Phosphorylated Peptides from Antarctic Krill (Euphausia superba

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Phosphorylated peptides from Antarctic krill (Euphausia superba) prevent estrogen deficiency-induced osteoporosis by inhibiting bone resorption in ovariectomized rats guanghua xia, jingfeng Wang, yingying tian, yiming wang, yanlei zhao, zhe yu, shanshan wang, and Changhu Xue J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04263 • Publication Date (Web): 11 Oct 2015 Downloaded from http://pubs.acs.org on October 15, 2015

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Phosphorylated peptides from Antarctic krill (Euphausia superba) prevent

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estrogen deficiency-induced osteoporosis by inhibiting bone resorption in

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ovariectomized rats

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Guanghua Xia, Jingfeng Wang∗, Yingying Tian,Yiming Wang, Yanlei Zhao, Zhe

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Yu, Shanshan Wang, Changhu Xue

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College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province

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266003, China

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∗

College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province

266003, China. E-mail: [email protected], Fax: +86 0532 82032468; Tel: +86 0532 82031948. 1


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ABSTRACT

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In the current study, we investigated the improvement of phosphorylated peptides

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from Antarctic krill Euphausia superba (PP-AKP) on osteoporosis in ovariectomized

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rats. PP-AKP was supplemented to ovariectomized Sprague-Dawley rats for 90 days.

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The results showed that PP-AKP treatment remarkably prevented the reduction of

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bone mass, improved cancellous bone structure and biochemical properties. PP-AKP

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also significantly decreased serum contents of tartrate-resistant acid phosphatase

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(TRACP),

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deoxypyridinoline (DPD), C-terminal telopeptide of collagen Ⅰ (CTX-1), Ca, and P.

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Mechanism investigation revealed that PP-AKP significantly increased the

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osteoprotegerin (OPG)/ receptor activator of nuclear factor κB ligand (RANKL) ratio

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in mRNA expression, protein expression and serum content. Further research

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suggested that NF-κB signaling pathways was inhibited by suppressing the mRNA

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and protein expressions of nuclear factor of activated T-cells (NFATc1) and tumor

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necrosis factor receptor-associated factor 6 (TRAF6), diminishing the mRNA

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expression and phosphorylation of nuclear factor κB p65 (NF-κB p65), three key

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transcription factors in NF-κB pathways. These results suggest that PP-AKP can

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improve osteoporosis by inhibiting bone resorption via suppressing the activation of

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osteoclastogenesis related NF-κB pathways.

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KEYWORDS: phosphorylated peptides; antarctic krill; bone resorption; bone

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mass; receptor activator of nuclear factor κB

cathepsin

K

(Cath-k),

matrix

metallo

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2


proteinases-9

(MMP-9),


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INTRRODUCTION

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Osteoporosis is a progressive bone disease that is characterized by bone mass

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reduction, increased bone fracture risk and potential bone architecture alterations. It

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mainly affects postmenopausal women and elderly people, and can result in hip and

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vertebral fracture.1-3 As the aging population increases, it has become a global healthy

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problem, and been listed as one of the three major age-related diseases by the World

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Health Organization.4 Conventional therapeutic agents that aim to stimulate bone

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formation or inhibit bone resorption fail to re-establish bone turnover equilibrium

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even after long-term usage. Furthermore, these agents have multiple side effects,

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including bisphosphonate-induced osteonecrosis of the jaw,5, 6 estrogen replacement

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therapy-induced malignancies.7-9 Therefore, it is necessary to develop a new natural

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functional factor factor with less undesirable side effects that minimize bone loss in

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postmenopausal women.

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Osteoporosis is caused by excessive activities of osteoclasts. Osteoclasts are highly

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specialized multinucleated bone-resorbing cells and play an essential role in

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regulating bone morphogenesis, bone remodeling and bone calcium homeostasis.10

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During osteoclastogenesis, RANKL specifically binds to the receptor activator of

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NF-κB (RANK), activates the NF-κB pathway,11 and thereby promotes the formation

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of osteoclasts. With a disruption of RANKL binds to RANK, as well as the

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subsequent activated NF-ĸB manifesting in severe osteopetrosis due to a lack of

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osteoclast.12 Therefore, RANKL and its associated signaling cascades are essential for

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osteoclastogenesis, and may serve as potential therapeutic targets for the treatment of

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osteoporosis.

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Antarctic krill (Euphausia superba) has an estimated biomass of 6.5-10 million and

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contains the largest amount of protein (over 65% of dry weight) among all the

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organisms worldwide.13 It offers a sustainable resource of protein with high quality.

