Anti-Osteoporotic Effects of Kukoamine B in Osteoblast and Osteoclast Cells and Ovariectomized Mice

1 Department of Medical Genetics, Ajou University School of Medicine, Suwon 16499, Republic of Korea 2 Department of Biomedical Sciences, Ajou University Graduate School of Medicine, Suwon 16499, Republic of Korea 3 Nine B Company, Daejeon 34121, Republic of Korea 4 Natural Products Research Institute, Gyeonggi Institute of Science & Technology Promotion, Suwon 16229, Republic of Korea 5Department of Biomedical Laboratory Science, College of Life and Health Sciences, Hoseo University, Asan 31499, Korea 6 International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea 7 Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming 650200, China 8 Korea Food Research Institute, Seongnam 13539, Republic of Korea 9 Dongwoodang Pharmacy Co., Ltd., Yeongchen 38819, Republic of Korea


Introduction
Bone is a living organ that is constantly remodeled and is maintained by a balance between bone formation and resorption [1,2].Bone remodeling is a physiological condition that is regulated by replacing new bone formations by osteoblasts and removing old or damaged bone by osteoclasts [1].Osteoblasts, which are differentiated from mesenchymal stem cells, play an important role in bone formation by synthesizing various bone matrix proteins and transporting mineral into the matrix.Osteoclasts are differentiated from mononuclear cells of the monocyte/macrophage lineage, and they break down bone tissue that is essential for osteoblast new bone formation.Bone formation includes the proliferation and differentiation of osteoblasts via the activation of alkaline phosphatase (ALP), collagen synthesis, and the mineralization of bone [3].Bone resorption involves the differentiation of osteoclasts via the activation of tartrate-resistant acid phosphatase (TRAP) [4,5].The balanced relationship between bone formation and bone resorption is critical for maintaining bone strength and preventing bone loss [6]; however, an imbalanced regulation of these bone-remodeling processes leads to serious bone loss with metabolic bone disease, called osteoporosis [1].
Osteoporosis is a serious condition that causes a loss of bone density and strength, leading to increased painful bone fractures [7].Osteoporotic fractures become a serious health problem, particularly for aging postmenopausal women [8].Currently, various pharmacological treatments for osteoporosis therapies are proven to reduce fracture risks, such as inhibitors of bone resorption by osteoclasts (bisphosphonates), selective estrogen receptor modulators, and the monoclonal antibody to the receptor activator of nuclear factor kappa-B ligand (RANKL) or stimulators of bone formation by cells of the osteoblast lineage (parathyroid hormone analogs) [9].Natural herbal plants have been widely used for alternative medicinal purposes due to the potential fewer side effects for long-term use [10][11][12].Studies have suggested that pharmacological bio-active compounds from plats have beneficial therapeutic effects on herbal medicines [13,14].For the treatment of osteoporosis, many herbal medicines derived from natural products have been reported as having a long tradition of use with fewer negative effects [10,15].In addition, bio-active components and related properties for alternative treatments of osteoporosis have been identified [16][17][18][19].Because long-term treatment is required for osteoporosis, herbal medicine is a good therapy for preventing and treating osteoporosis.The natural plant Lycii radicis cortex (LRC) has been used as traditional medicine in East Asia, and physiological bio-active properties, such as apigenin, luteolin, kaempferol, quercetin, oleanolic acid, dihydrophaseic acid, and urosolic acid, were identified in LRC extract [20,21].Our previous study suggested that LRC extract promoted osteoblast differentiation and inhibited the loss of bone mineral density (BMD) in an osteoporotic mice model without negative side effects [22].Furthermore, LRC prevented osteoclast differentiation induced by RANKL through the down-regulation of osteoclastogenesis-related markers [23].Recently, dihydrophaseic acid 3'-O-beta-d-glucopyranoside was identified as a candidate bioactive compound for the anti-osteoporotic effects of LRC extract [24]; however, because a small amount of this compound is contained in LRC extract, other component(s) responsible for the bone formation enhancing effect of LRC may still be unidentified.
The aim of this study was to identify the other bioactive component(s) for anti-osteoporotic effects in LRC extract.First, a fractionation of the ethanol LRC extract was performed, and then a single compound was isolated.Next, the anti-osteoporotic effects of the compound on in vitro and in vivo osteoporosis models were investigated.

