Introduction
============

Osteoporosis (OP) is a chronic bone metabolism disease that is clinically characterized by both bone mass reduction and bone architecture alteration. OP increases bone fragility and fracture risk worldwide ([@b1-ijmm-45-05-1543],[@b2-ijmm-45-05-1543]). With the ageing of the population, particularly in Asia, the number of individuals who are aged ≥65 is projected to be 9.3% in 2025 ([@b3-ijmm-45-05-1543]). Patients with OP and osteoporotic fracture (OPF) experience pain, inconvenience, low quality of life, high economic burdens and mortality ([@b4-ijmm-45-05-1543]--[@b6-ijmm-45-05-1543]).

For OP prevention, controlling the risk factors is necessary. Exercising, body mass index (BMI) monitoring, and taking vitamin D and calcium are effective methods. In addition, decreasing hyperkyphosis, and avoiding smoking, alcohol and OP-inducing medications are preventive measures for OP ([@b7-ijmm-45-05-1543],[@b8-ijmm-45-05-1543]). The primary purpose of osteoporotic therapy is to decrease the risk of fracture. The relevant medications are classified into two groups, antiresorptive agents and anabolic agents, and include bisphosphonates (BPs), estrogen replacement therapy (ERT) agents, selective estrogen receptor modulators, and calcitonin (CT) ([@b9-ijmm-45-05-1543]). However, the drugs available at present have a number of adverse effects. ERT, for example, is associated with coronary heart disease, breast cancer, stroke and dementia ([@b10-ijmm-45-05-1543],[@b11-ijmm-45-05-1543]), and CT leads to nausea and local inflammation ([@b12-ijmm-45-05-1543]).

Osteoking is a Traditional Chinese Medicine (TCM) compound originating from the Yi ethnic group in Yunnan that has been used to treat bone diseases for decades ([@b13-ijmm-45-05-1543]). Initially, osteoking was approved by the Chinese State Food and Drug Administration in 2002 to treat femoral head necrosis, lumbar disc herniation and osteoarthritis in the clinic ([@b14-ijmm-45-05-1543]). In our previous study, osteoking improved these bone diseases by upregulating the gene expression of Runt-related transcription factor 2 and vascular endothelial growth factor (VEGF) in osteoporotic rabbit models ([@b15-ijmm-45-05-1543]--[@b18-ijmm-45-05-1543]). In clinical settings, osteoking prevents fracture in humans ([@b19-ijmm-45-05-1543]). Osteoking improves osteoporotic fracture in rats ([@b20-ijmm-45-05-1543]). Prevention of OP/OPF may be a new application for osteoking. However, its exact mechanisms *in vivo* and *in vitro* remains unclear.

With the development of bioinformatics and network-pharmacology analyses, in particular component characterization of TCM compounds, researchers can easily obtain data, including chemical components, biological targets, and even metabolic processes *in vivo*, including absorption, distribution, metabolism and excretion. Researchers can analyze the relevant gene data from the Gene Expression Omnibus (GEO) database to obtain disease-associated targets ([@b21-ijmm-45-05-1543]). Investigating the components of osteoking and its OP-associated targets by network pharmacology may help elucidate the mechanisms of action of osteoking ([@b22-ijmm-45-05-1543]). The present study aimed to reveal the mechanism of osteoking and provide a new strategy for OP or OPF therapy.

