In human body, the homeostasis of bone is maintained by constant remodeling that was conducted by osteoclastic bone resorption and osteoblastic bone formation. In adulthood, osteoclasts are the only cell type for bone resorption that is derived from hematopoietic stem cells. Hematopoietic cytokines including macrophage-colony stimulating factor (M-CSF, encoded by
In this study, we predicted the possible biological functions of physalin A, B, D, and F by using BATMAN-TCM, an
The chemical formulas of physalin A, B, D and F are shown in Supplementary Fig. S1A. We first used the BATMAN-TCM system to predict the potential biological functions of different physalins. Significantly enriched KEGG pathways, Gene Ontology (GO) terms, and OMIM/TTD disease phenotypes, among the query potential targets together with corresponding adjusted P values and targets mapped to this term of different physalins, are showed in the supplementary documents. Ten of the most enriched GO terms of each physalin are shown in Supplementary Fig. S1B. Because of their similar chemical constructions, all four physalins analyzed share common enriched GO terms such as “Protein Binding, Bridging” and “Cell-Cell Signaling”, suggesting the similar biological effects they might have in common. Furthermore, the visualization of the molecular pathway and disease-correlation networks of the four physalins are shown in Supplementary Fig. S2-S5. The
BMMs are the most important osteoclast precursor pool in mammals. To study the effects of physalins on osteoclastogenesis, we first evaluated the cytotoxicity of physalins in BMMs treated with RANKL. To investigate the effects of physalins on cell apoptosis, we used flow cytometry to test the FITC-Annexin-V/PI stain (Supplementary Fig. S6A). BMMs (in the presence of RANKL) were treated with physalins A, B, D, and F with different concentrations (0, 10 μM, 30 μM, 50 μM). Quantification analysis showed that both the early and the late cell apoptosis rate significantly increased at the concentration of 10 μM for both physalins (Supplementary Fig. S6B). We then investigated the effects of physalin A, B, D and F on the cell viability of BMMs. We found that physalin A, D, and F began to decrease BMM cell viability significantly when the dosage reached 30 μM. For physalin B, the concentration of 40 μM started to show significant inhibitory effects on BMM cell viability (Supplementary Fig. S6C).
To further evaluate the effects of physalins (A, B, D and F) on RANKL-induced osteoclastogenesis
To confirm the inhibitory effects of physalin D on RANKL-induced osteoclastogenesis
To understand the details of the underlying mechanisms by which physalin D inhibits RANKL-induced osteoclastogenesis, we focused on the calcium signaling, which is crucial for osteoclast differentiation and maturation (15). NFATc1 is activated by intracellular calcium ([Ca(2＋)](i)) oscillation upon RANKL stimulation. We first detected the effects of physalin D on [Ca(2＋)](i) oscillation. We found that both average amplitude and frequency of [Ca(2＋)](i) oscillation were increased upon RANKL stimulation (1s-300s) compared with BMMs without RANKL treatment. Introduction of physalin D robustly alleviated [Ca(2＋)](i) oscillation stimulated by RANKL (Fig. 3A, Supplementary Table 2-5). RANKL-RANK signaling activates phospholipase Cγ2 (PLCγ2) and leads to an increase in [Ca(2＋)](i) via ITAM-harboring molecules DAP12 and FcRγ, followed by activation of Ca2＋/calmodulin-dependent protein kinases (CaMK)IV, which mainly contribute to activating cAMP-responsive element binding protein (CREB) (16, 17). We found that phosphorylation of PLCγ2, CaMKIV and CREB by RANKL were significantly suppressed by physalin D treatment (5 μM) in BMMs without a substantial change in total protein expression (Fig. 3B). In contrast, downstream
We then evaluated the
A recent study reported that physalin B has a relative long dwell time with a half-life of 321.2 ± 29.5 min and clearance of 175.4 ± 25.7 ml/min/kg in rats after intravenous injection (19). Although widely distributed in tissues, physalin B penetration was particularly high in the lungs. Another more recent study suggested that orally administered physalin A can further be metabolized to two sulphonate metabolites characterized from the feces (20). Nevertheless, the detailed function of these metabolites remains unknown. More