A Bioactive Component Promoting Osteoblast Differentiation Was Isolated from LRC Extract
Our previous study reported that the ethanol extract of LRC induced osteoblast differentiation in preosteoblast MC3T3-E1 cells and prevented the loss of bone mineral density in ovariectomized (OVX) mice [22].The study identified the 13 main constituents using a highperformance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry system [22].In the present study, a fractionation of the bioactive component from LRC ethanol was performed (Figure S1).Seventy percent of LRC ethanol extract was fractionated, and each fraction was investigated for a bone formation-enhancing effect using an alkaline phosphatase (ALP) activity assay in preosteoblast MC3T3-E1 cells for identifying the bio-active fraction.ALP is a homodimeric protein enzyme located in the cell membrane of osteoblasts and is a reliable marker of bone metabolism during osteoblast differentiation [25].
Therefore, an ALP assay was conducted to screen the bioactive fraction on bone formation.
Each fraction was treated in an osteoblastic cell line for three days, and osteoblast differentiation was measured by the ALP assay.Consequently, a bioactive single compound, Kukoamine B (KB), was isolated from LRC extract and identified by nuclear magnetic resonance (NMR) and mass spectrometry analyses (Figure 1 and Figure S2).KB is a natural spermine alkaloid compound that was first isolated from LRC [26].It has been reported that KB neutralizes both lipopolysaccharides (LPS) and oligodeoxynucleotides containing CpG motifs (CpG DNA) and inhibits LPS-or CpG DNA-mediated proinflammatory signal transduction and cytokine expression [27,28].The biological characteristics of KB on osteoporosis have not been studied.

KB Increased Osteoblast Differentiation and the Mineralized Nodule Formation of Preosteoblastic MC3T3-E1 Cells
First, the effects of KB on osteoblast differentiation in the MC3T3-E1 cell line were examined.Osteoblast differentiation is characterized by three stages: (1) cell proliferation, (2) matrix maturation, and (3) matrix mineralization [29].To evaluate the effect of KB on osteoblast differentiation, orthodox methods were used, including cellular proliferation, ALP activity, and mineralization.ALP activity plays a crucial role in the mineralization of newly formed bone and increases during osteoblast differentiation [30,31].Cells were cultured with three different concentrations of KB (5, 10, and 20 μM), and ALP activity was assessed at 2, 3, 4, and 5 days (Figure S3).ALP activities in the KB treated cells (5 and 10 μM) were increased at 3 and 4 days, but they decreased at day 5 (Figure S3).After 5 days incubation with KB (5, 10, and 20 μM), cell viability was analyzed using a water-soluble tetrazolium salt (WST) assay.
A higher ALP activity was observed for the treatment of 10 and 20 μM KB with increased ALP positive staining colonies in osteoblastic MC3T3-E1 cells compared to the control group (Figures 2A and 2C).KB of 5, 10, and 20 μM did not affect the proliferation of osteoclastic cells (Figure 2B).Next, the effects of KB on mineralized nodule formation in MC3T3-E1 cells were examined.Bone matrix is mineralized by osteoblast differentiation, leading to the induction of calcium and phosphate-based minerals.Consequently, bone mineralization with several matrix proteins develops [32].Many studies have suggested that the mineralized osteoblast is a characteristic method for testing the effects of drug treatments on calcium deposition and bone formation [33,34].Alizarin red S is a histochemical method that is commonly used for evaluating calcium-rich deposits in the mineralization of osteoblast cells [35].Calcium phosphate and osteoblast mineralization with positive alizarin red S staining revealed successful mineralized osteoblast cells in vitro [36].The MC3T3-E1 cells were treated with osteoblast induction reagents (ascorbic acid and β-glycerophosphate) for 21 days, resulting in the mineralized nodule formation and the osteoblast mineralization of MC3T3-El cells.Cotreatment of KB (10 and 20 μM) with induction reagents presented higher positive alizarin red S staining colonies than the non-treated control cells.These results demonstrated that KB enhanced the differentiation and mineralized nodule formation of bone-forming osteoblasts.

