EuOCP3 preparation
EuO cortex was obtained from Sichuan Province, China, and its components were systematically analyzed according to previously described method 44. The polysaccharide was purified from the cortex of EuO in accordance with our previous study18. Briefly, EuO cortex was extracted with boiled double-distilled (D.D.) water. The extraction was concentrated, deproteinized, dialyzed, and precipitated to obtain the crude polysaccharides. Then, the complex polysaccharides were further purified by a diethylaminoethyl cellulose-52 column (AI0034; Bestchrom Biosciences, Shanghai, China), a SephacrylTM S-400 column (28-9356-05; GE Healthcare, Uppsala, Sweden) and a Chromdex 200 pg column (EG007; Bestchrom Biosciences). Finally, the collected fraction was labelled as EuOCP3. The extraction and fractioning methods of EuOCP3 are described in detail in the supplementary material (Fig. S2).
Animal experiments
Sixty 6–8 weeks old male C57BL/6 mice, purchased from Changsheng Biotechnology (Liaoning, China), were fed under an animal facility (temperature: 23 ± 1 °C; humidity: 40–60%; 12 h light/dark cycle; specific pathogen-free) with free access to water and diet. After 10 days of acclimatization, these mice were randomized into six groups (n = 10/group): the control (CTRL) mice were administered with normal saline (intraperitoneally (i.p.) injection, daily) and D.D. water (intragastrically (i.g.), daily); the EuOCP3-treated mice were administered with normal saline (i.p., daily) and EuOCP3 (300 mg/kg, i.g., daily); the vehicle-treated OP mice were administered with Dex (30 mg/kg, i.p., daily) and D.D. water (i.g., daily); the positive control OP mice were administered with Dex (30 mg/kg, i.p., daily) and estradiol (E2; 15 μg/kg, i.p., daily); the EuOCP3-treated OP mice were administered with Dex (30 mg/kg, i.p., daily) and EuOCP3 (low dose: 100 mg/kg, high dose: 300 mg/kg, i.g., daily). After 49 days of administration, all experimental animals were fasted for 12 h, and euthanized after collecting blood from the tail vein. The femoral tissues of these experimental mice were quickly collected for subsequent studies. This study adhered to the ARRIVE guidelines American Veterinary Medical Association (AVMA) guidelines for anesthesia and euthanasia of animals.
All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Jilin University. The ethical approval number was NO. SY202103006. All animal experimental procedures in this study were conducted according to ARRIVE guidelines (https://arriveguidelines.org) and all methods were conducted in accordance with applicable guidelines and regulations. Prior to sacrifice, the mice underwent a 12 h fasting period and were euthanized under mild sevoflurane-based anesthesia, following by the Experimental Animal Center of Jilin University guidelines for anesthesia and euthanasia of animals.
Micro-CT
Micro-CT (NMC-200, PINGSENG Healthcare Inc., Shanghai, China) was used to examine the distal femur structure. The scanned images were reconstructed by Avatar software (version 1.6.5, PINGSENG Healthcare Inc.). The region of interest was set 1 mm from the epiphyseal growth plate of the distal femur and extended 0.5 mm. Analyze parameters included trabecular bone volume fraction (Tb.BV/TV), bone surface area/bone volume (Tb.BS/BV), and trabecular thickness (Tb.Th).
Immunohistochemistry (IHC)
The femoral tissues were decalcified by ethylene diamine tetraacetic acid solution for 7 days. The decalcified tissues were dehydrated, embedded in paraffin, and serially sectioned. IHC staining was conducted to detect the expression of Osterix, OCN, RUNX2, and OPN following the same experimental protocol as that in our previous study 45. Detailed information on the antibodies can be found in Table S1. OCN, a non-collagenous protein associated with bone matrix mineralization, is highly expressed during the terminal phase of osteoblast differentiation 39. Tartrate-resistant acid phosphatase (TRAP), specifically expressed in osteoclasts and their precursors, reflects the number and distribution of osteoclasts 46. Based on the OCN and TRAP (GP1049; Servicebio, Hubei, China) staining results, the osteoblasts and osteoclasts on the surface of trabecular bone in each group were recorded as osteoblast/osteoclast number per bone perimeter (N.Ob/B.Pm)/(N.Oc/B.Pm) and osteoblast/osteoclast surface per bone surface (Ob.S/BS)/(Oc.S/BS).
Cytokine detection
The levels of BMP-2 (ml002216), carboxyterminal propeptide of type I procollagen (PICP) (ml038003), and OCN (ml063317) (Enzyme-linked Biotechnology, Shanghai, China) in serum samples were determined on the basis of enzyme-linked immunosorbent assays.
Cell culture
The MC3T3-E1 cells (CRL-2593; American Type Culture Collection, Gaithersburg, MD, USA) were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin in a 37 °C/5% CO2 incubator.
For its osteogenic differentiation, MC3T3-E1 cells were induced at 80% confluence in osteogenic medium (50 µg/mL ascorbic acid and 10 mM β-glycerophosphate). MC3T3-E1 cells was co-cultured with EuOCP3 for 1, 7, and 14 days. The culture medium was changed every 3 days.
Cell viability assay
The MC3T3-E1 cells were seeded in 96-well plates and co-cultured with EuOCP3 (10, 20, 50, and 100 µg/mL) for 24 h. The methyl thiazolyl tetrazolium method was used to measure cell viability in accordance with our previous study 47.
Alkaline phosphatase (ALP) staining and activity measurement
The MC3T3-E1 cells were exposed to EuOCP3 (10 and 20 µg/mL) for 14 days for ALP staining (C3206; Beyotime Institute of Biotechnology, Shanghai, China). Cells treated with EuOCP3 (10 and 20 µg/mL) for 7 days were used for the ALP activity assay (A059-2-2; Jiancheng Bioengineering Institute, Nanjing, China).
Western blotting
The MC3T3-E1 cells were exposed to EuOCP3 (10 and 20 µg/mL) for 1, 7, and 14 days in osteogenic medium to detect the expression of osteogenic protein. The femur tissues were homogenized in tissue grinder (SCIENTZ-48; Xinzhi Biotechnology, Zhejiang, China) to extract bone tissue protein. The collected cells or femoral tissues were disrupted using radioimmunoprecipitation assay buffer (PC101; EpiZyme, Shanghai, China) for 20 minutes. A bicinchoninic acid assay kit (23225; Thermo Fisher Scientific) was used to measure the protein concentration of the supernatant in each group. The quantified protein was separated by 10-12.5% SDS-PAGE and transferred to polyvinylidene fluoride membranes (10600023; Cytiva, Marlborough, MA, USA). Then, membranes were blocked with closure solution (P30500; NCM Biotech, Jiangsu, China) for 20 minutes, incubated with primary antibodies for 14 h (Table S2) and secondary antibodies for 4 h at 4 °C (Table S2). Protein signals were detected using Ultra-High Sensitivity ECL kits (GK10008; GLPBIO, Montclair, CA, USA) with an imaging system (Tanon 5200; Tanon Science & Technology Co., Ltd., Shanghai, China) and analyzed using ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, USA). Data were qualified via densitometry and expressed as the fold of control mice or non-treated cells.
Statistical analysis
All data are shown as the mean ± the standard error of the mean (SEM). Multiple comparisons were performed using one-way ANOVA followed by Tukey's honest significant difference post hoc test with DSS software (version 25.0; IBM, Armonk, NY, USA). Statistical significance was set at P < 0.05.