Extraction, purification and anti-osteoporotic activity of a polysaccharide from Epimedium brevicornum Maxim. in vitro

Journal Pre-proof Extraction, purification and anti-osteoporotic activity of a polysaccharide from Epimedium brevicornum Maxim. in vitro

Hong Zheng, Bing He, Tianxiu Wu, Jie Cai, Jinsong Wei PII:

S0141-8130(19)36012-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.145

Reference:

BIOMAC 13926

To appear in:

International Journal of Biological Macromolecules

Received date:

31 July 2019

Revised date:

9 November 2019

Accepted date:

18 November 2019

Please cite this article as: H. Zheng, B. He, T. Wu, et al., Extraction, purification and anti-osteoporotic activity of a polysaccharide from Epimedium brevicornum Maxim. in vitro, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.145

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© 2019 Published by Elsevier.

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Extraction, purification and anti-osteoporotic activity of a polysaccharide from Epimedium brevicornum Maxim. in vitro

Hong Zhenga, Bing Heb, Tianxiu Wuc, Jie Caid, Jinsong Weia, * a

Department of Orthopedics Center, Affiliated Hospital of Guangdong Medical

Department of Nursing, The Affiliated Hospital of Guangdong Medical University,

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b

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University, Zhanjiang 524001, Guangdong, China

College of Basic Medical, Guangdong Medical University, Zhanjiang 524023,

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c

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Zhanjiang 524001, Guangdong, China

d

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Guangdong, China

College of Information Engineering, Guangdong Medical University, Zhanjiang

Corresponding author. Jinsong Wei, Department of Orthopedics Center, Affiliated

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*

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524023, Guangdong, China

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Hospital of Guangdong Medical University, 57 South Renmin Avenue, Xiashan District, Zhanjiang 524001, Guangdong, China. Tel.: +86 759-2369606; fax: +86 759-2369606. E-mail address: [email protected]

ABSTRACT Osteoporosis is the most widespread metabolic bone disease characterized by decreased bone mass and bone quality, and its diagnosis and treatment remains challenging. To date, medicinal plants have received increasing attention from

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researchers for researching effective and less toxic therapeutic ingredients, including polysaccharide, to treat osteoporosis. The present study aims to evaluate the osteoprotective effects of a polysaccharide (EBP) from Epimedium brevicornum in glucocorticoid-induced osteoporosis in vitro and investigate the underlying mechanism. EBP (25 and 100 μg/ml) pretreatment could significantly prevent

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decreased cell proliferation of osteoblasts (OBs) treated only with 100 μM of

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dexamethasone (Dex) via induction of apoptosis. The osteoblastic differentiation of

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EBP pretreatment on OBs at early and later phase was further confirmed by the

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increased alkaline phosphatase (ALP) activity and calcium content, respectively.

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Meanwhile, the increased expression of cleaved caspase-3 and Bax, as well as a decrease of Bcl-2 and the phosphorylation of PI3K, Akt and mTOR protein in

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Dex-treated OBs were totally reversed by EBP pretreatment. Moreover, the protein

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expression of Lrp-5, β-catenin, Runx2 and Osx were significantly up-regulated in the

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presence of EBP pretreatment. In conclusion, these results demonstrated that EBP pretreatment may be a potential therapeutic agent for patients with glucocorticoid-induced osteoporosis (GIO).

Keywords: Epimedium brevicornum; Polysaccharide; Anti-osteoporotic activity

1. Introduction Bone homeostasis is dynamically balanced throughout life via coordination by osteoclastic bone resorption and osteoblastic bone formation [1]. Any disturbance in 7

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the function of osteoclasts and osteoblasts (OBs) eventually results in metabolic bone disorders, including osteoporosis, suggesting that both cell types play an essential role in during bone remodeling [2]. Osteoporosis is characterized by structural deterioration of the bone tissue and a reduction in bone density, which can lead to increased bone fragility and fractures [3]. Postmenopausal women are at high risk of

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developing osteoporosis because of excessive bone resorption associated with

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estrogen deficiency [4]. However, glucocorticoid-induced osteoporosis (GIO), a

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drug‐induced secondary osteoporosis mainly resulted from the inhibition of bone

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formation [5], which is closely associated with the proliferation and function of OBs.

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For example, dysfunction and apoptosis of OBs caused by dexamethasone (Dex), a synthetic glucocorticoid (GC) hormone, has greatly contributed to the development of

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GIO [6]. Thus, promoting the proliferation and differentiation of OBs and inhibiting

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the apoptosis of OBs are potential strategies to handle this disease.