KB Decreased Osteoclast Differentiation of Primary-Cultured Monocytes
The imbalanced regulation of the bone-remodeling process promotes abnormal metabolic bone diseases, especially osteoporosis [1].Osteoporosis, particularly for postmenopausal women, increases the bone resorption process because osteoclast differentiation occurs rather than bone formation, resulting in an enhancement of the risk of bone fragility and fractures [37].To examine the effects of KB on bone resorption, whether KB promoted the cellular differentiation of osteoclast cells isolated from the bone marrow of six-week-old mice was investigated.Monocytes from mouse bone marrow were successfully isolated and confirmed by a fluorescence-activated cell sorting (FACS) analysis with monocyte-specific surface markers (CD11b antibody) (Figure 3A).Monocytes are capable of osteoclast differentiation under a suitable microenvironment [38].For the differentiation of primary-cultured monocytes to osteoclasts, cells were treated with macrophage colony-stimulating factor-1 (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL).During a period of osteoclast differentiation, the treatment of KB did not affect the proliferation of primary-cultured monocytes of mouse bone marrow (Figure S4).After the induction of osteoclast differentiation, KB (5, 10, and 20 μM) was treated in the primary monocytes for Bone remodeling is regulated by two processes: bone formation by the differentiation of osteoblasts and bone resorption by the differentiation of osteoclasts in the surface of the bone [32].The relationship between bone formation and bone resorption includes the interaction of various cell types during bone remodeling [39,40].Osteoclast and osteoblast cells communicate with each other through gap junctions [39].The effects of KB on bone formation under the more physiological conditions of a co-culture system of preosteoblast MC3T3-E1 cells and primary-cultured monocytes were investigated.We established the co-culture system of preosteoblasts and preosteoclasts based on previous co-culture studies [41][42][43].The osteoblast differentiation of co-cultured cells was induced by the treatment of ascorbic acid and β-glycerophosphate for 3 or 6 days, and the effects of co-treated KB (5, 10, and 20 μM) on osteoblast and osteoclast differentiations were examined.KB treatment increased ALP activity compared to the non-treated control cells, and a higher ALP activity was observed at 3 days of KB treatment rather than 6 days (Figure 4A and Figure S6A); however, KB did not influence TRAP activity in a co-cultured system at either 3 or 6 days of incubation (Figure 4B and Figure S6B).The homeostasis of bone remodeling is maintained by the balance of bone resorption and bone formation [1].A previous study demonstrated that a co-culture of preosteoblasts and preosteoclasts enhanced both osteoblast and osteoclast differentiation [41]; however, other studies have suggested that co-cultured cells enhanced osteoblast differentiation but reduced osteoclast differentiation [42,43].In the present study, KB treatment promoted osteoblast differentiation but did not affect osteoclast differentiation in the co-culture system, indicating that KB plays a key role in osteoblast differentiation during the bone remodeling process.

KB Inhibited BMD Loss in OVX mice
It is well-known that OVX mice present a reduced bone mass and quality with significantly decreased BMD and bone mineral content (BMC) [44].Based on the in vitro study, the antiosteoporotic effects of KB in OVX mice was investigated.Seven-week-old female ddy mice underwent either an ovariectomy or sham-operated surgery (Sham).After surgery, the mice were divided into four groups: Tb.Th, and Tb.N and increased Tb.Sp compared to the Sham group, indicating OVX-induced osteoporotic-bone loss was processed; however, BMC and food intake did not differ among the Sham, OVX control, or KB-administered groups (data not shown).KB administration inhibited the OVX-induced BMD loss in the right femur bone and restored the impaired bone structural properties of BV/TV, Tb.Th, Tb.N, and Tb.Sp compared to the OVX control group (Figure 5).These results demonstrated the anti-osteoporotic effects of KB in vivo.