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Since it has not been fully utilized, studying and converting it to a high-value

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commercial product is more and more significant and desirable. It is known to us that

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native phosphorylate groups in protein effectively contributes to their physiological

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functions of food protein.14,

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phosphorylated protein from Antarctic krill with trypsin.13 Two types of phosphate

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bonds were introduced into the peptide of Antarctic krill: tyrosine phosphate with O-P

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bonds and phosphodiesters or polyphosphates with O=P bonds. In our previous study,

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we found that PP-AKP could promote the proliferation of pre-osteoblastic MC3T3-E1

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cells and could be used as a functional factor against osteoporosis. Here, for the first

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time, we investigated the improvement activity of PP-AKP on osteoporosis in OVX

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rats, and the underlying mechanism was further researched by studying the NF-κB

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pathway.

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MATERIALS AND METHODS

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Wang et al. obtained PP-AKP by hydrolyzing the

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Materials. TRACP, calcium (Ca), phosphorus (P) and BCA were from Jiancheng

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Biotech (Nanjing, China). ELISA kits for DPD, Cath-K, CTX-Ⅰ, MMP-9 were

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obtained from R&D Co. (Shanghai, China). TRIzol reagent and Maxima SYBR Green

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qRT-PCR Master Mix were Invitrogen products (Hoffmann-La Roche Ltd, Basel,

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Switzerland). The primers of TRAF6, NFATc1, NF-κB, OPG, RANKL, MMP-9,

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Cath-K, TRACP, and β-actin were synthesized by ShengGong Ltd Co. (Shanghai,

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China). Rodent diet (containing 5-10mg/g K and 10-18mg/g Ca) was purchased from

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Beijing LiAoXieLi Ltd Co. (Beijing, china). Rabbit anti-rat OPG, RANKL, TRAF6,

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NFATc1, NF-κB p65, p-NF-κB p65, β-actin and horseradish peroxidase-conjugated

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secondary antibodies were CST products (Boston, MA, USA).

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Preparation of the PP-AKP. PP-AKP was prepared by Doc. Yanchao Wang,

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Ocean University of China. The protein content of PP-AKP was above 90%. Contents

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of organic phosphorus and fluorine in PP-AKP were 1.52% and 2.49 mg/kg. The

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molecular weight distributed between 2000 to 200 Da. 13

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Animal Treatments. Fifty female Sprague-Dawley rats, 3 months, 238˗262 g, were

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purchased from Vital River Laboratory Animal Center (Beijing, China; Licensed ID:

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SCXK2012-0001). The usage of animals was approved by the ethical committee of

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experimental animal care at Ocean University of China. They were housed at 23±1°C

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on a 12-12 h light-dark cycle with free access to food and water. The osteoporotic

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model rats were established by bilateral ovariectomy (OVX, n=40), and successful

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osteoporotic model rats were defined as rats with serum E2 concentration significantly

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decreased and serum TRACP activity markedly increased in comparison with sham

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group which underwent bilateral laparotomy (Sham, n=10).16,

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model rats were randomized into 4 groups of 8 animals each for some rats were died

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due to infection: OVX group (treated with physiological saline, 7 mL/kg · body

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weight); ALN-treated group (treated with alendronate as a positive control group, 1

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mg /kg · body weight); low-dose and high-dose PP-AKP-treated group (400 and 800

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The osteoporotic

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mg/kg · body weight). The sham group was treated with physiological saline (7 mL

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/kg · body weight). The volume of intragastric administration was 1 mL/100g · body

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weight.

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Each group was subjected to daily intragastric administration for 90 days. All rats

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were fasted for 10 hours and anesthetized with tribromoethanol. Blood was collected

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via abdominal aorta puncture, and serum was separated for measurement of bone

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resorption related indexes. Tibias were dissected, wrapped with wet gauzes to prevent

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dehydration, and stored at -20℃ for BMD measurement and biomechanical testing.

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Femurs were dissected and sliced into three sections (proximal part, middle part,

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telecentric part). Proximal parts were fixed in 10% methanal for histomorphology

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analysis. Middle parts and telecentric parts were wrapped with silver paper, put into

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liquid nitrogen, and then store in -80℃ for qRT-PCR and western blotting analysis,

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respectively.

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BMD and Bone Biomechanical Measurement. The BMD of tibias were measured

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using dual-energy X-ray absorptiometry scanning (GE Healthcare, Fairfield, CT, USA)

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with small animal measurement software. The measurements were expressed as grams

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of mineral contents per cm2 of surface area. Bone biomechanical parameters were

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measured using YLS-16A bone strength tester (Yiyan, China).

