Influence of calcium supplementation against fluoride-mediated

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Agricultural and Environmental Chemistry

Influence of calcium supplementation against fluoridemediated osteoblasts impairment in vitro: Involvement of canonical Wnt/#-catenin signaling pathway Jinming Wang, Jiarong Yang, Xiaofang Cheng, Fengfeng Yin, Yangfei Zhao, Yaya Zhu, Zipeng Yan, Forouzan Khodaei, Mohammad Mehdi Ommati, Ram Kumar Manthari, and Jundong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03835 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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Journal of Agricultural and Food Chemistry

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Influence of calcium supplementation against

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fluoride-mediated osteoblasts impairment in vitro:

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Involvement of canonical Wnt/β-catenin signaling pathway

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Jinming Wang&#*, Jiarong Yang&#, Xiaofang Cheng§, Fengfeng Yin&#, Yangfei Zhao&#,

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Yaya Zhu&#, Zipeng Yan&#, Forouzan Khodaei&#, Mohammad Mehdi Ommati※, Ram

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Kumar Manthari&#, Jundong Wang&#* Jinzhong, Shanxi, China

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8

&

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University. Taigu 030801, Shanxi, PR China.

College of Animal Science and Veterinary Medicine, Shanxi Agricultural

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#

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University. Taigu 030801, Shanxi, PR China.

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§

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Shanxi, PR China.

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PR China

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* For correspondence:

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Jinming Wang,

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Associate Professor, Shanxi Key Laboratory of Environmental Veterinary Medicine,

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Shanxi Agricultural University, Taigu 030801, Shanxi, PR China; E-mail:

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[email protected]

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Jundong Wang,

Shanxi Key Laboratory of Environmental Veterinary Medicine, Shanxi Agricultural

College of Arts and Sciences, Shanxi Agricultural University, Taigu 030801,

College of Life Sciences, Shanxi Agricultural University. Taigu 030801, Shanxi,

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Full Professor, Shanxi Key Laboratory of Environmental Veterinary Medicine,

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Shanxi Agricultural University, Taigu 03081, Shanxi, PR China; E-mail:

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[email protected]

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Abstract: Fluoride (F) is capable of promoting abnormal proliferation and

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differentiation in primary cultured mouse osteoblasts (OB cells), although; the

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underlying mechanism responsible remain rare. This study aimed to explore the roles

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of Wingless and INT-1(Wnt) signaling pathways and screen appropriate doses of

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calcium (Ca2+) to alleviate the sodium fluoride (NaF)-induced OB cells toxicity. For

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this, we evaluated the effect of Dickkopf-related protein 1 (DKK1) and Ca2+ on

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mRNA

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receptor-related protein 5 (LRP5), Dishevelled 1 (Dv1), glycogen synthase kinase 3β

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(GSK3β), β-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular

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myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells

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challenged with 10-6 moL/L NaF for 24 h. Data demonstrated showed that F

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significantly increased the OB cells proliferation rate. Ectogenic 0.5 mg/L DKK1

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significantly inhibited the proliferation of OB cells induced by F. The mRNA

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expression levels of Wnt 3a, LRP5, Dv1, LEF1, β-catenin, cMYC and Ccnd1 were

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significantly increased in F group, while significantly decreased in 10-6 moL/L

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NaF+0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5,

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β-catenin and cMYC were significantly decreased in 10-6 moL/L NaF+2 mmoL/L

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CaCl2 (F+CaII) group. The proteins expression levels of Wnt3a, Cyclin D1, cMYC

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and β-catenin were significantly increased in F group whereas decreased in the

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F+CaII group. However, the mRNA and protein expression levels of GSK3β were

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significantly decreased in the F group while significantly increased in the F+CaII

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group.

levels

of

wingless/integrated

3a

(Wnt3a),

low-density

lipoprotein

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In summary, F activated the canonical Wnt/β-catenin pathway, changed the related

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genes expression and β-catenin protein location in OB cells, promoting cell

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proliferation. 2 mmoL/L Ca2+ supplementation reversed the expression levels of genes

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and proteins related to the canonical Wnt/β-catenin pathway.