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Mounting evidences demonstrated that a number of medications, such as antiresorptive agents that prevent bone resorption and anabolic agents that help in new bone formation, have been reported to be effective for the prevention and treatment of osteoporosis [7,8]. Despite these advances, these pharmacological interventions generally come with serious side effects such as hypercalcemia, increased risk of malignancy, etc. [9,10]. Recently, oriental traditional medicines have been attracted more attention by researchers in medicine filed because of their far fewer side effects and more suitable long-term use compared with chemically synthesized medicines. Consequently, it is necessary to develop therapeutic agents that either increase the 8

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growth and proliferation of OBs or stimulate differentiation of OBs for GIO thearpy. It is well established that the polysaccharides derived from medicinal plants exhibited various important biological properties such as significant antitumor, antimicrobial, immunomodulatory, anticoagulant, anti-inflammatory and hypoglycemic effects [11,12]. Moreover, a growing body of evidence has shown that

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many polysaccharides extracted from various Chinese herbal medicines, such as

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Dipsacus asper, Achyranthes bidentata, and Polygonatum sibiricum, showed good

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anti-osteoporosis ctivity [13-15]. Epimedium brevicornum Maxim. (Yinyanghuo) is

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one of the representative traditional Chinese herbs, which displays kidney-toning and

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antiosteoporosis activities and has been widely used for bone disease prevention and therapy for thousands of years [16]. At present, this herb is commonly used alone or in

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conjunction with other herbs in traditional herbal drug prescriptions to prevent and

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treat osteoporosis [17]. Modern pharmacology verified that its water extract or

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flavonoids had the property to enhance bone healing and manage osteoporosis not only in experimental models, but also in clinical studies [18,19]. However, little is known about the anti-osteoporotic properties of the polysaccharide from E. brevicornum, despite its diverse biological activities, such as antioxidant, immunomodulatory and antimicrobial [20,21]. The aim of this study is to isolate the homogeneous polysaccharide from E. brevicornum and investigate the effects of this polysaccharide on the proliferation and differentiation of cultured primary mouse OBs in vitro, and elucidate the underlying mechanism.

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2. Materials and methods 2.1. Materials and chemicals E. brevicornum was purchased from the local Chinese medicine store in Guangzhou (China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), fetal bovine serum (FBS), trypsin-EDTA, 1-phenyl-3-methyl-5-pyrazolone

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(PMP), dextrans with different molecular weight (Mw: 9.6, 21.1, 36.3, 72.7, 158.1, 344.8 and 714.5 kDa), monosaccharide standards (ribose, glucose, mannose,

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rhamnose, arabinose, fucose, xylose, galactose, glucuronic acid, and galacturonic

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acid), penicillin, streptomycin, dimethyl sulfoxide (DMSO) and dexamethasone (Dex)

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was purchased from Sigma (St. Louis, MO, USA). Annexin V-FITC/PI Apoptosis Detection Kit and alkaline phosphatase (ALP) colorimetric assay kit were obtained

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from KeyGen Biotech (Nan-jing, China). Enhanced BCA Protein Assay Reagent was

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from Beyotime (Shanghai, China). An enhanced chemiluminescence (ECL) detection

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kit was from Millipore (Billerica, MA, USA). Primary antibodies against bax, bcl-2, cleaved caspase-3, PI3K, Akt, mTOR, Lrp-5, β-catenin, Runx2, Osx, β-actin and Horseradish Peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technologies Inc. (Beverly, MA, USA). Sephacryl S-400 gel and DEAE-cellulose 52 were rom Amersham Biosciences (Uppsala, Sweden). 2.2. Isolation and purification of polysaccharide EBP from the safflower Dried small pieces of dried E. brevicornum was soaked in 95% ethanol under constant stirring overnight at 40 °C to eliminate lipids, pigments and low molecular

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weight compounds. This mixture was centrifuged at 8000 rpm for 10 min to discard the supernatants and was repeated three times until the supernatant became colorless. Then the defatted sample was extracted with boiling water at a water/raw material ratio of 20:1 (ml/g) for three times and 1h for each time. The supernatant was filtered through muslin cloth followed by Whattman No.1 filter paper. The resulting filtrate

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was concentrated to about 1/4 of original volume under vacuum and precipitated by

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the addition of 95% ethanol until the final concentration of ethanol reached to 70%

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(v/v) overnight at 4 °C. The precipitate was then dissolved in distilled water and

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deproteinated using repeated freeze thawing and the method of Sevag [22], followed

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by dialyzation against distilled water for 3 days. The aqueous fraction inside was concentrated and dissolved in distilled water before lyophilization to gain crude

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CEBP.