Materials and methods
=====================

Drugs and reagents
------------------

Osteoking was prepared according to the Chinese Pharmacopeia (China Pharmacopeia Committee, 2002) and was supplied by Crystal Pharma Co., Ltd. (lot. no. 20160506) ([@b23-ijmm-45-05-1543]). Its formula was as follows: *Citrus reticulata Blanco*, *Carthamus tinctorius* L., *Panax notoginseng* (Burk) F. H. Chen Ex C., *Eucommia ulmoides Oliv*., *Panax ginseng* C. A. Mey., *Astragalus membranaceus* (Fisch.) Bunge, and *Trionycis Carapace* ([Fig. S1](#s1-ijmm-45-05-1543){ref-type="supplementary-material"}). The above materials were ground into a coarse powder, immersed in 10-fold distilled water for 12 h at room temperature and then boiled using a distillation apparatus for 1 h. This process was repeated twice, and for the second and third extraction, the residue from the previous extraction was filtered, and the same extraction procedures were applied. Thereafter, the combined extracts were filtrated and evaporated using a rotary evaporator at 50°C to a relative density of 1.03 g/cm^3^ and centrifuged (1,450 × g; 30 min; room temperature) and the obtained supernatant was centrifuged (1,450 × g; 30 min; room temperature) once again following precipitation for 12 h. Subsequently, 0.36 g/ml was the clinical concentration of crude osteoking used.

Recombinant human parathyroid hormone 1--34 (rhPTH 1--34) was obtained from Dailan Meilun Biotech Co., Ltd. (cat. no. MB1241), Chemical Abstracts Service (CAS ID: 52232-67-4).

The antibodies used were anti-β-actin clone AC-15 (1:5,000; cat. no. A1978; Sigma-Aldrich; Merck KGaA), anti-BMP-2 (1:2,000; cat. no. 18933-1-AP; ProteinTech Group, Inc.), anti-heat shock protein HSP 90-β (HSP90-β; 1:3,000; cat. no. 11405-1-AP; ProteinTech Group, Inc.), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L; 1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L; 1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.), and HRP-linked anti-Rabbit IgG (1:5,000; cat. no. G1215; 1:5,000; Wuhan Servicebio Technology Co., Ltd.).

OP model
--------

All animal experiments were approved by the Animal Study Committee of Kunming Medical University (approval no. KMMU 2015007) and were conducted according to the requirements of the National Institutes of Health Guidelines for care and use of laboratory animals ([@b24-ijmm-45-05-1543]). A total of 62 female 3-month-old Sprague-Dawley rats (270±15 g; Dossy Co.) were maintained in standard conditions with a controlled temperature (21--23°C) and a strict 12:12 h light: Dark cycle. All rats were fed standard rat chow and allowed *ad libitum* access to distilled water at all times during acclimation and experimental treatment periods. The health and behavior of rats was monitored every day. After 7 days of adaptation, animals were randomly divided into the bilateral ovariectomy (OVX) group (54 rats) and the sham-surgery group (8 rats). A total of 8 rats underwent bilateral adipose tissue resection, in which adipose tissue of a similar weight to the ovaries was removed (sham group), and the remaining 54 rats were subjected to bilateral ovariectomy (OVX rats). The animals were anesthetized with intraperitoneal injection of 30 mg/kg sodium pentobarbital (Servio Co.). Preoperatively, all animals were fasted for 12 h. Benzylpenicillin sodium (60,000 IU/kg; Harbin Pharmaceutical Co.) was administered for 3 consecutive days following the surgeries.

Experimental protocol
---------------------

A total of 54 OP rats underwent OVX surgery, randomly selected from the OP animals (450±20 g), were randomly divided into three groups: A positive control group treated with 0.33 μg/kg/2 days *subcutaneous* (*s.c*.) rhPTH; a negative control group treated with 0.59 ml/kg *intragastric* (*i.g*.) 0.9% NaCl (Baxter Medicine Co., Ltd.); and osteoking group treated with 0.59 ml/kg i.g. osteoking. The dosage used for each animal was dependent on the weight and body surface area of the rat, as well as the conversion coefficient of the human clinical dosage for rats ([@b25-ijmm-45-05-1543]). After 12 weeks of treatment, the rats were sacrificed via cervical dislocation under deep anesthesia (intraperitoneal injection of 30 mg/kg sodium pentobarbital), as described previously. When animals were sacrificed, serum and bone were collected and preserved at −20°C.

BMD analysis
------------

After 4, 8 and 12 weeks of treatment with osteoking, the whole-body BMD was measured by dual-energy X-ray absorptiometry using the Lunar Prodigy Advance (GE Healthcare). Specific software for small animals (GE Medical Systems, enCORE2004 software; v.8.80.001) was used ([@b26-ijmm-45-05-1543]), as described previously.