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Key words: Fluoride; Ca2+; Osteoblasts; Proliferation; Wnt/β-catenin signaling

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pathway

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Introduction

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Fluoride (F), the most reactive halogen, was found in the soil and drinking water all

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around the world with high levels.1 F can directly participate in bone metabolism and

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maintain the normal levels of calcium (Ca2+) and phosphorus metabolism. F has a

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strong affinity for bone,2 but excessive intake of F may lead to enamel and skeletal

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fluorosis.3 The fundamental cause of bone fluorosis is the dysregulation of bone

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metabolism, which caused by the active function, abnormal proliferation, and

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differentiation of osteoblasts (OB) cells that regulate bone formation.4 When OB cells

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proliferate and differentiate abnormally, a large amount of Ca2+ is transported to the

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skeletal system, which ensures the occurrence of bone metabolism at normal levels, so

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that the blood Ca2+ levels decreases significantly, and the bone tissue undergoes a

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serious Ca2+ deposition phenomenon.5 The previous study reported F could affect

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bone metabolism and stimulate osteoblastic bone formation due to an anabolic effect

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of F, in-vitro and in-vivo.6

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Canonical Wnt/β-catenin signaling pathway, which can regulate the proliferation

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and differentiation of OB cells and chondrocytes, is a new hotspot in the study of

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bone fluorosis. The change of expression or function of related factors in the

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canonical

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differentiation of OB cells, the formation and mineralization of bone matrix, and leads

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to the change of bone mass.7 It has been shown that Wingless/integrated 1, 2, 3, 3a, 8a

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and 8b (Wnt1, Wnt2, Wnt3, Wnt3a, Wnt8a and Wnt8b) activate the canonical

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Wnt/β-catenin signaling pathway, in which Wnt3a regulates the differentiation of OB

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cells.8 In canonical Wnt/β-catenin signaling, it has been shown that Wnt3a bind to

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low-density lipoprotein receptor-related protein 5 (LRP5), and Dishevelled 1 (Dvl)

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within the cytoplasm is activated, thereby inhibiting the activity of degrading

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complexes (APC-glycogen synthase kinase 3β (GSK3β)-axin-β-catenin).9,10 As a

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result, the unphosphorylated β-catenin avoids the degradation of the proteasome and

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thus accumulates in the cytoplasm. It recruits transcriptional activators to stimulate

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the expression of target genes cellular myelocytomatosis oncogene (cMYC) and

Wnt/β-catenin

signaling

pathway

affects

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development

and

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Ccnd1 (Cyclin D1) after translocation to the nucleus and combination with LEF1.11,12

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In addition, abnormal expression of the β-catenin gene in cells is closely related to the

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activation of the canonical Wnt/β-catenin signaling pathway and tumorigenesis.13 On

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the contrary, mutations on this gene (a family carrying loss-of-function) is linked to

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osteoporosis, which decreases the OB cells development and increases the numbers of

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OC cells.14

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One of specific inhibitors of the canonical Wnt/β-catenin signaling pathways is

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Dickkopf-related protein 1 (DKK1), competing with Wnt protein and binding LRP5/6

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receptor to inhibit the intracellular transduction of the Wnt protein.15 In order to

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examine whether sodium fluoride (NaF) activates the canonical Wnt/β-catenin

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signaling pathway in mouse OB cells and if the canonical Wnt/β-catenin signaling

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pathway is required to induce osteoblast proliferation, this study co-treated OB cells

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with NaF and DKK1. In addition, it has been reported that Ca2+ supplementation can

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relieve the symptoms of skeletal fluorosis,16 but the interpretation of its impact

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mechanism is still unclear. Ca2+ supplementation can antagonize the toxicity of F and

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prevent bone softening, but Ca2+ supplementation can alter the permeability of cell

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membrane, and cause Ca2+ to access the cell to trigger the change of cell activity.17

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Recently, we have demonstrated that the decreased OB cells proliferation, increased

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intracellular Ca2+ levels, and the mRNA and protein expression level in the

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endoplasmic reticulum (ER) stress apoptosis pathway related genes was observed at 9

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mg/L NaF group, whereas 0.5-1 mmoL/L CaCl2 can significantly reduce the

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perturbation caused by NaF, while 2-8 mmoL/L CaCl2 can enhance the damage of

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NaF to OB cells.18 Based on our previous investigations and MTT method results, this

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study aimed to investigate whether Ca2+ supplementation inhibits the abnormal OB

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cells proliferation induced by low dosage of NaF via the canonical Wnt/β-catenin

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signaling pathway or not.