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The crude CEBP (50 mg) was dissolved in 10 mL of distilled water, centrifuged,

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applied to a DEAE-52 cellulose column (7.0 × 30 cm) equilibrated with distilled water, and sequentially eluted stepwise with distilled water and NaCl aqueous solutions (0.5 and 1.0 M) at a flow rate of 1 ml/min, respectively. Each eluent fraction (5 mL/tube) was collected automatically and determined by tracking the absorbance at 490 nm using the phenol-sulfuric acid method [23]. As a result, three completely separated fractions, named as CEBP 1 (distilled water), CEBP 2 (0.5M) and CEBP 3 (1.0 M), were obtained respectively. In the present study, we mainly focused on the investigation of CEBP 1. Subsequently, CEBP 1 was further purified by gel-permeation chromatography on a column of Sephacryl S-400 gel column (2.5 × 11

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100 cm) on which it was eluted with 0.1 M NaCl at a flow rate of 0.5 ml/min, and the elution was monitored as described above. The resultant fraction (named EBP) was gathered, desalted and lyophilized for the subsequent assays on its structure characterization and anti-osteoporosis activity. The total carbohydrate, uronic acid and protein contents were quantified by the phenol–sulfuric acid [23], m-hydroxydiphenyl

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[24] and Bradford methods [25], respectively.

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2.3. Molecular weight and distribution determination and monosaccharide

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composition analysis

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The homogeneity and the molecular weight distribution of the polysaccharide

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were determined by high performance size-exclusion chromatography (HPSEC) on a SHIMADZUHPLC system (Shimadzu, Japan) fitted with a TSK-G3000 PWXL

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column (7.8mm×30 cm) and a RID-10A detector. Polysaccharide fractions (10 mg)

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were dissolved with distilled water (1 ml), and filtered through a 0.45μm hydrophilic

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membrane. The 0.1mol/mL NaNO3 was used as the mobile phase at a flow rate of 1.0m L/min. Injection volume was 20 μL. The T-series dextran standards with different molecular weights (9.6, 21.1, 36.3, 72.7, 158.1, 344.8 and 714.5 kDa) were used to calibrate the column and establish a calibration curve. The molecular weight of the polysaccharide was calculated by comparison with the calibration curve. Monosaccharide composition analysis was determined using precolumn derivation of High Performance Liquid Chromatography (HPLC) with PMP on a Shimadzu LC-2010A HPLC system equipped with a LC-10AT pump, a UV detector

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Journal Pre-proof and a reversed-phase C18 column (4.6 mm i.d. × 250 mm, 5 μm)[26]. 2.4. Isolation and culture of OBs Primary OBs were obtained from the calvarias of newborn Wistar rats (2–4 days old) from Experimental Animal Centre of Guangdong Medical University by enzymatic digestion as previously described [27]. Briefly, the fetal rats were sacrificed

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under anesthesia via intraperitoneal injection of pentobarbital and their skulls were dissected under sterile conditions. After the other connective tissues were completely

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removed, the skulls tissues were cut into 1x1‑mm sections and then transferred into 5

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ml of 0.25% trypsin-EDTA (w/v) at 37 °C in Hank’s buffer solution with agitation for

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10 min, followed by the addition of 5ml collagen II solution (200 U/ml) for four periods of 15-min digestion. The first digestive liquid was discarded and the cells

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from the second to fourth digestive liquid were collected and cultured in DMEM

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supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at

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37 °C in 5% CO2 cell incubator. Following incubation for 24 h, the unattached cells were discarded and the adherent cells were cultured in the same culture medium, whose medium was replaced every 3 days. When 80% confluence, cells were washed three times with D‑Hank's solution, trypsinized, passaged and made into OBs suspensions at 1×106/ml. 2.5. Cell proliferation and phase contrast microscopy Cell proliferation was measured by MTT assay. Briefly, the passaged OBs suspension was centrifuged at 3000 rpm for 5min, and the supernatant was discarded.

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Journal Pre-proof The remained sample was washed twice with D‑Hank's solution. Following counting, OBs were inoculated in 96-well culture plates at a density of 5×103 cells/well with three duplicate wells per treatment and maintained in DMEM containing 10% FBS for 24 h at 5% CO2 at 37 °C. After 48 h incubation, the culture medium was discarded and the cells were treated with phosphate-buffered saline (PBS, Normal control), 100 μM DEX (Negative control) alone, or pretreated with EBP (25mg/ml or 100mg/ml)

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for 2 h and then exposed to 100 μM DEX for 48 h in the 5% CO2 cell incubator. After

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exposure period, 20 μl of MTT (5mg/ml) solution was added to each well and the

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cells were further incubated for 4 h, followed by addition of 100μl DMSO. After

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shaking at room temperature for 10 min, the optical density (OD) of each well was measured at once by a micro-plate reader (Bio-Rad) in wave length of 570 nm with a

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reference wavelength of 630 nm. Meanwhile, morphology of OBs in different group

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was observed under an inverted phase contrast microscopy.