Identification of active ingredients and prediction of OP-associated targets by bioinformatics
----------------------------------------------------------------------------------------------

The Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database (<http://lsp.nwu.edu.cn/tcmsp.php>) was used to obtain the main active ingredients of osteoking, which were identified using the cut-off values of oral bioavailability ≥30% and drug-likeness ≥0.18. The target-prediction model of TCMSP was used to predict the associated targets. Significantly different genes were obtained through the secondary mining of the GEO database (Gene accession numbers: GSE35955; GSE35958 and GSE35959) ([@b27-ijmm-45-05-1543]).

Cell culture
------------

Rat bone mesenchymal stem cells (rBMSCs) were obtained from the Chinese Academy of Science Kunming Cell Bank of Type Culture Collection (Kunming Institute of Zoology, Chinese Academy of Sciences). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Biological Industries) supplemented with 10% FBS (Biological Industries). The cells (1×10^4^ in 200 ml/well) in 96-well plates and cultured in different concentration of osteoking. After 24 h of administration, the proliferations in each well were cultured with MTS colorimetric assay kit (Abcam Co., Ltd.) at 37°C for 1 h and measured using a microplate reader (Tecan Group, Ltd.) on OD=490 nm ([Fig. S2](#s1-ijmm-45-05-1543){ref-type="supplementary-material"}). Osteoking was filtered through a 0.22 mm filter and diluted 16 times for culture. The final concentration of osteoking was 22 g/l crude osteoking. There were two groups: The control group (DMEM+10% FBS) and osteoking group (22 g/l crude osteoking) for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The cells in 25 cm^2^ culture flask were induced for 4 weeks. Concomitantly, the cells (1×10^5^ in 1 ml/well) were divided into four groups: Control; 20 mmol/l HSP90-b inhibitor NVP-AUY922 (Selleck Chemicals); osteoking (22 g/l crude osteoking); and osteoking + HSP90-b inhibitor, and cultured in 6-well plates for 4 weeks. After 4 weeks of administration, the plates were stained by alizarin red at room temperature for 25 min and measured using a microplate reader.

Histopathology and immunohistochemistry
---------------------------------------

After 12 weeks of treatment, the 3rd lumbar vertebrae were harvested and fixed in 10% paraformaldehyde at room temperature for 14 days, dehydrated (60, 75, 95 and 100% ethyl alcohol) and decalcified (300 ml 30% HCL; 700 ml 4% paraformaldehyde; 140 g NaCl and 20 ml glacial acetic acid) gradually. Then, 5-μm-thick sections were prepared using a Leica RM2245 microtome (Leica Microsystems, Inc.). The anti-HSP90-β antibody (ProteinTech Group, Inc) was diluted 1:1,000 in blocking solution. After samples were washed 3 times in PBS, they were incubated with HRP-linked anti-Rabbit IgG (1:5,000; cat. no. G1215; Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1 h. The results were analyzed using ImageJ software (version 1.46R; National Institutes of Health). The cells in six-well plates were washed and fixed using 10% paraformaldehyde at room temperature for 15 min. Alizarin red S (100 ml/bottle; Wuhan Servicebio Technology Co., Ltd.) was used at room temperature for 25 min and staining intensities were measured by microplate reader at an absorbance wavelength of 560 nm (Tecan Group, Ltd.).

Micro computed tomography (micro-CT) analysis
---------------------------------------------

Following removal of adherent soft tissues, the bilateral femurs were preserved in 4% paraformaldehyde at room temperature as described previously ([@b28-ijmm-45-05-1543]). Micro-CT analysis was performed according to recent guidelines^56^ using a micro-CT imaging system (Bruker microCT N.V.) with a spatial resolution of 17.75 μm (X-ray source 70 KV/357 μA; exposure time 250 msec; magnification, ×15; 1.0 mm aluminum filter applied). Volumetric reconstructions and analyses were performed using built-in software NRecon 1.6 and CTAn 1.8 (Bruker microCT N.V.). The parameters measured were: Percent bone volume; bone surface/volume ratio (BS/BV); trabecular number (Tb.N); trabecular separation; trabecular thickness (Tb.Th); and structure model index ([@b29-ijmm-45-05-1543]).

