), rabbit anti- OPG-antibody (1:50, ab73400, Abcam) and anti-RANKL antibody (1:100, sc52950, Santa Cruz Biotechnology, USA)were added dropwise and reacted at 4 °C overnight. After rewarming, the sections were incubated with the horseradish peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG (Abcam, UK) at 37 °C for 1 h. DAB (Solarbio, Beijing, China) was used for color development, hematoxylin was lightly counterstained, dehydrated, transparent, and sealed. Three sections of each tissue were observed under a 100× optical microscope (Olympus, Japan) to analyze positive cell.
QRT-PCR
The right femur of each group of rats was extracted RNA by TRIzol Reagent (Invitrogen, USA) after grinding. RNA purity and integrity were detected by UV spectrophotometer (Thermo Fisher Scientific, USA) at A260 /280. Reverse transcription was performed by TaKaRa Primescript TM RT ReagentKit (TaKaRa, Japan). 10 μL cDNA configuration reaction system was used to perform the PCR reaction by the SYBR Premix Ex Taq (TaKaRa, Japan). The primer sequences were as follows: OPG: (Forward) 5'-TGGACAACCCAGGAAACCTTTCCTCCAAAA-3' (Reverse) 5'-TTTGCCTGGGACCAAAGTGAATGCAGAGAG-3'; RANKL: (Forward) 5'-GCCAGCCGAGACTACGGCAAGTACCTGCGC-3', (Reverse) 5'-GGCCAGGTGGTCTGCAGCATCGCTCTGTTC-3'; β-actin: (Forward) 5’-GTGG GGATAATGAACTTGCAG-3’, (Reverse) 5’-GGAACCCCTGGTAGAACAGT-3’. The PCR reaction was performed under the following conditions: 95℃ for 30 sec, 95℃ for 5 sec, 60℃ for 30sec, 30 cycles, and incubation at 72 °C for 10 min. Data were processed using the 2-ΔΔCt method17 and relative expression levels were calculated using β-actin mRNA as an internal reference.
Western blot assay
The right femur of each group of rats was extracted and the tissue protein was extracted after grinding. The concentrations of RANKL and OPG protein were sequentially determined according to the method of BCA Protein Quantitation Kit (Thermo Fisher Scientific, USA). Each group of sample was loaded with 40 μg, separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% skim milk for 1 h, then primary antibody anti-OPG antibody (1:1000, sc390518, Santa Cruz Biotechnology, USA), anti-RANKL antibody (1:1000, sc52950, Santa Cruz Biotechnology, USA) were added and incubated at 4 °C overnight. TBST (TBS, 1 ml/L Tween-20) was used to wash the membrane for 3 times, 5 min/time. HRP-labeled secondary goat anti-mouse IgG (1:5000, sc2005, Santa Cruz Biotechnology, USA) was added and incubated for 2 h at room temperature. The membrane was washed with TBST three times, 10 min/time; ECL chemiluminescence was developed in darkroom. The protein expression levels were normalized to β-actin and quantified by Image J 1.46 (National Institutes of Health, USA) software.
Statistical analysis
IBM SPSS statistical software 19.0 was used to perform the statistical analysis. All experimental data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to comparison between groups, subsequent analysis was performed by LSD test. Statistical significant difference was assumed at p < 0.05.