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Mar 29, 2016 - observed in men.7 Senile osteoporosis exhibits restrained bone ...... fourteen types of human bone morphogenetic proteins (BMPs). J. Bo...
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Sialoglycoprotein Isolated from Eggs of Carassius auratus Ameliorates Osteoporosis: An Effect Associated with Regulation of the Wnt/β-Catenin Pathway in Rodents Fei Wang, Yiming Wang, Yanlei Zhao, Qiping Zhan, Peng Yu, Jingfeng Wang,* and Changhu Xue College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China S Supporting Information *

ABSTRACT: In the current study, ovariectomized (OVX) rats and the senescence-accelerated mouse strain P6 (SAMP6) were employed to establish models of postmenopausal osteoporosis and senile osteoporosis, respectively. The effects of treatment with sialoglycoprotein isolated from the eggs of Carassius auratus (Ca-SGP) on these two types of osteoporosis were investigated in vivo. Results showed that Ca-SGP significantly increased bone mineral density, ameliorated trabecular bone microstructure, and improved bone biomechanical properties in both OVX rats and SAMP6. The osteogenesis related Wnt/β-catenin pathway was targeted to study the underlying mechanism of Ca-SGP activity. In postmenopausal osteoporosis, Ca-SGP suppressed the activation of Wnt/β-catenin signal via down-regulating the expression of key genes including LRP5, β-catenin, and Runx2, suggesting that overactive osteogenesis was controlled by Ca-SGP. The bone formation was sharply weakened in senile osteoporosis, whereas Ca-SGP treatment promoted osteoblast activity by stimulating the Wnt/β-catenin signal. In conclusion, Ca-SGP ameliorated these two types of osteoporosis by normalizing bone anabolism. KEYWORDS: Carassius auratus eggs, sialoglycoprotein, OVX, SAMP6, Wnt/β-catenin

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INTRODUCTION Bone is a dynamic tissue that undergoes continual remodeling to attain and maintain skeletal shape, size, and structural integrity. During the adult stage of vertebrate life, the preservation of bone microstructure and mineral homeostasis relies on physiological bone remodeling, which removes and repairs damaged bone.1 If the highly coordinated bone remodeling breaks down, various bone-related diseases may occur including osteoporosis, which is characterized by low bone mineral density, microstructure degeneration, increased fragility, and susceptibility to fracture.2−4 With the rapid development of an aging society, osteoporosis has become one of the most considerable public health problems. Both men and women start losing bone mass after the age of 40, although the rate of loss is faster in women due to menopause.5 Overactive bone resorption and bone formation, with the former exceeding the latter, is generally observed in postmenopausal osteoporosis.6 An initial sudden loss of bone mass in the peri-menopausal period is followed by a more progressive and gradual loss with age that has also been observed in men.7 Senile osteoporosis exhibits restrained bone remodeling, in which the activity of osteoblasts seems to be much lower than that of osteoclasts. To date, drugs for the treatment of osteoporosis have belonged to two categories: anabolic drugs, which induce osteoblast activity such as parathyroid hormone (PTH);8 and anti-resorptive agents, which inhibit osteoclast function such as bisphosphonates, estrogen, and estrogen analogues.9,10 Given the fact that these existing therapeutic options are limited due to their adverse effects, it is especially urgent to identify novel alternatives that can be used for either blocking bone resorption or activating bone formation. © XXXX American Chemical Society