Proteome analysis
-----------------

After 12 weeks of treatment with osteoking, the protein expression levels of the osteoking group and the control group were measured by label-free quantification (Genecreate). The basic principle was based on the extraction peak area of the peptide segment parent ion. Then, the peptide segments and proteins in the sample were identified, and the identified peptide fragments/proteins were quantitatively analyzed. The two groups were designated as the osteoking group and the control group. Following removal of redundant sequences, the data contained 36,728 protein sequences from the UniProt database ([@b30-ijmm-45-05-1543]). The proteins were quantitatively analyzed by Skyline software (version 3.5; SkylineGlobal Co.), and the differences were identified using the cut-off \|fold change\| ≥2.

RT-qPCR
-------

Total RNA from cells and tissue samples was separately isolated using TRIzol^®^ reagent (Thermo Fischer Scientific, Inc.) according to the manufacturer's protocol. The primer sequences refer to the National Center for Biotechnology Information database ([@b31-ijmm-45-05-1543]) and were assessed by Oligo Calc. (<http://biotools.nubic.northwestern.edu/OligoCalc.html>) ([@b32-ijmm-45-05-1543]). The primers were designed as following: β-actin, forward 5′-TCTGAACCCTAAGGCCAACC-3′, and reverse 5′-TACGTACATGGCTGGGGTGT-3′; Bmp-2, forward 5′-GTGCCCCCTAGTGCTTCTTAG-3′, and reverse 5′-CACCATGGTCGACCTTTAGGA-3′; Hsp 90-β, forward 5′-GCCCTGGACAAGATTCGGTA-3′, and reverse 5′-ATCTTCAGCTCTTTCCCGCTG-3′. The thermocycling conditions: Initial denaturation is 95°C for 30 sec; 30 of cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec and elongation at 72°C for 20 sec; and a final extension is 72°C for 1 min). For RT-qPCR, cDNA was prepared from 2 μg RNA using a Prime Script RT Reagent kit (Takara Biotechnology Co., Ltd.) and analyzed with SYBR Green Master Mix (Takara Bio, Inc.) in an LC480 real-time PCR system (Roche Diagnostics). The data were quantified using the relative quantitative 2^−ΔΔCq^ method and were normalized to β-actin expression data ([@b33-ijmm-45-05-1543]).

Western blot analysis
---------------------

Protein lysate (1 ml) with 10% PMSF (Dalian Meilun Biology Technology Co., Ltd.) was added into 100 mg bone or 1×10^6^ cells on the ice for 30 min. The samples were tested using BCA protein assay kit and adjusted as a same concentration 1 μg/ul. The loading quality of β-actin was 5 μl, and the loading quality of targets is 20 μl. Precast gel (4--20%) and PVDF were used. BSA (10%) blocked the PVDF at room temperature for 1 h. Anti-β-actin (1:5,000; cat. no. A1978; Sigma-Aldrich; Merck KGaA), anti-HSP 90-β (1:3,000; cat. no. 11405-1-AP; ProteinTech Group, Inc.) and anti-BMP-2 (1:2,000; cat. no. 18933-1-AP; ProteinTech Group, Inc.) were incubated at 4°C overnight, as described previously. Then, 1:5,000 HRP-anti-Mouse (H+L; 1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) or HRP-anti-Rabbit IgG (H+L; 1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) was incubated at room temperature for 2 h. One PVDF was added with 1ml super HRP-reagent (Dalian Meilun Biology Technology Co., Ltd.) and displayed using an automatic chemiluminiscence system (Tanon Science and Technology Co., Ltd.). The degree of gray density was analyzed using Image J software (version 1.46R, National Institute of Health.).