Fractionation, Isolation, and Structure Elucidation of the Bioactive Component
The dried LRC (1 kg) was extracted with 70% ethanol at room temperature, and the ethanol solution was then evaporated under reduced pressure.The 70% ethanol extract (97 g) was chromatographed on a Diaion HP-20 gel column and eluted with an H2O-Ethanol gradient system to obtain four fractions (A-D).Fraction B (11 g) showed that the most potent ALP activity was chromatographed on a RP-18 column (3×55 cm) and eluted with an H2O-methanol gradient (from 90:10 to 0:100, 800 mL for each step) to yield five fractions (B1-B5).Fraction B4 (98 mg) was further chromatographed on a RP-18 column (2×30 cm) and eluted with H2Omethanol (from 90:10 to 0:100, 400 mL for each step) to obtain a bioactive component (20 mg) (Figure S1).The structure of the bioactive component was elucidated by proton nuclear magnetic resonance ( 1 H-NMR), carbon-13 nuclear magnetic resonance ( 13 C-NMR), and mass spectrometry analyses (Figure S2) as well as by a comparison with the previously reported data [26] (Figure S2).

Cell Culture and Reagents
A mouse MC3T3-E1 cell line was purchased from the RIKEN Cell Bank (Tsukuba, Japan) and cultured in an α-modified minimal essential medium (α-MEM) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml).Osteoblast differentiation was induced by adding an osteogenic medium containing ascorbic acid (50ug/ml) and β- Glycerophosphate (10 mM) after allowing 24 h for cell adherence (day 0).For the primaryculture of monocytes, bone marrow cells were flushed from the femoral bones of 6-week-old mice with a fine-bore syringe in the presence of 1 mM ascorbate-2-phosphate (Sigma-Aldrich; St. Louis, USA).The isolated monocyte cells were identified by immunophenotypic analysis with a CD11b antibody (BioLegend; San Diego, USA) using an FACS Aria III cell sorter (BD Biosciences, San Jose, USA) and FACS Diva software (BD Biosciences).Monocyte cells were cultured in an α-MEM medium in the presence of 30 ng/ml of M-CSF (PeproTech; Rocky Hill, USA) and 50 ng/ml of RANKL (PeproTech) for the induction of differentiation from preosteoclasts to osteoclasts [45].For the co-culture system, MC3T3-E1 (2×10 4 cells/well) cells and primary monocytes (4×10 4 cells/well) were co-cultured in osteoblast differentiation media containing ascorbic acid (50 µg/ml) and β-glycerophosphate (10 mM).All cultured cells were incubated in a humidified atmosphere at 37°C and 5% CO2.The cells were used at passages 5-10 after purchase for all experiments.The medium was changed every three days.

WST Assay and ALP Activity Assay and Staining
The primary-cultured monocytes and MC3T3-E1 cells (3×10 3 cells/well) were incubated in a 96-well plate overnight and co-treated with different concentrations of KB (5, 10, and 20 μM).
Cell viability was determined by a WST assay using an EZ-Cytox Cell Viability Assay Kit (Daeil; Seoul, Korea).A WST solution (20 μl, 5 mg/ml in phosphate-buffered saline) was added to each well, the cells were incubated for another 4 h, and the media were carefully removed.Formazan crystals were dissolved in acidified isopropyl alcohol (40 mM HCl in isopropanol), and their absorbances were measured at 450 nm and 655 nm using a microplate reader (BIO-RAD; Hercules, CA, USA).Mouse MC3T3-E1 cells were lysed at 4°C in a buffer containing 1 mmol/l Tris-HCl (pH 8.8), 0.5% Triton X-100, 10 mmol/l Mg 2+ , and 5 mmol/L p-nitrophenylphosphate as substrates.After homogenization, ALP activity was measured at the absorbance of 405 nm (BIO-RAD; Hercules, CA, USA).For ALP staining, cells were fixed in cold 4% paraformaldehyde for 10 min and washed with PBS three times.The fixed cells were stained with a BCIP/NBT (Sigma-Aldrich; St. Louis, USA) for 30 min at room temperature.
ALP-positive cells were determined under a light microscope.