Extraction of bioactive components from natural products for the prevention and treatment of osteoporosis is becoming more and more popular. For example, casein phosphopeptides (CPP), egg-CPP, and phosphorylated peptides from Antarctic krill have been reported to promote osteoblast activity in vitro or increase bone mass in vivo.11−13 A new family of highly acidic sialoglycoproteins (SGPs), which are characterized by high carbohydrate (80−90%, w/w) and sialic acid contents, exists in the eggs of many vertebrates and has species-specific structural diversity.14 Recently, water-soluble yolk protein from chicken eggs was demonstrated to promote longitudinal bone growth in adolescent rats and prevent bone loss in ovariectomized (OVX) rats.15,16 However, its underlying mechanism is still unknown. Fish eggs are one of the major byproducts in fish processing and contain abundant bioactive substances such as polyunsaturated fatty acids, lecithin, and glycoproteins. Presently, the high potential value of fish eggs has not been fully developed. In our previous work, sialoglycoprotein isolated from Carassius auratus eggs (CaSGP) was screened from many aquatic animal eggs and hen egg yolk via tracing the proliferation activity of MC3T3-E1 preosteoblastic cells. Ca-SGP exhibited osteogenesis promotive effect by stimulating the proliferation, differentiation, and mineralization of MC3T3-E1 cells.17 Here, for the first time, both an OVX-induced postmenopausal osteoporosis model in rats and aging-induced senile osteoporosis model in senescence-accelerated mouse strain P6 (SAMP6) were applied to Received: December 28, 2015 Revised: March 22, 2016 Accepted: March 29, 2016

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DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

treatment, to obtain the sera, femurs, and tibiae used for the subsequent experiments. Determination of Biochemical Indicators. The bone formation indicators carboxy-terminal propeptide of procollagen type I (PICP), bone morphogenetic protein 2 (BMP2), and bone resorption indicator C-telopeptide of type 1 collagen (CTX-1) in serum were measured using commercial ELISA kits (R&D, Minneapolis, MN, USA). Bone Mineral Density (BMD) and Bone Biomechanical Testing. Dual-energy X-ray absorptiometry was performed to determine the BMD of femur using a GK99-UNIGAMMA X-RAY PLUS Bone Densitometer (L’ACN, Italy). Bone biomechanical properties of femur including ultimate load and stiffness were measured using a YLS-16A bone strength tester (Yiyan, China). Histological Analysis. For the postmenopausal osteoporosis model, the proximal femur was fixed in 10% neutral formaldehyde for 24 h and decalcified in decalcifying solution (formic acid/nitric acid/hydrochloric acid/acetic acid/methanol/distilled water, 10:3:4:2:10:70, v/v) for 4 weeks. For senile osteoporosis model, the distal femur was fixed in 10% neutral formaldehyde for 24 h and decalcified in 10% EDTA for 3 weeks. The decalcified tissues were then embedded in paraffin after dehydration with ascending grades of ethanol. The embedded samples were serially sectioned (5 μm thickness) and stained with hematoxylin and eosin (H&E) stain. Bone morphologic metrology parameters including trabecular area (Tb. Ar), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp) were determined using a BX41 microscope equipped with matching software (Olympus, Tokyo, Japan). Real-Time Quantitative Polymerase Chain Reaction (RT-PCR) Assay. The mRNA expression of key genes including BMP2, LRP5, Wnt10b, β-catenin, and Runx2 were measured by RT-PCR. The primers used in this study are shown in Table S1. RNA extraction and amplification were performed as previously described with slight modification.18 In brief, total RNA from femur was extracted with TRIzol reagent, and 1 μg of RNA was reverse transcribed to cDNA using M-MLV. RT-PCR analysis was performed on a Multicolor RealTime PCR Detection System (Bio-Rad, Hercules, CA, USA). Before PCR analysis, samples were diluted to contain comparable cDNA concentrations. Twenty-five microliters of reaction system was applied for RT-PCR consisting of 12.5 μL of FastStart Universal SYBR Green Master, 0.75 μL each of forward and reverse primers (10 μM), 6 μL of sterile Milli-Q water, and 5 μL of template. The thermal conditions consisted of an initial denaturation at 95 °C for 10 min followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s. The standard curves were constructed from PCR reactions using 5-fold serial dilutions of mixture samples. The housekeeping gene β-actin or GAPDH was used as a reference. The relative mRNA levels are expressed as the ratio of signal intensity for the target genes to that of β-actin or GAPDH. Western Blot Analysis. The protein levels of β-catenin and Runx2 were examined by Western blot. Tibia was ground into powder using liquid nitrogen. To obtain total protein, 0.1 g of powder was lysed in Western and IP lysis buffer (Applygen, China) and then centrifuged to remove precipitate. Nuclear proteins were collected using a nuclear extract kit (Solarbio, China). The obtained proteins were separated on a 10% SDS-PAGE gel and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against β-catenin and Runx2 overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP) conjugated IgG secondary antibody. Protein bands were visualized using an ECL stain kit (Applygen, China) and quantified with the ImageJ program. The β-actin and TBP were analyzed as internal controls for total protein and nuclear protein analysis, respectively. Statistical Analysis. All data were submitted to one-way analysis of variance (ANOVA) with SPSS statistical program (version 17.0), and significant differences among treatments were compared by the least significance difference (LSD) method multiple-range test. The results are presented as means with their standard deviation. In statistical analysis, values of P < 0.05 were considered as significant.