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MATERIALS AND METHODS

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Animals

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24 h-old neonatal healthy Kunming (KM) male mice were purchased from the

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Experimental Animal Center of Shanxi Medical University (Taiyuan, China). Animal

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experimentation was performed according to the regulations of laboratory animal care

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approved by the Ethics Committee of Shanxi Agricultural University.

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Chemicals and Instruments

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NaF and MTT were purchased from Sigma-Aldrich (Shanghai, China). Dulbecco's

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Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and fetal calf serum

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(FCS)

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penicillin/streptomycin were purchased from Solarbio (Beijing, China). Trizol

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Reagent, PrimeScriptTM RT reagent and SYBR® Premix Ex Taq™ Kit were obtained

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from Takara Biotechnology Company (Dalian, China). Recombinant human DKK1

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was purchased from MCE (Shanghai, China). Bicinchoninic acid (BCA) protein assay

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kit, alkaline phosphate (ALP) staining kit, phenylmethylsulfonylfluoride (PMSF), and

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Radio-Immunoprecipitation Assay (RIPA) were obtained from Beyotime Institute of

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Biotechnology (Shanghai, China). Mouse anti-rabbit β-catenin antibody and mouse

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anti-rabbbit GSK3β antibody were provided by Proteintech (Wuhan, China). Mouse

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anti-rabbit Wnt3a antibody was purchased from Biogot Technology, Co, Ltd (Nanjing,

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China). Mouse anti-rabbit cMYC antibody, mouse anti-rabbit Cyclin D1 antibody and

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goat anti-rabbit secondary antibody were purchased from Boosen (Beijing, China).

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FITC-conjugated goat anti-rabbit β-catenin, Wnt3a, and Cyclin D1 antibodies were

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purchased from Sangon (Shanghai, China).

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Primary isolation and culture of OB cells

were

obtained

from

Gibico

(Grand

Island,

NY,

USA),

1

%

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Primary mice OB cells were isolated from the calvaria of 1-day-old KM male mice

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by sequential enzymatic digestion.19 1 mm3-sized fine broken bone slices were

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digested with 0.25 % trypsin at 37  °C water bath for 30 min, by shaking for every 10

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min. Debris released were discarded and cells were incubated with preheated 0.1 %

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type II collagenase at 37  °C for 1 h digestion. The supernatant was centrifuged at

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1000 r/min for 10 min, and the precipitated cell mass was dissolved in DMEM

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containing 15 % FBS and 1 % penicillin/streptomycin to make 1 × 105 cells/mL

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suspension and cultured in an incubator maintained at 37  °C with 5 % CO2.

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Passage and identification of OB cells

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In order to purify the OB cells isolated and cultured, the mice OB cells were

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filtered out by differential velocity adherent procedure.20 When the cells in the culture

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flask grow to about 90 %, add 1 mL of pre-heated 0.25 % trypsin digest at 37 °C.

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Some semi-confluent cells supplemented with 5 % FCS medium were fixed with 4 %

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paraformaldehyde and then routinely stained with hematoxylin and eosin (H&E),

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others were fixed with methanol for 10 min and stained with Giemsa for 15 min. The

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cultured OB cells were stained according to the procedures in the instructions of the

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ALP staining kit. The fully confluent cells were fixed in 95 % ethanol for 10 min,

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washed with distilled water, and then stained with 0.1 % alizarin red (pH 7.4) at 37 °C

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for 10 min.21,22 For all the steps, primary OB cells used were of 3rd passage.

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Screening the F concentration

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200 μL cell solution were seeded at a density of 5×104 /mL in a 96-well culture

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plate. After the cells were attached, 10-8, 10-7, 10-6, 10-5, and 10-4 moL/L NaF were

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added for 24 h, 48 h, and 72 h culture, and 10 replicate wells were set for each

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concentration. 20 μL of 0.5 % MTT was added to each well, and the culture was

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continued for 4 h. The liquid in the culture plate was added with 100 μL of dimethyl

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sulfoxide (DMSO) per well, and incubated at 37 °C for 30 min to fully dissolve the

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intracellular crystallization. The OD value was detected at 570 nm to determine the

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optimal NaF concentration for promoting OB cells proliferation with an EIA

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Analyzer.

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Screening the Ca2+ concentration

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OB cells were treated with 0 moL/L NaF+0 mmoL/L CaCl2, 10-6 moL/L NaF+0

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mmoL/L CaCl2, 10-6 moL/L NaF+0.5 mmoL/L CaCl2, 10-6 moL/L NaF+1 mmoL/L

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CaCl2, 10-6 moL/L NaF+2 mmoL/L CaCl2, 10-6 moL/L NaF+4 mmoL/L CaCl2, and

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10-6 moL/L NaF+8 mmoL/L CaCl2 respectively for 24 h, 48 h, and 72 h. MTT

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method was applied to screen the Ca2+ concentration.