We found that physalin D can regulate the calcium signaling during RANKL-induced osteoclastogenesis. Calcium ions (Ca2＋) affect nearly every aspect of cellular life; they also play a crucial role during osteoclastogenesis. Cytoplasmic Ca2＋ oscillations in RANKL-induced osteoclast differentiation provide a digital Ca2＋ signal that stimulates osteoclasts to upregulate and autoamplify the expression of NFATc1 (2). RANKL introduction induced a continuing intracellular Ca2＋ increase in osteoclasts cultured from human monocytes. However, the extracellular Ca2＋ influx seemed to be the main source for the Ca2＋ flux (23). It has also been shown that the CaMKIV-CREB pathway is critical for osteoclastogenesis (24). Inhibition of CaMKs as well as the genetically ablation of
In this study, we looked at the
We purchased female C57BL/6 mice from the animal center of Army Medical University. All procedures involving mice and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Army Medical University, and were performed according to the guidelines on laboratory animal care and use. We tried to reduce the suffering of the animals as much as possible. We established a model of sRANKL-induced rapid bone loss by injecting sRANKL (2 mg/kg) intraperitoneally at 24 h intervals for 3 days into 7-wk-old female mice. Physalin D was intraperitoneally injected with low (10 mg/kg) and high (100 mg/kg) doses daily for 2 weeks. Bone-marrow cells were separated and cultured with M-CSF (50 ng/ml) for 72 h to obtain bone-marrow macrophages (BMMs), which were cultured in α-minimal essential medium (MEM) containing 10% FBS and 1% Penicillin-streptomycin solution.
BMMs and preosteoclasts were cultured in α-MEM containing 10% FBS. For TRAP staining, cells were cultured in a 96-well plate at a density of 5 × 103 cells per well with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 5 days. Cells were then fixed in preheated 4% paraformaldehyde (PFA) of 37℃ for 2 min and then stained with TRAP staining solutions according to the manufacturers’ instructions. Mature osteoclast was counted as TRAP positive with 3 or more nuclei for identification. We measured relative TRAP activity by colorimetric analysis using software Image J2. For actin cytoskeleton and focal adhesion stain, cells were cultured on glass sheet in 48-well plates at a density of 1 × 104 cells/well. Cells were stimulated with M-CSF (50 ng/ml) and RANKL (100 ng/ml) for 5 days for osteoclastogenesis. Detailed procedures were described in a previous study (26). For bone resorption pit-formation tests, cells were incubated in 48-well plates covered with bovine bone slices of 1 × 104 cells/well, as described previously (27). In brief, cells were induced with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 3 days. Bleach solution was added to remove cells followed by light microscope observation.
We used a Bruker MicroCT Skyscan 1272 system (Kontich, Belgium) for μCT analysis. Isotropic voxel size of 10.0 μm was used to scan the whole femur. We did scans in 4% paraformaldehyde and used an x¬ray tube potential of 60 kV, an x¬ray intensity of 166 μA, and an exposure time of 1700 ms. For distal femur trabecular bone analysis, a 3¬mm region started at 0.8 mm proximal to the central epiphysis of the femur was contoured. For femur cortical bone analysis, a 0.5¬mm region was started at 4.5 mm proximal to the central epiphysis of the femur. Trabecular and cortical bones were thresholded at 86-255 (8-bit grey-scale bitmap). Reconstruction was accomplished by Nrecon (Ver. 1.6.10); 3D images were obtained from contoured 2D images by methods based on distance transformation of the original grey-scale images (CTvox, Ver. 3.0.0). We did 3D and 2D analysis using software CT Analyser (Ver. 220.127.116.11).
All data presented are representative of three or more independent assays performed in triplicate unless otherwise indicated. All data in the figures are presented as mean ± SD unless otherwise indicated. One-way ANOVA was used with later on Newman-Keuls methods to evaluate the significance of result differences. Significance was regarded as *P < 0.05, and **P < 0.01. The detailed methods are shown in the supplemental materials.
This work was supported by a grant from the Nature Science Foundation of China (NSFC) (No.81802166), Nature Science Foundation of Liaoning Province (20170540938, 2019-BS-264), and TMMU funding for young investigators (2017MPRC-04).
The authors have no conflicting interests.