Crataegus pinnatifida Bunge Inhibits RANKL-Induced Osteoclast Differentiation in RAW 264.7 Cells and Prevents Bone Loss in an Ovariectomized Rat Model

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.


Introduction
Osteoporosis is characterized by a decrease in bone microarchitecture and an increased risk of fracture [1]. Bone remodeling is balanced between bone formation by osteoblasts and bone resorption by osteoclasts [2]. However, the excessive activity of osteoclasts induces osteoporosis, rheumatoid arthritis, and periodontitis. us, the inhibition of the osteoclast differentiation and its activity plays a role in the treatment strategy of osteoporosis.
Bisphosphonate and selective estrogen receptor modulators (SERMs) are frequently used as treatments. However, long-term treatment of these agents causes side effects such as Paget's disease of bone, breast cancer, prostate cancer, hot flashes, and night sweats [7][8][9][10]. erefore, there is a need for integrating complementary and alternative medicines for osteoporosis based on natural products with few side effects. Consequently, the importance of developing an alternative treatment for osteoporosis has increased currently.
Crataegus pinnatifida Bunge (CPB) is the dried fruit of Crataegus pinnatifida Bung, called "Sansa" in Korea [11]. Previous studies have shown that CPB has antioxidant and anti-inflammatory effects [12,13]. Chlorogenic acid is the major component of Crataegus pinnatifida Bunge and has an inhibitory effect on osteoclast differentiation induced by RANKL [14]. It has also been linked to anti-inflammatory and antioxidant effects [15,16]. Osteoporosis is caused by endocrine, metabolic, and mechanical factors. Furthermore, recent studies have shown that the risk of developing osteoporosis is increased in inflammatory conditions [17,18]. erefore, we hypothesize that CPB may have a positive effect on bone metabolism.
In this study, we investigated the in vitro effects of CPB on RANKL-induced osteoclast differentiation. In addition, we also investigated the in vivo effects of CPB on bone loss in an ovariectomized (OVX) model. e sample extract was prepared by decocting 600 g dried herb with 6 L boiling distilled water (dH 2 O) for 2 h. Next, the filtrate was evaporated using a vacuum evaporator and freeze-dried into powder. e yield from the dried herbs was 39.6% (freezedried powder: 237.8 g), and the powder was subsequently stored at −20°C.

High-Performance Liquid Chromatography Analysis.
Quantitative analysis of main components in CPB was performed using an A Waters 2695 system equipped with a Waters 2487 Dual λ absorbance detector and X-bridge C18 Column (250 mm × 4.6 mm, 5 μm). CPB dissolved in dH 2 O. CPB was passed through a 0.2-μm membrane filter and 10 μL volume of the filtrate was injected into the HPLC column.
e mobile phases are composed of solvent A (acetonitrile) and solvent B (H 2 O (1% acetic acid)). e detection time was 0-30 min. e flow rate was 1.0 mL/min.

Cell Culture and Cell Viability.
e RAW 264.7 cells were purchased from Korean Cell Line Bank (Seoul, Korea). RAW 264.7 cells were cultured in DMEM supplemented with 1% P/S and 10% FBS. e cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 ( ermo Fisher, Waltham, MA, USA). e MTS assay was performed to examine the toxicity of CPB on RAW 264.7 cells. RAW 264.7 cells were seeded at a density of 5 × 10 3 cells/well in a 96-well plate.
e CPB was administered at 125, 250, 500, and 1000 μg/mL for 24 h. Afterwards, 20 μL MTS solution was added to the wells for 2 h. e absorbance (490 nm) was measured by an enzyme-linked immunosorbent assay (ELISA) reader. Results were indicated as a percentage of the control. Cytotoxicity was considered as cell viability less than 90% of the control.