Bio-layer interferometry (BLI)
------------------------------

Biomolecular interactions were measured by the BLI method *in vitro* ([@b34-ijmm-45-05-1543]). All experiments were performed in 37°C PBS and AR2G biosensors were used in ForteBio Octet Red 96 (ForteBio Inc.). All samples were added into the black 96-well plate (Greiner Bio-One Co., Ltd.). Before protein immobilization, the baseline was established with PBS prewetted AR2G biosensor. Then, HSP90-β and MGP (Matrix gla protein) were fixed to the AR2G biosensors at 0.1 mg/ml. The binding time was 600s and the dissociating time was 1,200 sec. The curve was fitted globally with a 1:1 model (Octet Red system; version 7.0).

Statistical analysis
--------------------

All the experimental data were assessed using SPSS v21.0 statistical software (SPSS, Inc.), and the values are expressed as the mean ± standard deviation. Data distribution was determined by measuring kurtosis and skewness. Statistical significance was determined by a Student's t-test or ANOVA with Tukey's post hoc test. P\<0.05 was considered to indicate a statistically significant difference.

Results
=======

HSP90-β is the key target of osteoking in OP
--------------------------------------------

In our previous studies, osteoking was identified to have positive effects on OP/OPF rats ([@b19-ijmm-45-05-1543],[@b23-ijmm-45-05-1543]). The components of osteoking and its possible targets were obtained from the TCMSP database ([Fig. 1](#f1-ijmm-45-05-1543){ref-type="fig"}). Notably, based on these targets, functional enrichments suggested that osteoking was associated with drug metabolic processes, blood circulation, hemostasis, circulatory system processes and VEGF ([Fig. 2](#f2-ijmm-45-05-1543){ref-type="fig"}). These targets are indirectly associated with osteogenesis, but the related functions, including growth, repair and angiogenesis, may benefit OP/OPF. Based on gene data from the GEO database, the association between OP and osteoking focused on HSP90-β ([Fig. 3A](#f3-ijmm-45-05-1543){ref-type="fig"}). Importantly, HSP90-β was also confirmed to be highly expressed, as determined by proteomic results in OP rat models ([Fig. 3B](#f3-ijmm-45-05-1543){ref-type="fig"}). Thus, HSP90-β may be a key effector molecule of osteoking and serve an important role in the pathophysiological process of OP, as indicated by network-pharmacology analysis of the components of osteoking and the *in vivo* proteomic results.

Osteoking leads to calcification
--------------------------------

Postoperatively, the total BMD values of rats in the OVX and sham groups were assayed using dual-energy X-ray. At the 23rd week, there was a significant difference (P\<0.05) in BMD between the OVX group (0.177±0.006 g/cm^3^) and the sham group (0.188±0.008 g/cm^3^), suggesting that the OP rat model was successfully established. *In vivo*, the rat osteoporotic model was divided into three groups as aforementioned. Following treatment with osteoking, changes in BMD, which is the gold standard for evaluation of OP, were observed in each group ([Fig. 4A](#f4-ijmm-45-05-1543){ref-type="fig"}). The BMD in the osteoking group significantly increased at the 8 and 12th weeks compared with that of the negative control group (P\<0.05); however, the BMD in the osteoking group was decreased compared with that in the positive control group (P\<0.05). After 12 weeks of administration, the micro-CT results indicated that the number of bone trabeculae increased in the osteoking group ([Fig. 4B](#f4-ijmm-45-05-1543){ref-type="fig"}). The BS/BV ratio, Tb.Th and Tb.N in the osteoking group increased significantly compared with those of the negative control group (P\<0.05; [Fig. 4C](#f4-ijmm-45-05-1543){ref-type="fig"}). However, these parameters were all decreased compared with those in the positive control group (P\<0.05). At the initial time, all osteoporotic rats in the three groups exhibited the same baseline bone alkaline phosphatase (BALP; 21.55±1.54 U/l), PINP (280.63±75.27 pg/ml) and tartrate-resistant acid phosphatase-5β (TRACP-5β; 0.71±0.01 mIU/ml) values. BALP and PINP were used as bone formation markers. The osteoking group demonstrated significantly increased BALP and PINP after 12 weeks of treatment with osteoking compared with those of the negative control group (P\<0.05; [Fig. 4D](#f4-ijmm-45-05-1543){ref-type="fig"}). In addition, in the osteoking group, TRACP-5β, a bone resorption marker, was decreased compared with in that of the other two groups.