evaluate the ameliorative effect of Ca-SGP on osteoporosis in vivo. Furthermore, the osteogenesis-related Wnt/β-catenin signaling pathway was investigated in an effort to reveal the underlying mechanism, which may provide some theoretical basis for the use of Ca-SGP as a potent anti-osteoporotic functional food.

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MATERIALS AND METHODS

Materials and Reagents. Fresh female C. auratus was purchased at a seafood market in Qingdao, China. The ovaries were quickly taken out under an ice bath. After removal of the egg envelope, the fish eggs were obtained and stored at −80 °C until use. RNase free water, dNTPs, moloney murine leukemia virus reverse transcriptase (M-MLV), random primer, and PageRuler prestained protein ladder were from TaKaRa Bio Inc. (Otsu, Shiga, Japan). FastStart Universal SYBR Green Master (Rox) came from Roche (Germany). The primers of bone morphogenetic protein 2 (BMP2), β-actin, GAPDH, Wnt10b, low-density lipoprotein receptor-related protein 5 (LRP5), β-catenin, and Runt-related transcription factor 2 (Runx2) were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). Rabbit anti-rat β-catenin, Runx2, β-actin, TATA box binding protein (TBP) polyclonal antibodies, and goat-anti rabbit antibody IgG-HRP were from Cell Signaling (Beverly, MA, USA). Preparation of Ca-SGP. Ca-SGP was prepared as previously described.17 The purity was 94.76%, and the molecular weight was 195.35 kDa. Ca-SGP contained 14.33% protein, 62.81% hexose, and 19.72% N-acetylneuraminic acid (Neu5Ac). Monosaccharide composition analysis showed the presence of mannose, glucosamine, galactosamine, and galactose with a molar ratio of 1:2.98:2.65:5.09. Animals and Experimental Design. This study was approved by the Ethical Committee of Experimental Animal Care at Ocean University of China (certificate no. SYXK20120014). Three-monthold female Wistar rats (200 ± 20.0 g) were purchased from Vital River Laboratory Animal Center (Beijing, China; license ID SCXK20120001). The animals were housed two or three per cage at 23 ± 1 °C with a 12 h light/12 h dark cycle and given food and water ad libitum. Five-month-old male SAMP6 and SAMR1 (28−30 g) mice were obtained from First Teaching Hospital of Tianjin University of TCM, and they were housed alone per cage to avoid fighting with each other. The body weight of these animals was recorded every 3 days. For the postmenopausal osteoporosis model, the rats were acclimatized for 7 days and then were sham operated (sham, n = 8) or subjected to bilateral ovariectomy (n = 32) and left untreated for 4 weeks to allow for postoperative recovery. The OVX animals were randomly distributed among four groups (n = 8 per group) as follows: OVX-C group (treated with physiological saline), ALN group (treated with alendronate sodium as a positive control, 1 mg/kg body weight), Ca-SGP-L group (treated with Ca-SGP, 200 mg/kg body weight), and Ca-SGP-H group (treated with Ca-SGP, 400 mg/kg body weight). The sham operated group was treated with physiological saline. Animals in each group were given intragastric administration of physiological saline or respective drugs (1 mL/100 g body weight) once a day. After 90 days of treatment, blood was collected from the abdominal aorta, and serum was isolated to determine bone turnoverrelated biochemical markers. Femurs and tibiae were quickly isolated after sacrifice, followed by an evaluation of bone mineral density, bone biomechanical properties, trabecular microarchitecture, and gene expression. For the senile osteoporosis model, SAMP6 mice (n = 32) were randomly divided into four groups (n = 8 per group): model group (treated with physiological saline), ALN group (treated with alendronate sodium as a positive control, 1 mg/kg body weight), Ca-SGP-L group (treated with Ca-SGP, 300 mg/kg body weight), and Ca-SGP-H group (treated with Ca-SGP, 600 mg/kg body weight). The control group (SAMR1, n = 8) was treated with physiological saline. Animals in each group were given intragastric administration of physiological saline or respective drugs (1 mL/100 g body weight) once per day. These animals were sacrificed after 150 days of B

DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Effects of Ca-SGP on Bone Turnover Markers in Seruma group sham (control) OVX-C (model) ALN Ca-SGP-L Ca-SGP-H

PICP in rats (ng/mL) 8.691 11.466 9.054 10.445 9.036

± ± ± ± ±

0.953 0.867aa 0.770bb 0.386aa,b 0.515bb

CTX in rats (ng/mL) 3.426 4.213 3.613 3.907 3.639

± ± ± ± ±

0.178 0.374aa 0.277bb 0.286aa,b 0.227bb

PICP in mice (ng/mL) 11.613 9.348 10.419 9.547 10.340

± ± ± ± ±

0.725 0.956aa 0.910a,b 0.835aa 1.149aa,b

CTX in mice (ng/mL) 4.818 5.705 5.151 5.352 5.046

± ± ± ± ±

0.556 0.831aa 0.252b 0.451 0.414b

Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. a, P < 0.05; aa, P < 0.01 versus sham or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C or model group.

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Figure 1. Effects of Ca-SGP on femur BMD: (a) femur BMD of rats; (b) femur BMD of mice. Femurs were collected and treated with dual-energy X-ray absorptiometry. Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. aa, P < 0.01 versus sham or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C or model group.

Figure 2. Effects of Ca-SGP on trabecular microarchitecture of OVX-induced postmenopausal osteoporosis model. The proximal femur was embedded in paraffin after 4 weeks of decalcification. Five micrometer paraffin sections were stained with H&E (10× magnification). The parameters of trabecular area (Tb. Ar), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp) were selected for quantitative analysis. Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. a, P < 0.05; aa, P < 0.01 versus sham group or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C group or model group.

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RESULTS Ca-SGP Improved Bone Turnover Status. Loss of coordination of bone turnover is closely associated with osteoporosis. As a result, changes in bone turnover markers can predict bone loss and the risk of osteoporotic fractures.19 Serum PICP and CTX-1 have been identified as specific bone formation and resorption indicators, respectively. Results showed that there were increased PICP and CTX-1 levels in the OVX-C group, suggesting that OVX caused excessively active bone formation and bone resorption compared with sham control (Table 1). Treatment with Ca-SGP distinctly inhibited the OVX-induced high bone turnover in a dosedependent manner. High doses of Ca-SGP reduced the levels of both PICP and CTX-1 by 21.9 and 13.54%, respectively (P < 0.01).

In the senile osteoporosis model, the osteoblast activity in the model group was sharply decreased compared with the control, whereas bone resorption activity was significantly increased (Table 1). This undesirable decline of bone turnover was remarkably reversed by the intervention of Ca-SGP. For instance, Ca-SGP-H inhibited the elevation of CTX-1 by 11.54% and promoted secretion of PICP by 10.61%, respectively (P < 0.05). In conclusion, Ca-SGP prominently ameliorated the adverse status of bone turnover in both OVX rats and SAMP6 via inhibiting osteoclast-mediated bone resorption and regulating osteoblast-induced bone formation. Ca-SGP Increased Bone Mineral Density. Because there are no apparent symptoms in early-stage osteoporosis, measurement of BMD has been regarded as an effective C

DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Effects of Ca-SGP on metaphysis microarchitecture of senile osteoporosis model. The distal femur was embedded in paraffin after 3 weeks of decalcification. Five micrometer paraffin sections were stained with H&E (10× magnification).