TRAP Staining and Pit
Assay. RAW 264.7 cells were seeded at a density of 5 × 10 3 cells/well in 96-well plate. After 24 h, RAW 264.7 cells were differentiated with α-MEM supplemented with 1% P/S, 10% FBS, and RANKL (100 ng/ mL). e media was changed every 2 days. After 5 days, the osteoclast cells were fixed with 10% formalin for 10 min and then stained using a TRAP staining kit, according to the manufacturer's instructions. Afterwards, cells were rinsed with dH 2 O and dried at room temperature. Multinucleated osteoclasts were considered as TRAP-positive cells with three or more nuclei (red color). To measure TRAP activity, differentiation medium was transferred to new 96-well plates and TRAP solution (4.93 mg pNPP + 850 μL 0.5 M acetate solution + 150 μL tartrate solution) was added to 96-well plate at 37°C for 1 h. TRAP activity was measured at 405 nm by an ELISA reader. To examine pit formation, RAW 264.7 cells were seeded at a density of 5 × 10 3 cell/well in a multiple well osteo assay surface plate and incubated for 5 days.
ereafter, the cells were removed using NaClO. e pit area was measured by ImageJ version 1.46 (National Institutes of Health, Bethesda, MD, USA).
ereafter, total protein quantification was done using BCA assay according to the manufacturer's instructions. e protein samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. e membranes were blocked (5% skim milk) for 1 h and incubated overnight at 4°C with primary antibodies for β-actin (1 : 1,000), NFATc1 (1 : 1,000), and c-Fos (1 : 1,000). After 24 h, the membranes were incubated with secondary antibodies (1 : 10,000) for 1 h at room temperature. e protein was visualized by enhanced chemiluminescence (ECL) (Whatman plc; GE Healthcare) and protein band densitometry was measured by ImageJ version 1.46. All data were normalized to the β-actin density.

Reverse Transcription-Quantitative PCR (RT-qPCR).
RAW 264.7 cells were incubated with CPB (125, 250, 500, and 1000 μg/mL) for 4 days and the RANKL (100 ng/mL). Total RNA was extracted from RAW 264.7 cells using TRIzol reagent, according to the manufacturer's instructions. en, cDNA was synthesized using the reverse transcription kit (Invitrogen, Carlsbad, CA, USA). RT-qPCR was performed with a C1000 Touch ™ thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) and Taq polymerase. e PCR cycling conditions were initial denaturation cycle at 95°C for 5 min, followed by 30-40 cycles of amplification at 94°C for 30 sec, annealing at 53-58°C for 30 sec, and extension at 72°C for 30 sec. Primers for osteoclast-related genes are described in Table 1. e reaction was electrophoresed on 1-1.2% agarose gels stained with SYBR. e agarose gel was visualized using NαB I ™ (Neoscience, Suwon, Korea). e expression level of mRNA in the analyzed gene was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using ImageJ version 1.46.

Animal Experiments and Induction of OVX Rats.
Animal experiments were conducted in accordance with Guidelines for the Care and Use of Laboratory Animals approved by the Committee on Animal Experimentation of Kyung Hee University (KHUASP (SE)-15-101). Female Sprague-Dawley (SD) rats (12 weeks of age) were purchased from Nara Biotech (Seoul, Korea). SD-rats were housed at 22 ± 2°C, with a relative humidity of 53-55% in a 12 h light-dark cycle. In this study, all animals had ad libitum access to water and food. e SD-rats were acclimatized for one week before surgery. To establish an OVX model, the rats were anesthetized with 100% oxygen and 5% isoflurane to remove the ovaries. e sham group did not have their ovaries removed but received the same stress.
e rats were divided into five groups (n � 8 per group) as follows: (1)  OVX-induced, treated with 132 mg/kg CPB. e dose of CPB was calculated as follows: In Korean medicine, the recommended single dose for an adult is 8 g/60 kg, effectively equating to 3.168 g (yield, 39.6%) CPB powder of 8 g dried herbs.
erefore, the CPB-L group was administered 13.2 mg/kg CPB. Since the metabolism of rodents is faster than that of humans, the high-dose group was administered 10 times the concentration of the lowdose group [19].
us, CPB-H group was administered 132 mg/kg CPB. To prevent infection at the surgical site, all rats received injections with 4 mg/kg gentamicin for 3 days after surgery. E 2 and CPB were dissolved in dH 2 O and administered orally once per day for 8 weeks. Body weights were measured once a week. During the experiments, all animals showed no side effects and exhibited no abnormal behavior. After 8 weeks, the experimental animals were anesthetized with 100% oxygen and 5% isoflurane and sacrificed by lethal cardiac puncture and cervical vertebrae dislocation.