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Bone Histomorphometry Evaluation. Proximal part of femurs were fixed in 10%

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methanal for 48 h, decalcified in decalcifying solution (formic acid: nitric acid:

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hydrochloric acid: acetic acid: methanol: distilled water at a ratio of 10:3:4:2:10:70)

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for 4 weeks, and then embedded in paraffin after dehydrated with ethyl alcohol. The

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paraffin sections were deparaffinized and stained with hematoxylin and eosin (H&E

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stain). Sections with the widest marrow cavity near the growth plate of the metaphysis

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of the femurs were selected for bone histomorphology analysis with a BH-2

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microscope (Olympus, Tokyo, Japan).

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Real-Time Quantitative Polymerase Chain Reaction. The mRNA expression of

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key genes that regulate the osteoclastogenesis and bone resorption, such as OPG,

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RANKL, TRAF6, NFATc1, NF-κB, MMP-9, TRACP and Cath-k were examined by

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qRT-PCR. The method of RT-PCR was performed as described previously.18 Brieﬂy,

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total RNA from femur was extracted with TRIzol reagent. The concentration and

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purity of total RNA were determined by a spectrophotometer. RNA (1 µg) of each

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sample was reverse transcribed to cDNA by M-MLV reverse transcriptase. The

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real-time quantitative PCR was performed in the iCycler iQ5 system (Bio-Rad

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Laboratories Inc., Hercules, CA, USA). Twenty-five microliters of the reaction

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volume was used for the qRT-PCR analysis that consisted of 12.5 µL of Maxima

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SYBR Green qPCR Master mix (Hoffmann-La Roche Ltd, Basel, Switzerland), 10

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µM of primers (0.3 µL each of forward and reverse primer), 5.9 µL of nuclease-free

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water, and 6 µL of template. For the reactions, a qRT-PCR was programmed as

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follows: pre-denaturation 10 min at 95℃, 45 cycles of 15s at 95℃, 20 s at 60 °C, 30 s

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at 72 °C, and 10 min at 72 °C for the final extension step. The data normalization was

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accomplished using the endogenous references β-actin. The gene expression levels

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were analyzed by relative quantification using the standard curve method. The

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sequences of the primers used in this study are described in the Table 1. All the primer

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pairs were tested by preliminary experiments with amplification efficiencies of about

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80-120%, and linear R2>98%.

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Western Blotting Analysis. The protein expression levels of OPG, RANKL,

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TRAF6, NFATc1, NF-κB p65, and p-NF-κB p65 were determined by Western blotting.

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The method of western blotting analysis was carried out as described previously.19

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Femur was grinded into powder using liquid nitrogen. To obtain total protein, 0.1 g

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powder was lysed in western and IP lysis buffer (Applygen, China) and then

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centrifuged to remove precipitation. The proteins (30µg per well) were then

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fractionated by SDS-PAGE with 10% gels, and transferred to PVDF membrane. The

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PVDF membranes were blotted incubated with OPG, RANKL, TRAF6, NFATc1,

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NF-κB p65, p-NF-κB p65, or β-actin primary antibodies respectively, and

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subsequently incubated with horseradish peroxidase-conjugated secondary antibodies.

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The protein bands were visualized using an ECL detection kit (Applygen, Beijing,

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China). Normalization of protein expression was carried out using β-actin as control.

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Statistical Analysis. All data were presented as mean ± standard devation of at

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least three independent experiments. Statistical significance between the means of the

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individual groups was assessed by one-way analysis of variance (ANOVA) followed

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by least significant difference (LSD) test. A P-value of less than 0.05 was considered

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statistically significant.

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RESULTS

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PP-AKP Improved the Bone Microarchitecture of OVX Rats. The

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histomorphology of femur was investigated with H&E staining. As shown in Fig.1A,

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the trabecular region in the OVX group was small, thin, and sparse compared with

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that of sham group. It was prominently ameliorated by H-PP-AKP. Whereas,

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L-PP-AKP exhibited poorly effective in improve bone microarchitecture. Further

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verification by quantitative analysis indicated that the trabecular separation increased

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to 243.14%, the trabecular number and trabecular thickness decreased 22.66% and

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36.43%, respectively, in OVX group (Fig.1B, C, D). However, H-PP-AKP markedly

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reversed these indexes to 111.52%, 63.5% and 71.3%, respectively.

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PP-AKP Improved the Tibia BMD of OVX Rats. BMD is an important indicator

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for the diagnosis of osteoporosis and a reflection of bone loss.20 As shown in Fig.2A,

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ovariectomy caused significant BMD reduction in the rat tibia compared to that in the

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sham operated group (P

Phosphorylated Peptides from Antarctic Krill (Euphausia superba

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