HSP90-β improves OP by enhancing BMP-2 expression in vivo
---------------------------------------------------------

After 12 weeks of treatment, osteoking led to calcification compared with that of the negative control group, and its mechanism was associated with HSP90-β ([Fig. 5A](#f5-ijmm-45-05-1543){ref-type="fig"}). Osteoporotic rats in the osteoking group exhibited significantly increased HSP90-β expression levels ([Fig. 5B](#f5-ijmm-45-05-1543){ref-type="fig"}). Moreover, increased BMP-2 expression levels were observed in the osteoking group compared with those of the negative control group.

Osteoking leads to high expression of HSP90-β and BMP-2 after 4 weeks of culture in vitro
-----------------------------------------------------------------------------------------

After 4 weeks of culture with osteoking, rBMSCs demonstrated similar *in vitro* results ([Fig. 5C](#f5-ijmm-45-05-1543){ref-type="fig"}). HSP90-β expression was significantly increased in the osteoking group compared with that of the control group, and BMP-2 expression was also increased.

Blocking HSP90-β decreases calcium deposition
---------------------------------------------

After 4 weeks of culture, inhibiting HSP90-β *in vitro* decreased calcium deposition in the osteoking + inhibitor group compared with the osteoking group (P\<0.05; [Fig. 6](#f6-ijmm-45-05-1543){ref-type="fig"}). The number of calcified nodules increased in the osteoking group compared with those of the control group (P\<0.05). In addition, the osteoking + inhibitor group exhibited more calcified nodules compared with those of the control group (P\<0.05).

Discussion
==========

At present, bioinformatics has contributed to the development of TCM. Proteomics has validated the predicted results of network pharmacology. Moreover, this method accurately demonstrated marked changes between the osteoking and control groups.

In the present study, osteoking improved OP in rats, through increasing the BMD and enhancing the bone intensity. The micro-CT results demonstrated improved micro-structure in the osteoking group, which suggests an improved resistance to biomechanical force in OP rats, which would help to avoid fracture. Measurement of resistance to biomechanical force should have been used in this study instead of the micro-CT. Previous studies have indicated similar improvements of the OP/OPF model ([@b13-ijmm-45-05-1543],[@b15-ijmm-45-05-1543],[@b18-ijmm-45-05-1543],[@b19-ijmm-45-05-1543],[@b20-ijmm-45-05-1543],[@b23-ijmm-45-05-1543]) and this is a limitation of the present study. Measurement of biomechanical force could be better to explain the effects of osteoking.

The results of the present study suggest that the key protein involved in osteoking-mediated effects on and the physiological process of OP is HSP90-β both *in vivo* and *in vitro*. After 12 weeks of administration, osteoporotic rats in the osteoking group had increased expression of HSP90-β compared with that of the negative control rats. The results demonstrated increased calcification and transiently increased osteogenic activity in the osteoking group compared with that of the negative control group. RBMSCs expressed increased HSP90-β levels *in vitro*, and inhibition of HSP90-β decreased calcium deposition. These data suggest that high HSP90-β is associated with increased calcification in the osteoking group, inferring that osteoking likely improves OP by regulating HSP90-β.

HSP90-β has been reported to contribute to the cell cycle, proliferation, migration and apoptosis ([@b35-ijmm-45-05-1543]). HSP90-β affects endothelial cells and their isoforms to promote angiogenesis ([@b36-ijmm-45-05-1543],[@b37-ijmm-45-05-1543]). By contrast, an HSP90-β inhibitor inhibited tumor growth through apoptosis, inducing cell cycle arrest and downregulating target proteins ([@b38-ijmm-45-05-1543],[@b39-ijmm-45-05-1543]). In addition, HSP90-β has been demonstrated to regulate bone metabolism ([@b40-ijmm-45-05-1543]).