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2.6. Cell differentiation analysis 2.6.1. Crystal violet staining

The crystal violet staining assay was performed as previously established by Ho et al. [28]. Briefly, OBs were treated with different drug as described in MTT assay. After treatment, the plates were washed with PBS and the cells were stained with 0.5% crystal violet solution dissolved in 10% ethanol for 10 min. Subsequently, the excess crystal violet stain was washed with PBS thrice prior to being observed under an inverted phase contrast microscopy. Moreover, the cell viability of OBs was also

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evaluated quantitatively by measuring OD of the crystal violet that stained the cells at a wavelength of 590 nm. 2.6.2. Measurement of ALP activity After incubation for 48 h, the medium in the wells was discarded and the cells were washed twice with PBS, followed by cells lysis with 0.05% Triton X-100 for

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5min at room temperature. After the cells was centrifuged at 13,000g for 3 min to remove insoluble material, the amount of ALP released from the cells was measured

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with a ALP colorimetric assay kit, according to the manufacturer’s instruction. The

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OD value was measured at 405 nm using a microplate reader.

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2.6.3. Estimation of calcium content assay

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The calcium contents of OBs following drug exposure were quantitatively

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measured. After 10 days’ treatment, OBs was treated with lysis buffer at room

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temperature for 3 min and centrifuged at 3000 rpm for 5min. The OD of the resulting cell supernatant was then recorded at 612 nm using a micro-plate reader (Bio-Rad) and the calcium concentration was referred to standard calibration curve made from serial dilution of calcium standard solutions. Each experiment was repeated at least three times to obtain the mean values. 2.7. Measurement of apoptosis with flow cytometry Cell apoptosis was determined using the Annexin V-FITC/PI Apoptosis Detection Kit according to the instructions of the manufacturer. In brief, after treatment with different drug for the indicated time periods, OBs were washed twice 15

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2.8. Western blot analysis After treatment with different drug, total proteins of OBs cells were extracted

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using lysis buffer, and protein content was determined using Enhanced BCA Protein

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Assay Reagent. Then, equal amounts of protein (40 μg) in resulting cell lysates were

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separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to a polyvinylidene difluoride membrane. After blocking

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with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (pH

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7.4) for 1 h at 37 °C, the membranes were incubated with 1:1,000 diluted homologous

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primary antibodies (bax, bcl-2, cleaved caspase-3, PI3K, Akt, mTOR, Lrp-5, β-catenin, Runx2, Osx, β-actin ) in TBS-T at 4˚C overnight, and then washed thrice with TBS containing 0.1% Tween‑20. The washed membrane was further labeled with HRP-conjugated secondary antibody (1: 2500 dilution) in TBS-T for 2 h at room temperature, and subsequently visualized using an ECL detection kit. β-actin was performed as a loading control. 2.9. Statistical analysis Data are expressed as the mean ± standard deviation (SD) of triplicate

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measurements. The values were evaluated by one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

3. Results and discussion

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3.1. Isolation, purification and structural analysis of polysaccharide EBP

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The crude water-soluble polysaccharides (CEBP) were successfully extracted

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from dried E. brevicornum by ethanol-defatting, hot water extraction and precipitation

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by ethanol, and the yield was 5.3 % based on the dry material (1000 g). Then CEBP

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was fractionated on a DEAE-52 cellulose anion-exchange column and eluted stepwise in turn with distilled water, 0.5 and 1.0 M NaCl, each respectively giving a single

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fraction, namely CEBP 1, CEBP 2 and CEBP 3. The fraction CEBP 1 (10.52 g) eluted

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with distilled water was further fractionated by Sephacryl S-400 gel filtration

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chromatography based on molecular mass, affording a purified polysaccharide (EBP) (1.45 g, 13.78% of CEBP 1). EBP contained 94.20% of carbohydrates content, but no protein and uronic acid was detected in it. Moreover, UV scanning profile of EBP showed that no absorption at 280 and 260 nm, indicating the absence of the protein and nucleic acid in the purified polysaccharide. The elution profile of EBP on HPSEC showed a single and symmetric sharp peak (Fig. 1A), indicating that it was a homogeneous polysaccharide. According to the calibration curve equation (log Mw = −0.1868t + 6.8516, r = 0.9991, t was the retention time, min), its molecular weight was estimated to be about 2.0×104 Da. The monosaccharide composition of EBP was