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Screening the optimal time for DKK1 treatment

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OB cells were treated together with 0.5 mg/L DKK1 and 10-6 moL/L NaF for 24 h,

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48 h, and 72 h.22 The average optical density (OD) of each group was measured by

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MTT method under 570 nm, and the proliferation of OB cells were detected by

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calculating the proliferation rate of OB cells in each group. Thus, the optimal time for

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DKK1 inhibiting osteoblast proliferation under 10-6 moL/L NaF treated were

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screened.

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OB cells treatment with extracellular NaF, DKK1, and Ca2+

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Primary OB cells were passaged every 7 days and done up to 3rd passages in a total

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of 42 flasks. 42 flasks containing OB cells were randomly divided into seven groups

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of six flasks each and were maintained on the tissue culture solution as shown in

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Table 1.

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Total RNA extraction and quantitative RT-PCR (qRT-PCR)

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Following the manufacturer’s instruction, total RNA in the OB cells was extracted

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using Trizol Reagent. The list of primers was given in Table 2 by Primer 3.0 plus

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software. The 250 ng/μL total RNA was reversed and diluted to make 10 ng/μL

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cDNA by real-time reverse transcription-PCR (RT-PCR). The mRNA expressions of

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the

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GSK3β, β-catenin, LEF1, cMYC, Ccnd 1 and β-actin were evaluated using the SYBR

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Premix Ex TaqTM II kit on the PTC-200 QRT-PCR system (Bio-Rad, USA).

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QRT-PCR cycling conditions were 95  °C for 30  s, 45 PCR cycles of 95 °C for 5 s,

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60 °C for 30 s and 72 °C for 30 s, finally, 95 °C for 15 min, 60 °C for 1 s, and 95 °C

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for 15 s. All experiments were done in triplicate. The 2-∆∆Ct method was used to

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evaluate the relative expression levels of genes by comparing sample expression

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relative to a housekeeping gene (β-Actin).

canonical Wnt/β-catenin pathway related genes, including Wnt3a, LRP5, Dvl,

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Immunofluorescence staining for β-catenin nuclear translocation

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In order to make cell climbing tablets, 5×104 cells/mL were inoculated in a 6-well

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cell culture plate with a small sterilized cover wave plate in a 5 % CO2-containing

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saturated humidity with 37 °C constant temperature incubator. OB cells were grown

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on glass coverslips and incubated with NaF, Ca2+, and DKK1 for 24 h. After the cell

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culture solution was discarded, the cells were washed 3 times with PBS and fixed in 4

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% cold formaldehyde for 18 min. OB cells were permeabilized with 0.2 % Triton

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X-100 for 20 min and blocked with 10 % FBS for 30 min. Add mouse anti-rabbit

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β-catenin-antibody (1 : 200) to the cell slide and incubate overnight at 4 °C. Avoiding

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light, FITC-conjugated secondary antibody was diluted with PBS to a concentration

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of 1:150 and added to the cells for two hours’ incubation. Cells were sealed with

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anti-fluorescence quenching tablets containing DAPI working solution. The signal

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was visualized by FV 1000 confocal fluorescence microscopy (Olympus, Japan).

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Immunofluorescence staining for Wnt3a and Cyclin D1

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For previous steps, please refer to method 2.10. Finally, samples were incubated

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with mouse anti-rabbit Wnt3a/Cyclin D1 antibody (1 : 200) overnight at 4 °C

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followed by incubation with FITC-conjugated secondary antibody (1 : 150) for 120

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min at 37 °C in dark. Cells were sealed with anti-fluorescence quenching tablets and

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observed under a fluorescence microscope (Japan, OLYMPUS).

