Animals and Reagents

8–10-week-old male and pregnant C57BL/6 J mice were purchased from CLEA Japan (Tokyo, Japan). Male mice were randomly assigned into four groups for subcutaneous supracalvarial reagents injection, and they were the source of bone marrow-derived macrophages (BMMs) and peritoneal macrophages. Newborn mice aged 5–9 days provided primary osteoblasts from calvariae. All animal caring and experimental procedures complied with the Regulations for Animal Experiments of Tohoku University. (Certification of conformity: No. 2018DnA-049-12). LPS (Sigma-Aldrich, St. Louis, MO, USA), recombinant murine TNF-α (R&D system, Minneapolis, MN), RANKL (PEPROTECH, Cranbury, NJ), and Angiotensin-(1–7) trifluoroacetate salt (Bachem, Bubendorf, Switzerland) were used in this study. Ang-(1–7) powder was dissolved in distilled water at a stocking concentration of 100 mg/mL. Recombined mouse M-CSF was provided by an mM-CSF-producing cell line as described previously [27].

Histological Analysis

Mice were divided into four injection groups: 1 × phosphate-buffered saline (PBS), LPS (100 μg/day), LPS + Ang-(1–7) , and Ang-(1–7) (100 μg/day) only. Each group contains at least 4 samples. The mice received a subcutaneous supracalvarial injection according to the assigned group for five consecutive days. On the sixth day, mice were sacrificed by cervical dislocation after anesthetized by isoflurane inhalation (Pfizer, New York, NY). The calvariae were dissected and fixed in 10% formalin for 3 days at 4 ℃. Micro-computed tomography (Micro-CT) scanning using ScanXmate (Comscan, Kanagawa, Japan) and 3-dimensional reconstruction for comparing the destructed areas by TRI/3D-Bon (RATOC System Engineering, Tokyo, Japan) had been conducted after fixation. Photoshop (Adobe, San Jose, CA) is used for specifying the absorption area in the calvariae images, while ImageJ (NIH, Bethesda, MD) is used to calculate the proportion of this area in a specified area. Then decalcification in 14% ethylenediaminetetraacetic acid (EDTA) was processed at room temperature for 1 week. The sagittal sutures of calvariae were divided into three equal parts from the coronal plane and subjected to a dehydration in a tissue processer (TP1020, Leica, Wetzlar, Germany). Dehydrated bone pieces were embedded in paraffin and cut into 5-µm sections perpendicular to the sagittal suture with a microtome (Leica). Sections were stained with tartrate-resistant acid phosphatase (TRAP) solution. To make the TRAP solution: Fast Red Violet, Naphthol AS-MX phosphate and N, N′-Dimethylformamide were incubated in a mixture of 0.5 M sodium tartrate dihydrate and 0.1 M sodium acetate (pH 5.0). The nucleus was counterstained with hematoxylin. All observation and picturing were performed with a digital camera DP-72-SET-B (OLYMPUS, Tokyo, Japan) and cellSens Standard (OLYMPUS). TRAP-positive multinucleated cells (with ≥ three nuclei) closely adjacent to the red-stained bone wall in the sagittal suture mesenchyme were counted manually (Supplementary Fig. 1). Statistical analysis was based on an average of 10 sections from one sample [28].

Preparation of Primary Osteoclast and Osteoclastogenesis

Bone marrow was flushed from femora and tibiae with pre-cooled sterilized 1 × PBS by centrifuge (3000 rpm, 3 min, 4 ℃). Cells were filtered by a 40-μm cell strainer (Falcon: Corning, Corning, NY) in minimum essential medium α(α-MEM) supplied with 10% fetal bovine serum (FBS), 100U/mL penicillin, and 100 μg/mL streptomycin (Wako, Osaka, Japan), cultured with 100 ng/mL M-CSF using a petri dish (Asnol: AS ONE, Osaka, Japan) in 37 ℃ for 3 days. On the fourth day, adhesive cells were harvested as BMMs using 0.05% trypsin-EDTA (Gibco: Thermo Fisher Scientific, Rockford, IL). BMMs were seeded in a 96-well plate at a concentration of 5 × 104 cells/200μL/well, and reagents were added as follows: 100 ng/mL M-CSF (negative control), M-CSF + RANKL or TNF-α (positive control), the second group plus 10 μg/mL Ang-(1–7) (about 10−5 M, experimental group), and M-CSF + Ang-(1–7), n = 4. The medium and reagents were changed and added every 2 days and cultured for 5 days in total. Cells were fixed in 10% formalin for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. After staining with TRAP solution, TRAP-positive cells were observed under a light microscope and counted manually [29].