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determined by HPLC by comparing the retention times with those of PMP-labelled standard monosaccharide. As illustrated in Fig. 1B, EBP was mainly composed of glucose. Figure 1 insert here

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3.2. Effects of EBP pretreatment on the cell proliferation and morphological features

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of OBs

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The different EBP pretreatment on the cell viability of murine OBs was determined by the MTT assay. As shown in Fig. 2A, pretreatment with EBP (25 and

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100 μg/ml) can significantly prevent cell loss induced by DEX as compared with

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negative control (P<0.01 or P<0.001), and even has a noticeable

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dose (P<0.01).

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proliferation-promoting action on OBs than the normal control, especially at the high

In support of MTT assay, the morphological features of OBs was observed using phase contrast microscopy and the photographed micro-image were depicted in Fig. 2B. The micrographs of negative control cells clearly indicated that a decrease in the numbers of OBs when compared with normal control and EBP pretreated cells. Conversely, an escalating increase in the density and thickness of cells was observed in OBs treated with EBP at both concentrations compared to the negative control group, which were also in agreement with MTS proliferation assay. These findings further confirmed the EBP can facilitate OBs proliferation in vitro. 18

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Figure 2 insert here

3.3. Effects of EBP pretreatment on crystal violet staining of OBs The purpose of examining the effect of EBP pretreatment on the growth pattern during the differentiation phase of murine OBs was conducted using crystal violet

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staining assay. The image in Fig. 3A indicated that a dramatic increase of cell growth was observed in OBs pretreated with EBP at the concentration of 25 and 100 μg/ml

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compared to those in OBs treated with 100 μM DEX. Quantitative analysis of resulted

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data in Fig. 3B also demonstrated evident similar pattern of cell growth as described

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in MTT and morphological observing of OBs, and showed a high cell proliferation

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rate of OBs pretreated with EBP compared with negative control (P<0.05 or P<0.01).

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The OD value in low concentration of EBP was comparable to that of normal control

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cells and the maximum value of cell growth was reached especially at 100 μg/ml, which is statistically different from that of normal control (P<0.01). This result further suggested that EBP has a beneficial promoting effect on cell growth of OBs compared with negative control group during differentiation phases. Figure 3 insert here

3.4. Effects of EBP pretreatment on ALP activity and calcium content of OBs As shown in Fig. 4A, both the normal control and EBP (25 and 100 μg/ml)

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pretreated cells expressed the higher ALP activity (P<0.05 or P<0.01) compared with model cells at 48h, however, the significance of EBP pretreatment in enhancing ALP activity was more pronounced (P<0.05) than that of normal control. Similarly, calcium content assay revealed that there was a dramatic increase in the magnitude of calcium content in both the normal control (P<0.05) and EBP-pretreated cells at both

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doses (P<0.01) for 10 days when compared with that of negative control cells (Fig.

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4B). Collectively, this increase of ALP activity and calcium content observed in OBs

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pretreated with EBP clearly supported the data presented in Fig. 2 and 3, indicating

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that an early and later stage of differentiation occurred in OBs induced by EBP

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pretreatment.

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Figure 4 insert here

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3.5. Effects of EBP pretreatment on the apoptosis of OBs The flow cytometry analysis indicated that a large number of OBs underwent apoptosis after treatment with 100 μM of DEX. However, following the pre-addition of EBP (25 and 100 μg/ml), only a small amount of apoptotic cells was observed in OBs, and the apoptotic rate is comparable to normal control cells, suggesting that EBP pretreatment can facilitate OBs proliferation and reduce apoptosis (Fig. 5A and B). Figure 5 insert here

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3.6. Effects of EBP pretreatment on the expression of apoptosis related protein in OBs In order to investigate the potential mechanism of EBP‑induced anti-apoptosis action, the levels of apoptosis regulatory proteins (Bax, Bcl-2, cleaved caspase-3) in OBs pretreated with EBP or not was analyzed in the present study using western blotting (Fig. 6). The results demonstrated that pre-exposure of OBs with EBP (25 and

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100 μg/ml) significantly down-regulated cleaved caspase-3 protein expression

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compared with those of the DEX (100 μM)-treated cells. Furthermore, the levels of

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Bax decreased (P<0.01) and the Bcl‑2 levels increased (P<0.01) when compared with

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those of negative control cells resulting in an increase in the

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anti‑apoptotic/pro‑apoptotic (Bcl‑2/Bax) protein ratio in OBs following EBP

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Figure 6 insert here

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pretreatment.