Mineralized Nodule Formation in Osteoblast Cells
Preosteoblast MC3T3-E1 cells were cultured in a 48-well plate overnight and treated with 50 μg/ml of ascorbic acid and 10 mM of β-glycerophosphate with or without treatment with KB (10 and 20 μM) for three weeks.The cells were fixed with cold 70% ethanol for 10 min at room temperature and washed with water.Calcium deposits in the mineralized cells were determined by Alizarin red S (Sigma-Aldrich; St. Louis, USA) staining.Positive Alizarin red S staining was determined using a light microscope.

Co-Culture System of MC3T3-E1 cells and Primary Monocytes
For the co-culture system, MC3T3-E1 (2×10 4 cells/well) cells were cultured in a 48-well plate overnight, and the isolated monocytes (4×10 4 cells/well) from the mouse bone marrow were added to the MC3T3-E1 cells and incubated for 1 day.The co-culture of MC3T3-E1 cells and primary-cultured monocyte cells was co-incubated with an osteoblast differentiationinduction media containing ascorbic acid (50 µg/ml) and β-glycerophosphate (10 mM) with or without treatment with KB (10 and 20 μM) for either three or six days.The differentiations between osteoblast and osteoclast were measured by ALP activity and TRAP activity, respectively.

TRAP Staining and Activity Test
After the induction of the primary-cultured monocytes, the medium was removed and gently washed with PBS.The differentiated osteoclast cells from the monocytes were measured by a TRAP activity assay and were further stained using an Acid-Phosphatase Kit (Sigma-Aldrich; St. Louis, USA).The absorbance was measured at 405 nm (BIO-RAD; Hercules, CA, USA), and TRAP activity was expressed as the percentage of the untreated control.For TRAP staining, the cells were fixed in cold 4% paraformaldehyde for 10 min and washed with PBS.
The fixed cells were stained with an Acid phosphatase and Leukocyte kit (Sigma-Aldrich; St. Louis, USA), for 2 h at room temperature.TRAP-positive multinucleated cells containing three or more nuclei were counted under a light microscope.

Measurement of BMD and Micro-CT Imaging in Bones
After anesthetization using tiletamine/zolazepam (Zoletil; Virbac Laboratories, Carros, France), the mice were placed on the specimen tray for measurements.The right femur of BMC and BMD were measured using a PIXI-mus bone densitometer with on-board PIXI-mus software (GE Lunar, Madison, USA) for small animals, and it was adjusted in relation to body weight.Transverse micro-CT images of the right femur were scanned using a high-energy spiral scan micro-CT, the BrukermicroCT (Skyscan 1173, Belgium), with a voltage of 60kV, a current of 400uA, an exposure time of 400 ms, a magnification of M-H, a CCD readout of 1280×1280, and rotation steps of 360.Reconstruction was performed using NRecon software (BrukermicroCT).Two-dimensional axial and three-dimensional images were reconstructed for qualitative and quantitative analyses.For the region of interest (ROI) measurements and analyses, axial images were displayed using Inveon Research Workplace (Siemens).Axial reformats were performed to allow slice-by-slice, manual tracing of the contours of the trabecular bone.For the cross-sectional study of the trabecular bone, a region was analyzed approximately 300 μm below the growth plate.The analysis provided information about the main histomorphometric parameters, including bone volume (BV/TV, %), trabecular number (Tb.N, 1/mm), thickness (Tb.Th, mm), and spacing (Tb.Sp, mm).All the experiments were repeated at least three times unless stated otherwise, and the results were presented as the means ± standard deviation, as indicated.A statistical software package (SPSS 11.0 for Windows, SPSS Inc., Chicago, IL, USA) was used to perform the statistical tests.The statistical significance of the differences was assessed by the Student's ttest.A probability value (p) less than 0.05 (p<0.05) was considered statistically significant.The results were expressed as mean ±SEM.

Conclusion
This is the first study to evaluate the anti-osteoporotic effect of Kukoamine B (KB) isolated

Figure 1 .
Figure 1.Chemical structure of the isolated Kukoamin B.