Preparation of Primary Osteoblasts and Osteoblasts Culture

5–9-day-old neonatal mice were euthanized by inhalation of overdose isoflurane and sterilized in 70% ethanol. The calvariae were dissected between anterior and posterior fontanelles then digested and fractionated to bone chips using a collagenase isolation buffer containing 70 mM NaCl, 10 mM NaHCO3, 60 mM sorbitol, 3 mM K2HPO4, 1 mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose, and 25 mM HEPES and collagenase powder. Fractionation was carried out in a BioShaker (TAITEC, Saitama, Japan) (300 rpm 20 min, 37 ℃) using 0.2% (w/v) collagenase solution and 5 mM EDTA in 1 × PBS with 0.1% BSA. The sequence of digestion and fractionation is as follows: collagenase (Fraction 1), EDTA (Fraction 2), and collagenase (Fractions 3–4) [30]. Cells harvested from fractions 2–4, which are rich in primary osteoblasts, were seeded in a treated dish (Corning). After culture in α-MEM in 37 ℃ for 3 days, cells were harvested as mature osteoblasts and treated with LPS ± Ang-(1–7) to investigate the RANKL expression. 3-h and 48-h cultures were conducted to study the effects of short-and long-term stimulation, respectively.

Isolation and Culture of Peritoneal Macrophages

Mice were euthanized and the peritoneum was exposed without breaking the serous membrane. A 21G needle and syringes were used to inject 6 mL ice-cold sterilized 1 × PBS beneath the peritoneal viscera over the inner side of the femur. Generally, 5 mL peritoneal lavage fluid was collected. Cells were harvested by centrifuging (1500 rpm, 8 min, 4 ℃) in 1 × PBS. Filtered cells were seeded in 12-well plates, and non-adhesive cells were washed two hours later. Then adherent cells were incubated in 37 ℃ overnight and cultured with LPS ± Ang-(1–7) for 3 days to study the effect of Ang-(1–7) on macrophages TNF-α expression.

RNA Extraction and Real-Time Reverse Transcription-Polymerase Chain Reaction

Calvariae tissues were homogenized by stirring with ceramic beads in TRIzol reagent (Ambion: Thermo Fisher Scientific) using Micro-Smash MS-100R (TOMY SEIKO, Tokyo, Japan). Chloroform (Wako, Osaka, Japan) was added to the mixture at one-fifth volume of the TRIzol and centrifuged (14,000 rpm, 10 min, 4 ℃) to collect the supernatant containing RNA. RNA was then extracted following manufacturer’s instruction using RNeasy Kits (QIAGEN, Hilden, Germany). The cells were lysed with a 100:1 mixture of RLT and 2-mercaptoethanol and then transferred to the QIAShredder for homogenization followed by centrifugation (14000 rpm, 2 min, 4 ℃). Total RNA was dissolved in RNase-free water and stored at − 80 ℃. RNA concentration and quality was checked using a Nano-Photometer (Implen, Munich, Germany). cDNA was made by Super-script IV Reverse Transcriptase system using Oligo dT primer (Invitrogen, Thermo Fisher Scientific). Primers utilized in real-time PCR are listed in Table 1. GAPDH was selected as an internal control gene, and TB Green® Premix Ex Taq™ II was used (TaKaRa, Shiga, Japan).

Table 1 Primer sequences used for real-time PCRWestern Blot

Peritoneal macrophages stimulated by LPS ± Ang-(1–7) for 0, 5, 15, and 30 min were lysed in radioimmunoprecipitation assay buffer consisting of 1% protease and phosphatase inhibitor after 3 h of starvation in serum-free α-MEM. QIAShredder was used to remove cell pellets (14,000 rpm, 2 min, 4 ℃) and homogenize total protein [30]. Protein concentration was quantified with a BCA assay kit (Thermos fisher Scientific) and a microplate reader was used to measure the absorbance at 595 nm. Protein was stored in -80℃ or mixed with sample buffer and denatured at 95 ℃ for 5 min. The sample buffer was prepared by combining 9 parts of 4 × Laemmli with 1 part of 2-mercaptoethanol, and each protein sample was mixed with the sample buffer in a 3:1 volume ratio. Denatured protein samples were stored in – 20 ℃.

Protein was loaded on Precast Gels (Biorad, Hercules, CA) in 1 × Tris/Glycine/SDS buffer (120 V,1 h) and transferred to the PVDF membrane by the Trans-Blot Turbo Transfer System (Biorad). The membrane was blocked with a mixture of 4 g block ace powder and 20 mg sodium azide dissolved in 100 mL 1 × Tris -buffered saline with 1% Triton X-100 (TBS-T). Primary and secondary antibodies (Table 2) were diluted in immunoreaction enhancer solution (TOYOBO, Osaka, Japan) in specific dilutions. Membranes were washed in 1 × TBS-T for 10 min twice and 1 × TBS once prior to and after secondary antibody incubation. Membrane reacted with West Femto Maximum Sensitivity Substrate was detected by FUSION FX, captured by Evolution-capt Edge (VILBER, Collégien, France), and analyzed by ImageJ using a inserted band quantification Macro [31].

Table 2 Antibodies and specific dilutions used for Western blottingStatistical Analysis

All data are expressed as the mean ± standard deviation. Scheffe’s test and paired t-test were used to assess the differences between groups. Statistical significance was defined as p < 0.05. Scheffe’s test is a one-way ANOVA post hoc test, and it corrects alpha for complex mean comparisons with a narrower confidential interval. Data were analyzed using the Excel statistics Statcel 3 (Microsoft, Seattle, WA).