3.7. Effects of EBP pretreatment on PI3K/Akt/mTOR signaling pathway in OBs To uncover the role of EBP pretreatment on modulating the apoptosis of OBs, we performed western blotting to detect the PI3K/AKT/mTOR signaling pathway in OBs pretreated with EBP or not. Western blot analysis results (Figu. 7A and B) revealed that the phosphorylation of PI3K, Akt and mTOR were significantly suppressed in negative control cells treated with DEX (100 μM) as compared with those in normal control cells (P<0.01), while PI3K, Akt and mTOR remain unchanged (P> 0.05). Interestingly, following EBP pretreatment, the phosphorylation of PI3K, Akt and 21

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mTOR was increased to the same level as those in the normal control cells, but no significant change was observed on the protein levels of PI3K, Akt and mTOR (P> 0.05). All the above data indicated that the activation of the PI3K/AKT/mTOR pathway strengthened the anti-apoptotic activity of EBP pretreatment toward OBs.

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Figure 7 insert here

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3.8. Effects of EBP pretreatment on the expression of osteogenic protein markers in

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OBs

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Considering the above findings, we considered that EBP pretreatment has a proliferation promoting effect on the cell growth of OBs and stimulating osteogenesis

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may be one of the mechanisms. Then a further study was performed to elucidate if

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EBP pretreatment regulated the process of osteogenesis via canonical Wnt/β- catenin

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signaling pathway. After exposure of OBs with EBP (25 μg/ml or 100 μg/ml) for 2 h and then exposed to 100 μM DEX for 48 h, the relative level of the Lrp-5, β-catenin, Runx2, and Osx protein expression was significantly up-regulated than that of negative control cells (P<0.01), which was comparable to normal control and even higher than normal control level at high dose of drug for β-catenin and Runx2 (P <0.05, Fig. 8A and B), suggesting that EBP pretreatment could alleviate osteonecrosis, at least in part, through up-regulation of the canonical Wnt/β-catenin signaling pathway of OBs.

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Figure 8 insert here

4. Discussion and Conclusions During the previous decade, the apoptosis theory concerning the pathological changes associated with osteocytes and OBs in early GIO has received increased

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research attention, and several studies have already proved that the apoptosis of

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osteocytes and OBs are considered to be closely associated with this disorder, which provide new understanding of the pathogenesis of this disease [29]. OBs, as the main

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target of Dex and other GCs, play pivotal roles in promoting bone formation [30].

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Once the balance between OBs and osteoclast was break down, osteoblastogenesis

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would be inhibited and OBs apoptosis would be initiated, finally causing

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osteonecrosis [31]. It has also been demonstrated that a deficiency in the function of

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OBs along with a dramatic elevated apoptotic rate of OBs was observed in mice suffering excessive use of glucocorticoids [32]. Therefore, identification of novel agents aiming at inhibiting the apoptosis of OBs induced by Dex and other GCs or promoting the proliferation and differentiation of OBs will open novel therapeutic avenues for GIO. In the present study, we isolated and purified one polysaccharide from E. brevicornum and aimed to evaluate its pro-proliferation and anti-apoptosis effect on OBs so as to theoretically support the application of EBP in clinic. The growth, development and maintenance of bone are a complex process, which is highly regulated by bone-forming cells (OBs) and the bone-resorbing cells

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(osteoclasts)[33]. OBs are derived from bone marrow mesenchymal stem cells and retained at the surface of bone responsible for bone formation [34]. Differentiation and proliferation of OBs are closely associated with active bone formation [35], indicating the role of the number of OBs in patients with GIO. MTT assay and morphological observation using inverted phase contrast microscopy showed that

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EBP (25 and 100 μg/ml) pretreatment could significantly facilitate osteoblastic

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proliferation, cause a consistent increase in the density of cells and exhibit a thicker

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appearance with well-defined cell membrane compared to those in negative cells.

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These findings indicated that EBP pretreatment has a promising tendency to improve

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the OBs proliferation.