Figure 2 .
Figure 2. Effects of Kukoamin B (KB) on cellular differentiation, proliferation, and mineralized nodule formation of the preosteoblast MC3T3-E1 cells.(A) Assessment of the alkaline phosphatase (ALP) activity in the KB-treated MC3T3-E1 cells.After induction of osteoblast differentiation with 50 μg/mL of ascorbic acid and 10 mM of β-

Figure 3 .
Figure 3. Effects of Kukoamin B (KB) on osteoclast differentiation of primary-cultured monocytes.(A) Validation of successful isolation of monocytes from mouse bone marrow.Primary-cultured monocytes were identified by an immunophenotypic analysis with a monocyte-specific surface positive marker (PE-conjugated CD11b antibody) using a fluorescence-activated cell sorting (FACS) analysis.(B, C) Assessment of tartrateresistant acid phosphatase (TRAP) activity in the KB-treated monocyte cells.After induction of osteoclast differentiation, cells were treated with KB (5, 10, and 20 µM) for 6 days, and then TRAP activity was assessed (B).The cells were also stained with a TRAP staining kit, and the differentiated osteoclast cells were visualized under a microscope (C).Control: non-induction of osteoclast differentiation.Induction: induction of osteoclast differentiation with 30 ng/mL of a macrophage colony-stimulating factor (M-CSF) and 50 ng/ml of a receptor activator of nuclear factor kappa-B ligand (RANKL).All of the experiments were repeated three times.*: p < 0.05 vs. Induction.

Figure 4 .
Figure 4. Effects of Kukoamin B (KB) on osteoblast and osteoclast differentiation in the co-culture of preosteoblasts and primary monocytes.Co-cultured MC3T3-E1 and primary monocyte cells were treated with osteoblast differentiation reagents, ascorbic acid and βglycerophosphate, and then co-treated with KB (5, 10, and 20 μM) for 3 days.Alkaline

( 1 )Figure 5 .
Figure 5. Effects of Kukoamin B (KB) on the improvement of bone mineral density (BMD) and bone structural properties in ovariectomized (OVX) osteoporosis model mice.The OVX mice were administered with KB (2 or 5 mg/kg/day) for 12 weeks.Sham: sham operated group (n=5), OVX: KB non-administered mice group (n=5).(A) BMD of right femur was measured using a PIXI-mus bone densitometer at 0, 6, and 12 weeks.(B) Transverse microcomputed tomography (micro-CT) images of the right femur were scanned at the end of the experiment.(C) Trabecular bone structural properties, including bone volume (BV/TV), trabecular thickness (Tb.Th), number (Tb.N), and spacing (Tb.Sp), were analyzed at the end of the experiment.*: p < 0.05 vs. OVX group.
from LRC in vitro and in vivo.The in vitro experiments demonstrated that KB enhanced the differentiation and mineralized nodule formation of preosteoblastic MC3T3-E1 cells and inhibited the osteoclast differentiation of preosteoclast-lineage monocytes isolated from mice bone marrow.In a co-culture of MC3T3-E1 cells and primary-cultured monocytes, KB treatment promoted osteoblast differentiation but not osteoclast differentiation.The in vivo experiments demonstrated that KB significantly improved OVX-induced bone mineral density loss and impaired bone structural properties in osteoporosis model mice.The results suggest that KB may be a good therapeutic candidate for the treatment and prevention of osteoporosis.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 October 2017 doi:10.20944/preprints201710.0196.v1
The OVX and sham-operated 8-week-old female ddY mice were purchased from Shizuoka Laboratory Center Inc. (Hamamatsu, Japan).The mice were acclimated for 10 days prior to experimentation.The mice were maintained on a diet (5.0 g/day) of Formula-M07 (Feedlab The BMD and BMC were measured at 0, 6, and 12 weeks during the experiment.The animal research procedures were approved by the Animal Care and Use Committee of the Ajou University School of Medicine (IACUC No. 2014-0066), and all experiments were conducted in accordance with the institutional guidelines established by the committee.All efforts were made to minimize animal suffering and to reduce the number of mice used.