Hematoxylin and Eosin (H&E) Staining.
e fixed femur samples were decalcified in ethylenediaminetetraacetic acid (EDTA) for 4 weeks at room temperature. Afterwards, femur samples were dehydrated and embedded in paraffin. Femur samples were sectioned using a rotary microtome (5 μm-thick, ZEISS, Oberkochen, Germany), then dried and stained with H&E. Changes in tissue parameters, such as femoral head area, were observed using an inverted light microscope (magnification, 40x and 100x; Olympus Corporation, Tokyo, Japan). e trabecular area was measured by ImageJ version 1.46.
2.11. Immunohistochemistry Staining. Sectioned tissues were paraffinized and rehydrated to prepare for immunohistochemistry (IHC). Femur tissue slides were treated with 0.3% hydrogen peroxide-methanol to inhibit endogenous peroxidase. Subsequently, nonspecific reactions were blocked with normal serum for 1 h at room temperature. After washing thrice with PBS, sections were incubated with primary antibody at 4°C overnight and then incubated with secondary antibodies for 1 h at room temperature. e tissues were incubated with an ABC kit for 30 min at room temperature, followed by staining with DAB solution and counterstaining with hematoxylin. Histological changes were analyzed using a light microscope (magnification, 100x and 200x).
Evidence-Based Complementary and Alternative Medicine 2.12. Statistical Analysis. Data are presented as mean-± standard error (SEM) of the mean for three replicates. Differences between the control and CPB treatment groups were analyzed using one-way ANOVA followed by a Dunnett's post hoc in GraphPad PRISM version 5.01 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was determined at p < 0.05.

Quantitative Analysis of the CPB Extract.
HPLC was used to confirm the main component of CPB [20]. As shown in Figure 1, the retention times of CPB are identical to the retention times of the chlorogenic acid standards.

Effect of CPB on RANKL-Induced TRAP Activity and Pit
Formation. To determine the cytotoxic effect of CPB, RAW 264.7 cells and osteoclast were treated with CPB concentrations from 125, 250, 500, and 1000 μg/mL. In this study, none of the CPB concentrations affected cell viability in either RAW 264.7 cells or osteoclasts (Figures 2(a) and 2(b)).
To investigate the effect of CPB on RANKL-induced osteoclast differentiation and pit formation, TRAP staining and pit assay was used. RANKL increased the number of TRAPpositive cells and TRAP activity compared with the untreated control group, confirming osteoclast differentiation. CPB treatment of differentiated osteoclasts decreased the number of TRAP-positive cells and TRAP activity in a dosedependent manner. In addition, the pit area was increased with RANKL treatment, compared to untreated controls, and decreased by CPB treatment in a dose-dependent manner (Figures 2(c)-2(f )).

Effect of CPB on RANKL-Induced Expression of NFATc1 and c-Fos.
To examine the expression of NFATc1 and c-Fos, we performed western blotting (Figures 3(a) and 3(b)). NFATc1 and c-Fos expression were significantly increased in the RANKL-induced cells compared to the nonstimulated control group. erefore, the expressions of NFATc1 and c-Fos were suppressed by CPB in a dose-dependent manner.

Effect of CPB on RANKL-Induced of Osteoclast-Related
Genes. To investigate the effect of CPB on osteoclast-related genes in RANKL-induced RAW 264.7 cells, RT-qPCR was performed. Treatment with RANKL increased the mRNA levels of Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In contrast, CPB reduced these mRNA levels in a dose-dependent manner, the most effective dose in all instances (Figures 4(a) and 4(b)).

Effect of CPB on OVX-Induced Models.
To analyze the effect of CPB on OVX-induced postmenstrual osteoporosis, we orally administered E 2 , CPB-L, and CPB-H to the OVXinduced rats daily for 8 weeks. As shown in Figure 5(a), the body weight of both treated and untreated OVX-induced rats significantly increased after 3 weeks as compared to that of the sham group. However, there was no significant difference in body weight between the OVX group and E 2 , CPB-L, and CPB-H, respectively. e uterus weight decreased in the OVX group as compared with that in the sham group ( Figure 5(b)). Furthermore, the uterus weight increased in the E 2 group as compared with that in the OVX group, with no effect observed in CPB-L and CPB-H, femur weights significantly decreased in the OVX group compared to that in the sham group ( Figure 5(c)). However, there was no difference in femur weight between the E 2 , CPB-L, and CPB-H groups compared to the OVX group. Tibia weight and ash were decreased in the OVX group compared with the sham group (Figures 5(d) and 5(e)), though there were no significant differences between E 2 , CPB-L, and CPB-H, compared to the OVX group.