Firstly, the present study revealed that osteoking directly stimulated rBMSCs, and that the increased HSP90-β expression was a reaction to the drug ([@b41-ijmm-45-05-1543]). These results suggest that high HSP90-β expression was not just a response to the drug, as osteoking promoted rBMSC proliferation *in vitro* ([Fig. S3](#s1-ijmm-45-05-1543){ref-type="supplementary-material"}).

Hsp 70 promotes calcium deposition by binding to matrix Gla protein (Mgp), and Pro64 and Gla residues are required for this binding ([@b42-ijmm-45-05-1543]). HSP90-β has similar domains to those of Hsp 70, and HSP90-β binding to Mgp was investigated. Notably, the molecular interaction results did not suggest that HSP90-β binds to Mgp ([Fig. S4](#s1-ijmm-45-05-1543){ref-type="supplementary-material"}).

Bioinformatics analysis indicated that the BMP signaling pathway was associated with the mechanism of action of osteoking. *In vivo* and *in vitro* data demonstrated that high HSP90-β was accompanied by high BMP-2 levels. High BMP-2 levels led to calcium deposition and osteogenesis. Low-intensity pulsed ultrasound, heating and cyclic tension have been reported to promote osteogenesis through upregulation of the HSP90 and BMP signaling pathways. High expressions of HSP90-β and BMP-2 was a common phenomenon of physical treatments for OP. HSP90-β promotes osteogenesis by enhancing BMP-2 ([@b43-ijmm-45-05-1543],[@b44-ijmm-45-05-1543]).

In clinical settings, HSP90-β inhibitors have demonstrated potential effects on disease, in particular HSP90-β inhibitor-based suppression of tumor angiogenesis ([@b45-ijmm-45-05-1543]). HSP90-β inhibitors have been studied for the treatment of atherosclerosis, due to their effects on decreasing calcification ([@b46-ijmm-45-05-1543]). Following the inhibition of HSP90-β, decreased calcium deposition was observed in the osteoking + inhibitor group compared with that of the osteoking group. HSP90-β is an effective target of the osteoking-mediated effects on OP, and high HSP90-β expression promoted calcium deposition. However, calcium nodes were still observed following the inhibition of HSP90-β. These data suggest that other components of osteoking also promote calcium deposition.

In the *in vitro* experiments, a limitation of the present study was the lack of a positive drug group. It has been previously reported that osteoking promoted the differentiation of BMSCs into osteoblasts, lipoblasts and chondroblasts ([@b47-ijmm-45-05-1543]). In the present study, high expression levels of HSP90-β promoted the differentiation of rBMSCs into osteoblasts. High BMP-2 expression levels leads to an improvement of the symptoms of OP ([@b48-ijmm-45-05-1543]). However, the mechanism between high expressions of HSP90-β and BMP-2 is unknown. A positive drug group could be helpful to explain the mechanism of action and further study is required. HSP90-β was an effective osteoking target that improved OP by enhancing BMP-2.

In addition, HSP90-β expression is also associated with blood circulation. Osteoblasts are more effective compared with osteoclasts in accelerating the exchange of oxygen-carbon dioxide, nutrient-toxic substances and serum, and increasing BMD ([@b49-ijmm-45-05-1543]--[@b51-ijmm-45-05-1543]). HSP90-β improves the bone microenvironment by activating angiogenesis, which should be investigated in future studies.

In conclusion, osteoking is a potential treatment for OP. Osteoking contains 7 components, and its mechanism is associated with the regulation of HSP90-β. Osteoking enhances HSP90-β expression levels and improves OP by upregulating BMP-2. Further examination of the association of HSP90-β and BMP-2 in OP would be valuable.

Supplementary Information
=========================

Not applicable.

Funding
=======

The present study was supported by National Science Foundation of China-Yunnan Province Joint Fund (grant no. U1502227).

Availability of data and materials
==================================

The data used to support the findings of this study are available from the corresponding author upon request.

Competing interests
===================

The authors declare that they have no competing interests.