means to diagnose this disease.20 In this study, the BMD of the femur was determined using dual-energy X-ray absorptiometry. Compared with control, both OVX and aging models showed a significant reduction of BMD in femur (Figure 1). However, Ca-SGP treatment rescued the decreased BMD. Quantitative analysis demonstrated that high doses of Ca-SGP improved femur BMD by 19.92 and 9.97% in OVX rats and SAMP6, respectively. Ca-SGP Ameliorated Cancellous Bone Microarchitecture. BMD does not necessarily indicate changes in cancellous microarchitecture. Bone histomorphometric analysis can be helpful to assess changes in bone quality. In the postmenopausal osteoporosis model, H&E stain was applied to paraffin sections of proximal femur to examine the changes of trabeculae. Figure 2 shows that OVX induced the deterioration of trabecular microarchitecture as evidenced by sharply decreased Tb. Ar and Tb. Th and increased Tb. Sp. Fortunately, the treatment of Ca-SGP conversely exhibited well-developed trabecular structure. Morphometry data indicated that compared with the OVX-C group, Ca-SGP-H reduced Tb. Sp by 31.19% and increased Tb. Th and Tb. Th by 59.25 and 86.59%, respectively (P < 0.01). In the senile osteoporosis model, paraffin sections of distal femur were used for the evaluation of cancellous microstructure in metaphysis. As shown in Figure 3, compared with control, the trabecular structure in the model group showed severe destruction. Furthermore, aging triggered serious loss of bone marrow stroma, which contributes to new trabecula formation. Nevertheless, treatment with Ca-SGP remarkably ameliorated the adverse changes of cancellous structure and bone marrow stroma in aging mice. Ca-SGP Enhanced Bone Biomechanical Properties. Bone loss and microstructure degeneration lead to bone fragility and subsequently increased risk of fractures. In the current study, ultimate load and stiffness were determined by a three-point bending test to evaluate the biomechanical properties of the femur. As shown in Figure 4, both OVX and aging resulted in reduced ultimate load and stiffness compared to control. This loss was moderated upon treatment with Ca-SGP. Quantitative analysis indicated that Ca-SGP-H rescued the stiffness and ultimate load in OVX rats by 27.40 and 20.24%, respectively. In SAMP6, it improved the above biomechanical parameters by 41.00 and 109.10%, respectively. Ca-SGP Regulated the Secretion and Expression of BMP2. Bone formation is a crucial aspect of remodeling, which is severely compromised in osteoporosis. Bone morphogenetic proteins (BMPs) remain the most potent inducers of bone formation in vivo.21 In particular, BMP2 is widely recognized to be one of the most powerful osteoinductive factors for bone regeneration. In OVX-induced osteoporosis, abnormal increases in bone formation were observed, as evidenced by

Figure 4. Effects of Ca-SGP on femur stiffness and ultimate load: (a) stiffness and ultimate load of femur in rats; (b) stiffness and ultimate load of femur in mice. Three-point bending test was applied to evaluate the biomechanical properties of femur. Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. a, P < 0.05; aa, P < 0.01 versus sham group or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C group or model group.

up-regulated BMP2 expression at the mRNA, protein, and secretion levels (Figure 5a−c). In contrast, the expression of BMP2 was distinctly decreased in the age-related osteoporosis model, indicating that the osteoblast activity was weakened (Figure 6c−e). In OVX rats, Ca-SGP-H significantly downregulated BMP2 expression at the mRNA, protein, and secretion levels by 37.27, 24.55, and 24.48%, respectively. In SAMP6, Ca-SGP-H treatment up-regulated BMP2 expression at mRNA, protein, and secretion levels by 93.53, 44.75, and 30.20%, respectively. The eventual result was that the treatment of Ca-SGP promoted the normalization of bone anabolism in both the postmenopausal and senile osteoporosis models. Wnt/β-Catenin Pathway Was Involved in Osteogenesis Regulation. Activation of the Wnt/β-catenin signaling pathway stimulates osteogenesis.22,23 The binding of Wnt ligands with frizzled (FZD) and LRP5/6 receptors induces the stabilization of cytoplasmic β-catenin, which is subsequently D

DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Effects of Ca-SGP on the expression of BMP2: (a) BMP2 content in serum of rats; (b) mRNA expression level of BMP2 in rats; (c) protein expression level of BMP2 in rats; (d) serum BMP2 content in mice; (e) mRNA expression level of BMP2 in mice; (f) protein expression level of BMP2 in mice. Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. a, P < 0.05; aa, P < 0.01 versus sham group or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C group or model group.