Effect of CPB on Bone Loss in OVX-Induced Models.
In the micro-CT image, the bone density in the femoral head of OVX group was decreased compared with the sham group ( Figure 6(a)). Furthermore, E 2 and CPB-H significantly increased bone density in the femoral head compared with the OVX group. From the results of the bone microstructure

Evidence-Based Complementary and Alternative Medicine
CPB-H groups had increased BV/TV but not significantly. In addition, SMI was increased in the OVX group compared to the sham group (Figure 6(d)). In contrast, SMI was reduced in all three groups: E 2 , CPB-L, and CPB-H, compared to the OVX group.

Effect of CPB on Trabecular Area and Expression of CTK in the Femoral Head.
To measure the trabecular area, bone tissues were stained with H&E (Figure 7(a)). To determine the effect of CPB treatments on the CTK in OVX-induced rats, we perform the IHC staining (Figure 7(b)). e trabecular area was decreased in the OVX group when compared with that of the sham group. Treatments with E 2 , CPB-L, and CPB-H inhibited the loss of the trabecular area compared with that of the OVX group (Figure 7(c)). Furthermore, OVX groups significantly increased CTK compared to the sham group. Concurrently, E 2 , CPB-L, and CPB-H groups reduced CTK compared to the OVX group (Figure 7(d)).