BMD

:   bone mineral density

BMC

:   bone mineral content

BALP

:   bone alkaline phosphatase

BPs

:   bisphosphonates

ERT

:   estrogen replacement

OVX

:   ovariectomized

PINP

:   procollagen I N-terminal peptide

rBMSC

:   rat bone mesenchymal stem cell

SERM

:   selective estrogen receptor modulator

TCM

:   Traditional Chinese Medicine

Authors' contributions
======================

HBZ, WHL and ZQS designed this study. YS, DZ and RC performed all the experiments. YS and WHL drafted the manuscript. HBZ and ZQS gave suggestions and corrected the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
==========================================

This study is approved by Animal Experimental Ethical Committee of Kunming Medical University (approval no. KMMU 2015007).

Patient consent for publication
===============================

Not applicable.

![Components of osteoking. A total of 7 natural plants constitute osteoking.](IJMM-45-05-1543-g00){#f1-ijmm-45-05-1543}

![Functional enrichments of the relative targets of osteoking. The top 20 enriched signaling pathways from the Gene Ontology database.](IJMM-45-05-1543-g01){#f2-ijmm-45-05-1543}

###### 

HSP90-β is a key target of the osteoking-mediated effects on osteoporosis. (A) The association between osteoporosis and osteoking, as determined by the Traditional Chinese Medicine Systems Pharmacology and Gene Expression Omnibus databases. (B) The difference in HSP90-β protein levels between the osteoking and negative control groups after 12 weeks of treatment with osteoking. The y-axis represents the fold change. The values are expressed as the mean ± standard deviation (n=6). ^\*^P\<0.05. Neg-control, negative control.

![](IJMM-45-05-1543-g02)

![](IJMM-45-05-1543-g03)

###### 

Osteoking leads to calcium deposition in osteoporotic rats. (A) Bone mineral density after 4, 8, and 12 weeks of administration. The values are expressed as the mean ± SD (n=6). ^\*^P\<0.05. (B) Micro-computed tomography results of femurs. Scale bar=1 mm. (C) Bone composition parameters. The values are expressed as the mean ± SD (n=6). ^\*^P\<0.05. (D) ELISA results of BALP and PINP after 12 weeks of administration. The values are expressed as the mean ± SD (n=6; ^\*^P\<0.05; SD, standard deviation; BALP, bone alkaline phosphatase; PINP, procollagen I N-terminal peptide). (E) ELISA results of TRACP-5β after 12 weeks of administration. \#, represents the value is out of the ELIASA sensitivity. The values are expressed as the mean ± SD (n=6 ^\*^P\<0.05; TRACP-5β, tartrate-resistant acid phosphatase-5β).

![](IJMM-45-05-1543-g04)

![](IJMM-45-05-1543-g05)

![](IJMM-45-05-1543-g06)

![](IJMM-45-05-1543-g07)

![](IJMM-45-05-1543-g08)

###### 

High HSP90-β expression enhances BMP-2 expression *in vivo* and *in vitro*. (A) IHC staining for anti-HSP90-β in the 3rd lumbar vertebra. The arrows denote the positive HSP90-β staining sections in the third lumbar vertebra. (B and C) The RT-qPCR and western blot analysis results after (B) 12 and (C) 4 weeks of administration *in vivo*. The values are expressed as the mean ± standard deviation (n=6). ^\*^P\<0.05. HSP90-β, heat shock protein HSP 90-β; BMP, bone morphogenic protein; IHC, immunohistochemical; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Neg-control; negative control.

![](IJMM-45-05-1543-g09)

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![Inhibition of HSP90-β decreases calcium deposition. The alizarin red staining intensities in osteoblasts was measured using a microplate reader. The values are expressed as the mean ± standard deviation (n=6). ^\*^P\<0.05. Scale bar=5 μM. HSP90-β, heat shock protein HSP 90-β; BMP, bone morphogenic protein; OD, optical density.](IJMM-45-05-1543-g12){#f6-ijmm-45-05-1543}

[^1]: Contributed equally