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translocated into the nucleus to regulate gene expression.24 Here, we measured the expression levels of key factors in this pathway including Wnt10b, LRP5, and β-catenin, as well as osteogenesis marker Runx2. In the postmenopausal osteoporosis model, higher mRNA expression of LRP5, β-catenin, and Runx2 was observed compared to the sham control. In addition, the elevated protein expression of total β-catenin, intranuclear β-catenin, and Runx2 in the OVX-C group further demonstrated that the Wnt/β-catenin pathway was activated (Figure 6a,b). This signaling pathway was remarkably inhibited by treatment with Ca-SGP. High doses of Ca-SGP decreased the mRNA levels of LRP5, β-catenin, and Runx2 by 36.19, 26.45, and 46.39%, respectively. Furthermore, Ca-SGP-H down-regulated the protein levels of total β-catenin, intranuclear β-catenin, and Runx2 by 42.21, 45.68, and 58.70%, respectively. In the senile osteoporosis model, the sharply decreased mRNA levels of Wnt10b, β-catenin, and Runx2, as well as the protein levels of total β-catenin, intranuclear β-catenin, and Runx2 in the model group indicated that the Wnt/β-catenin pathway was suppressed due to aging (Figure 6c,d). However, Ca-SGP treatment significantly promoted the activation of this pathway by increasing the expression of those corresponding genes. Quantitative data suggested that Ca-SGP-H elevated the mRNA expression of Wnt10b, β-catenin, and Runx2 by 40.46, 66.73, and 49.55%, respectively. In addition, the protein levels of total β-catenin, intranuclear β-catenin, and Runx2 were upregulated by 56.23, 160.97, and 116.54% with the treatment of Ca-SGP-H.

DISCUSSION In this study, we demonstrated that Ca-SGP significantly increased bone mineral density, improved trabecular microarchitecture, and enhanced biomechanical properties in both OVX-induced postmenopausal osteoporosis and aging-induced senile osteoporosis. Further study revealed that Ca-SGP ameliorated these two types of osteoporosis by regulating the osteogenesis-related Wnt/β-catenin signal pathway. Bone modifies its structure and material composition to accommodate loading circumstances by adaptive modeling and remodeling.25 The accelerated bone loss that occurs in midlife in women is mainly due to estrogen deficiency and is associated with overactive bone remodeling.26 Age-related osteopenia is generally characterized by the undesirable suppression of bone formation. Our results demonstrated that Ca-SGP controlled high bone turnover in OVX rats but elevated bone anabolism in SAMP6, as evidenced by the normalization of bone turnover related biochemical indicators in serum. Our previous study demonstrated that Ca-SGP inhibits osteoclastic bone resorption in these two types of osteoporosis models (data not shown). Here, osteo-anabolism was targeted to further explore the underlying mechanism. BMP2 is a critical autocrine and paracrine growth factor that directs osteoblast differentiation and bone formation.27 In the postmenopausal osteoporosis model, the expression of BMP2 was remarkably increased at mRNA, protein, and secretion levels, suggesting that osteoblasts were overactive in OVX rats. In the senile osteoporosis model, the sharp decline of BMP2 levels demonstrated that the osteogenic activity in SAMP6 was E

DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. Effects of Ca-SGP on mRNA and protein expressions of key genes in Wnt/β-catenin signal: (a) mRNA expression including LRP5, βcatenin, and Runx2 in rats; (b) protein expression including total β-catenin, intranuclear β-catenin, and Runx2 in rats; (c) mRNA expression including Wnt10b, β-catenin, and Runx2 in mice; (d) protein expression including total β-catenin, intranuclear β-catenin, and Runx2 in mice. Data are presented as the mean ± SD (n = 8 per group). Multiple comparisons were done using one-way ANOVA analysis. a, P < 0.05; aa, P < 0.01 versus sham group or control group; b, P < 0.05; bb, P < 0.01 versus OVX-C group or model group.