The osteoblastic phenotype differentiation included two phases, namely early

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phase and later phase, during bone formation. The early phase of differentiation is

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characterized by the maturation of ECM and the relative expression of ALP and

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collagen [36, 37]; the later phase of differentiation was evidenced by calcium and phosphate deposition to facilitate ECM mineralization, evuatually resulting in the formation of compact bone [38]. Thus, in the present study, early differentiation marker ALP and the late differentiation marker calcium content of treated OBs were examined to evaluate the effect of EBP (25 and 100 μg/ml) pretreatment on OBs's differentiation. However, prior to assessment of phenotype differentiation, the potential effect of EBP pretreatment on cellular growth pattern during the differentiation stages of OBs was also conducted using crystal violet staining. In line with MTT and morphological change assay, the higher OD value was observed in 24

Journal Pre-proof OBs pretreated with EBP (25 and 100 μg/ml) compared to those in negative control group, indicating a dramatic increase of the cell population induced by EBP during cell differentiation. In an attempt to assess cellular differentiation, ALP activity and calcium content were measured in the current experiment and the results showed that EBP (25 and 100 μg/ml) pretreatment remarkably enhance ALP activity and calcium

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content in OBs when compared with those in negative control, which were even all

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statistically higher than normal control. Comparatively this increase of ALP activity

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and calcium content observed in OBs pretreated with EBP was in accordance with the

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status of cell proliferation in OBs during an early and later stage of differentiation

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induced by EBP pretreatment. Hence, it was evident that EBP pretreatment could promote bone formation by strengthening the proliferation and osteogenic

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differentiation of pre-OBs at both early and later stage.

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Osteoblast apoptosis induced by glucocorticoids has been considered the critical

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factor in the pathogenesis of GIO [39,40]. Badly, excessive GC use history is able to disturb the proliferation and differentiation of bone cells, and promote the apoptosis of OBs and bone cells, thus delaying bone formation and resulting in bone loss [41]. The flow cytometry results demonstrated that the apoptosis rate of OBs was significantly increased following treatment with 100 μM of DEX, and this tendency was reversed after pretreatment with EBP (25 and 100 μg/ml), which was comparable to the normal level. These observing proposed that EBP pretreatment can facilitate OBs growth via suppressing apoptosis.

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Although the exact mechanism underlying the apoptosis implicated in GIO remains unclear, it has been well accepted that that Caspase-3 might play a crucial role in the development of this disease [42]. Kim et al. observed elevated caspase-3 expression occurred in GC-treated murine osteoblastic MC3T3-E1 cells [43] and a study by Chua et al. found that GCs can activate caspase 3 via endogenous and

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exogenous apoptosis pathways to execute apoptosis of MC3T3-E1 cells [44]. Bcl-2

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family proteins (the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2)

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and the change in the ratio of Bcl-2 to Bax are also typically involved in the apoptosis

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and cellular proliferation associated with GIO [45]. In this study, the increased protein expression of Bax and cleaved caspase-3, and decreased Bcl-2 protein expression

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were detected in OBs treated with DEX (100 μM). However, after EBP (25 and 100

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μg/ml) pretreatment, this tendency turned to the opposite, as evidenced by decreased

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caspase-3 and Bax expression, and the elevated Bcl-2 protein expression, thus leading

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to a high Bcl‑2/Bax ratio. These result suggested that anti- apoptosis activity of EBP pretreatment for OBs was in part mediated by modulating caspase-3, Bax and Bcl-2 protein expression.

To gain more depth insight into the mechanisms by which EBP pretreatment promoted OBs proliferation and inhibited apoptosis, we evaluated the signaling event underlying it. It is well documented that PI3K/AKT/mTOR signaling pathway represents important signal transduction mechanisms involved in the process of apoptosis [46]. Present study aimed to elucidate the role of this pathway in the anti-apoptotic effect of EBP (25 and 100 μg/ml) pretreatment on OBs. Our results

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showed that EBP pretreatment could restore the suppressed phosphorylation of PI3K, Akt and mTOR protein expression in DEX (100 μM)-treated control cells to the normal level, but the protein levels of PI3K, Akt and mTOR remained unchanged always, revealing that anti-apoptotic activity of EBP pretreatment toward OBs was mainly dependent on suppressing PI3K/Akt-mTOR signaling pathway.

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Based on the above findings, we concluded that EBP pretreatment has a potential

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therapeutical effect on GIO and stimulating osteogenesis may be one of the

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mechanisms. Evidence suggests that the Wnt/β-catenin pathway has been involved in

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the pathogenesis of early stage osteonecrosis and have been reported to positively

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regulate OBs differentiation and inhibit adipocyte differentiation [47]. Jullien et al. demonstrated that GCs treatments can stimulate osteocyte apoptosis and bone mass

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loss by negative regulation of Wnt/β-catenin signal-related molecules in OBs [48].