Discussion
According to recent studies, various side effects have been reported with the administration of bisphosphonate and SERM, which are currently used for the treatment of osteoporosis.
is has prompted many researchers to search for safer alternative medicinal agents with fewer side effects for osteoporosis treatment [7][8][9][10]. In this study, we examined the osteoclastogenesis and antiosteoporosis effects of CPB on RAW 264.7 cells. In vitro, CPB demonstrated an inhibitory effect on osteoclast differentiation by inhibiting transcription factors and osteoclast-related genes. In vivo, CPB also prevented bone loss in OVX-induced rat models.
TRAP is a known osteoclast phenotype marker, and TRAP staining is a standard method used to determine osteoclast expression and activation [21,22]. In the present study, TRAP staining results showed a significant decrease in TRAP-positive cells and TRAP activity following CPB treatment. Pit formation is commonly used to measure the osteoclasts' differentiation and bone resorption ability [23,24]. As a result of the experiment, CPB significantly Evidence-Based Complementary and Alternative Medicine 9 suppressed the pit area. It is unclear whether CPB reduces pit formation by inhibiting the ability of osteoclasts to bone resorption, or it controls pit formation by inhibiting osteoclast differentiation, but the TRAP staining and pit assay results, CPB, seem to regulate bone resorption by inhibiting osteoclast differentiation. Transcription factors, such as NFATc1 and c-Fos, are essential in osteoclast differentiation [25,26]. In a previous study, c-Fos-deficient cells were not able to differentiate into osteoclasts [11]. In contrast, excessive expression of c-Fos causes osteosarcoma and chondrosarcoma [27]. Furthermore, NFATc1-deficient mice develop osteopetrosis due to blocked osteoclast differentiation [28]. It has also been reported that embryonic stem cells deficient in NFATc1 cannot differentiate into osteoclasts upon RANKL stimulation [29]. erefore, c-Fos and NFATc1 are important factors for osteoclast differentiation [25,26].
e present study showed that CPB significantly decreased the expression of c-Fos and NFATc1 and subsequent osteoclast differentiation.
c-Fos regulates bone resorption markers, such as CA2, which acidifies the bone surface during bone resorption [30][31][32]. Furthermore, NFATc1 regulates the expression of osteoclast-specific genes such as TRAP, MMP-9, CTK, ATP6v0d2, and OSCAR [25]. MMP-9 and CTK are involved in the process of osteoclast differentiation and play an important role in osteoclast precursors and bone resorption [6,33,34]. MMP-9 has a negative correlation with BMD, and overexpression of MMP-9 attenuates osteoclast formation [35,36]. CTK is a cysteine proteinase mainly expressed in osteoclasts. CTK is known to play an important role in breaking down the organic phases of bone during bone resorption [37]. According to previous studies, a deficiency of CTK indicates an osteoporosis phenotype [38]. erefore, it was found that the deficiency of CTK is associated with the inhibition of osteoclast activity, and CTK is an effective target in the treatment of osteoporosis. ATP6v0d2 is an essential factor required for cell-cell fusion. Previous studies found ATP6v0d2-deficient mice present with an osteopetrosis phenotype due to abnormal osteoclast maturation [39,40]. OSCAR regulates osteoclast differentiation and cell maturation and is a costimulatory receptor for osteoclast differentiation through activation of NFATc1. It is known that OSCAR may contribute to the etiology and severity of osteoporosis and rheumatoid arthritis [41,42]. e present study showed that CPB significantly decreased the expression of osteoclast-related genes (Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2) in RANKL-induced osteoclast differentiation in RAW 264.7 cellsvia regulation of c-Fos and NFATc1 signaling.
OVX-induction is widely used in postmenopausal osteoporosis research. According to a previous study, OVXinduced rats share similar symptoms to human osteoporosis, such as the increase in body weight [43]. In addition, loss of uterus weight demonstrates that the postmenopausal osteoporosis model had been successfully established [44,45]. In this study, all OVX-induced rats, including CPB and E 2 treatment, increased body weight from week 4, while all groups, except for the E 2 -treatment, had decreased uterus weight.
ese results confirm previous studies that E 2 treatment reverses the effect of postmenopausal changes to uterus weight, while CPB had no effect in this regard.
Micro-CT is used to analyze the structural properties of bones in three dimensions [46,47]. Bone density and bone microstructure are indicators used to evaluate bone quality [48]. BV/TV represents the volume of bone within the volume of interest (VOI), whereas SMI refers to the structural morphology index of the cancellous bone [48,49]. According to a recent study, increased BMD is not sufficient to improve or prevent osteoporosis [50]. erefore, SMI is a complimentary representative index used for accurate bone quality assessment. In this study, the reduction in BMD, BV/ TV, and SMI was improved by the administration of E 2 and CPB-H. ese results suggest that CPB can be a treatment for postmenopausal osteoporosis through the prevention of bone loss.
As a result of histological examination, we showed that E 2 and CPB treatment prevented the decrease in the trabecular area, indicating that CPB inhibits bone loss of postmenopausal osteoporosis. IHC staining was used to measure the expression of bone-related factors in tissues. In this study, E 2 , CPB-L, and CPB-H groups suppressed the expression of CTK induced by OVX. Furthermore, this also correlates with the findings of the in vitro experiments. ese results further suggest that CPB inhibits bone resorption. In summary, CPB has antiosteoporotic effects on OVX-induced rats by suppressing BMD and bone resorption markers such as CTK. e limitations of this study are as follows: (i) MAPK and NF-κB signaling pathways play an important role in NFATc1 and c-Fos activation. In this study, CPB significantly inhibited the expression of NFATc1 and c-Fos, but MAPK and NF-κB pathways were not studied. erefore, it is still necessary to correlate the inhibitory effect of CPB to the MAPK and NF-κB signaling pathways. (ii) As patients with osteoporosis have already lost a certain amount of bone density, it is important to also promote osteoblast activity to restore the lost bone mass, along with osteoclast activity inhibitors to prevent disease progression. erefore, future studies should focus on the effects of CPB on promoting osteoblast differentiation. (iii) Treatment of osteoporosis remains focused on postmenopausal osteoporosis in type 1 osteoporosis. However, interest in male osteoporosis and senile osteoporosis is also increasing. erefore, studies on CPB in other osteoporosis models are also required.

Conclusion
In this study, CPB effectively inhibited osteoclast differentiation in vitro and prevented bone loss in vivo. e mechanisms of inhibition were via suppression of osteoporosis-related protein expression (NFATc1 and c-Fos), gene expression (Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2), and inhibited bone loss induced in the OVX model. ese results indicate that CPB may be useful in the treatment of metabolic bone diseases such as osteoporosis. 10 Evidence-Based Complementary and Alternative Medicine