weakened. The treatment of Ca-SGP rescued the adverse loss of bone formation via down-regulating BMP2 expression in OVX rats but up-regulating BMP2 expression in SAMP6. Wnt signaling is widely recognized as a key mechanism regulating bone metabolism.28,29 Increasing studies of gain or loss of LRP5/6, β-catenin, and many other molecules that participate in the Wnt signaling cascade identify the canonical Wnt signaling pathway as a crucial pathway in the skeleton that controls osteoblast differentiation and bone formation.27,30 In essence, canonical signaling is triggered by the binding of Wnt ligands to the dual receptor complex composed of FZD and LRP5/6.31 This results in the subsequent accumulation of βcatenin in the cytoplasm, which then translocates into the nucleus, where it associates with transcription factors to control the transcription of target genes.32,33 Our results indicated that Ca-SGP inhibited the activation of Wnt/β-catenin pathway through suppressing the expression of LRP5, β-catenin, and Runx2 in OVX rats, suggesting that the excessive increase of osteoblastic bone formation was down-regulated to normal levels by Ca-SGP. In the case of SAMP6, Ca-SGP stimulated the activation of this signaling pathway, which resulted in the up-regulation of osteogenesis. Curiously, the intervention of Ca-SGP on osteoblast activity was different in each of the osteoporosis models. However, in both cases the eventual outcome of Ca-SGP treatment was that bone anabolism in postmenopausal osteoporosis and senile osteoporosis model was regularized. Because existing therapeutic drugs have potential or real undesirable effects, the development of natural active alternatives for reducing osteoporosis risks is booming. We

fortunately identified a promising candidate in sialoglycoprotein extracted from eggs of C. auratus, which exhibited remarkable anti-osteoporotic activity in postmenopausal osteoporosis and senile osteoporosis models. It is worth noting that Ca-SGP was identified as a macromolecular compound and seems to be restricted to the gastrointestinal tract. Actually, there are other bioactive components with high molecular weight such as shark protein, egg yolk peptide, and milk basic protein that could exhibit bone protective effects by the oral route.16,34,35 However, the mechanism by which they act on bone metabolism is still unclear. In our preliminary study, the content of Neu5Ac in serum was determined after intragastric administration to evaluate the digestive absorption of Ca-SGP. Results showed that serum Neu5Ac reaches the maximum after oral administration for about 5 h (data not shown), suggesting that Ca-SGP could be at least partly absorbed in the intestine. Another possibility is that it could improve the composition of intestinal flora in the large intestine, which may mediate effects on bone loss. 36 Increasing numbers of studies have demonstrated that gut microbiota may regulate bone metabolism via interaction with the immune system, intestinal calcium absorption, and secretion of endogenous serotonin.37−40 Recently, we conducted a preliminary study of the influence of Ca-SGP on gut microbiota in OVX rats. The results of 16S r-DNA high-throughput sequencing indicated that Lactobacillus was significantly increased with the treatment of Ca-SGP (data not shown). Intriguingly, Lactobacillus is a probiotic organism that has been reported to exhibit antiosteoporotic activity.41−43 However, these speculations need our further research. F

DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b06132. Table S1, primers used for determination of mRNA expression (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(J.W.) E-mail: [email protected]. Fax: +86 0532 82032468. Phone: +86 0532 82031948. Funding

This study is financially supported by the National Natural Science Foundation of China (No. 31371876, 31571771, and U1406402). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED SAMP6, senescence-accelerated mouse strain P6; Ca-SGP, sialoglycoprotein isolated from eggs of Carassius auratus; ALN, alendronate sodium; PICP, carboxy-terminal propeptide of procollagen type I; CTX-1, C-telopeptide of type1 collagen; BMP2, bone morphogenetic protein 2; LRP5, low-density lipoprotein receptor-related protein 5; Runx2, Runt-related transcription factor 2

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DOI: 10.1021/acs.jafc.5b06132 J. Agric. Food Chem. XXXX, XXX, XXX−XXX