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The canonical Wnt/β-catenin signaling is extremely complicated and subject to a

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variety of feedback control. In brief, the binding of Wnt proteins with membrane-bound frizzled receptors and LRP5/6 co-receptor initiated a signaling cascade to inhibit the ubiquitination and degradation of β-catenin and accelerate its translocation into the nucleus. This nuclear β-catenin binds to and co-activates members of the T-cell factor/ lymphoid-enhancing factor family of transcription factors (TCF/LEF) to activate downstream target gene, such as Runx2 and Osx, thus leading to a promotion for differentiation and proliferation of OBs [49]. In line with this, in the current study we found the decreased protein expressions of members in Wnt pathways, including Lrp-5, β-catenin, Runx2 and Osx, in DEX (100 μM)-treated

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control cells when compared with the normal control. In contrast, these attenuated protein expressions in the model control were significantly reversed by pretreatment with EBP for 2h at both concentrations. In this respect, we speculated that EBP pretreatment could induce osteoblastogenesis through activation of the canonical Wnt/β-catenin signaling pathway of OBs.

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In summary, our present study offers the first convincing evidence that EBP

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pretreatment has a positive protective effect on osteoblastogenesis during the

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progression of GIO, which can be mediated by alleviating OBs apoptosis via the

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up-regulation of PI3K/AKT/mTOR and Wnt/β-catenin signaling pathway and

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inactivation of the mitochondrial apoptosis in OBs. However, further in vivo study should be performed to decipher the detailed mechanism of EBP in protecting against

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imbalance of osteoblastogenesis and support its clinical application for patients who

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are in need of GCs treatments and are at risk of developing GIO.

Acknowledgements

This research was financially supported by Competitive Allocation of Special Funds for Science and Technology Development in Zhanjiang (2018A01031) and Competitive Allocation of Special Funds for Science and Technology Development in Zhanjiang (2016A01009).

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FIGURE CAPTIONS Figure 1 (A) The HPSEC profile of EBP. (B) The HPLC chromatograms of PMP

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derivatives of 10 standard monosaccharides mixture and EBP hydrolysate.

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Figure 2 (A) Effect of EBP pretreatment on the proliferation of OBs assayed by

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MTT. (B) Observation of EBP pretreatment on morphological and proliferative

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changes of OBs using inverted phase contrast microscopy (original magnification,

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×100). Results are presented as means±S.D. (n = 3). *P < 0.05; **P < 0.01 compared with normal control; ##P < 0.01; ###P < 0.001 compared with negative control.

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Figure 3 (A) Effect of EBP pretreatment on the cell growth changes of OBs

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assayed by crystal violet staining under inverted phase contrast microscopy

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(original magnification, ×100). (B) Quantitative assessment of the cell growth changes of OBs. Results are presented as means±S.D. (n = 3). *P < 0.05 or **P < 0.01 compared with normal control; #P < 0.05 or ##P < 0.01 compared with negative control. Figure 4 (A) Effect of EBP pretreatment on ALP activity of OBs. (B) Effect of EBP pretreatment on calcium content of OBs. Results are presented as means±S.D. (n = 3). *P < 0.05 compared with normal control; ##P < 0.01 compared with negative control.

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Figure 5 (A) Effect of EBP pretreatment on the percentages of apoptotic OBs analyzed using flow cytometry analysis. (B) Quantification analysis of flow cytometry analysis. Results are presented as means±S.D. (n = 3). **P < 0.01 compared with normal control. Figure 6 (A) Effect of EBP pretreatment on the protein expression of Bax, Bcl-2,

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and cleaved caspase-3 in OBs using western blotting assay. (B) Quantification

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analysis of the protein expression of Bax, Bcl-2, and cleaved caspase-3 in OBs

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following EBP pretreatment. (C) The anti‑apoptotic/pro‑apoptotic (Bcl‑2/Bax)

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protein ratio in OBs following EBP pretreatment. Results are presented as

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means±S.D. (n = 3). *P < 0.05, **P < 0.01 or ***P < 0.001 compared with normal control; ##P < 0.01 or ###P < 0.001 compared with negative control.

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Figure 7 (A) Effect of EBP pretreatment on the protein expression of PI3K,

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p-PI3K, Akt, p-Akt, mTOR and p-mTOR in OBs using western blotting assay. (B)

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Quantification analysis of the protein expression of B PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR in OBs following EBP pretreatment. Results are presented as means±S.D. (n = 3). **P < 0.01 compared with normal control; ##P < 0.01 compared with negative control. Figure 8 (A) Effect of EBP pretreatment on the protein expression of Lrp-5, β-catenin, Runx2 and Osx in OBs using western blotting assay. (B) Quantification analysis of the protein expression of Lrp-5, β-catenin, Runx2 and Osx in OBs following EBP pretreatment. Results are presented as means±S.D. (n =

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Journal Pre-proof 3). *P < 0.05, **P < 0.01 compared with normal control; ##P < 0.01 compared with

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negative control.

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