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Western blotting

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The protein levels of β-catenin, GSK3β, cMYC, Wnt3a, Cyclin D1, pGSK3β and

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β-actin were detected by western blot. The cells were washed three times with PBS,

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lysed by RIPA and PMSF at a ratio of 99:1, collected with a cell scraper, and

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centrifuged (13,000 rpm/min, 10 min, at 4 °C). After measuring the protein

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concentration, 50 ng of the protein sample was mixed with the loading buffer (1 : 5)

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and denatured at 100 °C metal bath. Preparation of concentrated gel and separation

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gel, electrophoresis conditions were 80V, 30 min and 150V, 90 min, respectively,

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polyacrylamide gel electrophoresis on Bio-Rad equipment. Then, the separated

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proteins were determined in the gel and transferred onto polyvinylidene fluoride

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membrane (CA, USA) at 35 V for 70 min. Seal the membrane in the 5% skimmed

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milk powder, put it on the shaking table for an hour. Then the membrane was

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incubated with the primary antibodies (1 : 100; for β-catenin, GSK3β, cMYC, Wnt3a,

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Cyclin D1, pGSK3β and β-actin) in antibody dilution overnight at 4 °C. After three

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times washes with TBST, the membrane was incubated with secondary antibody for 2

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h at 37 °C (1 : 5000). The target protein bands were visualized with enhanced

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chemiluminescent (super ECL, KeyGEN BioTECH, Beijing, China). Optical density

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was calculated on the FluorChem Q system (AlphaInnotech, CA, Santa Clara, USA).

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Statistical analyses

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The data were checked for normality and transformed when appropriate. One-way

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analysis of variance was applied to data using the Prism software (CA, USA). The

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mean ± SD of at least six independent experiments are reported in the text. Mean

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separation was performed using the Tukey multiple range test. The level of

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significance was set at P < 0.05.

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Results

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Establishment of OB cells culture

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The subcultured mouse osteoblasts were observed under an inverted phase contrast

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microscope. After 24 h, all of them were attached to the wall, which was fusiform or

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triangular, and the nucleus was oval and large. After the cells were stained with H&E,

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the cytoplasm was stained pink, and the oval-shaped nucleus with clear outline was

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stained purple-blue, and the cell processes were cross-linked (Fig. 1a). After the cells

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were stained with Giemsa, the cytoplasm showed light blue and uniform staining, and

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the nucleus was stained blue or bluish-purple (Fig. 1b). Black particles or flocculent

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precipitates were shown in the cytoplasm and protrusions, indicating that the

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cytoplasm and processes of the cells are rich in ALP (Fig. 1c). The cytoplasm of

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osteoblasts stained with alizarin red has orange-red calcified nodules with clear

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boundaries and exhibits calcium salt deposition ability (Fig. 1d).

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Determination of appropriate NaF concentration

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The effects of F on the OB cells proliferation rate were shown in Fig. 2(A). After

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24 h and 48 h treatment, the proliferation ability of OB cells at 10-6 moL/L NaF was

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significantly higher than that of 0 moL/L NaF (p < 0. 01, p < 0. 05). However, the

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proliferation rate of OB cells at 10-4 moL/L NaF was lower than that of 0 moL/L NaF.

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Therefore, 10-6 moL/L NaF promoted the proliferation of mouse OB cells in vitro.

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Determination of appropriate Ca2+ concentration

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The effects of Ca2+ on the OB cells proliferation rate were shown in Fig. 2(B). The

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proliferation rate of OB cells at 10-6 moL/L NaF+0 mmoL/L CaCl2 group (F group)

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was in line with the previous result, increasing significantly compared with OB cells

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at 0 moL/L NaF (C group). As the concentration of supplemented Ca2+ increases, the

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proliferation rate of OB cells decreases first and then increases compared with the F

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group. Therein, the significant decreases in the OB cells proliferation rate were noted

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in the 10-6 moL/L NaF+1 mmoL/L CaCl2 group (F+CaⅠgroup), 10-6 moL/L NaF+2

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mmoL/L CaCl2 group (F+CaⅡgroup) and 10-6 moL/L NaF+4 mmoL/L CaCl2 group

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(F+Ca Ⅲ group) compared with the F group. According to this result, 1, 2, and 4

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mmoL/L Ca2+ for reducing OB cells proliferation rate were considered, and were

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represented as the CaⅠgroup, CaⅡgroup, CaⅢ group respectively, shown in Table

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1.

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Determination of duration for NaF and Ca2+ exposure

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Compared to the C group (Fig. 3), the OB cells proliferation rate in the F group was

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markedly higher (p < 0.01) at 24 h, while the difference was significant at 48 h and 72

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h (p < 0.05). The proliferation rate was decreased significantly in the 10-6 moL/L

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NaF+0.5 mg/L DKK1 (FY) group at 24 h (p < 0.01), 48 h, and 72 h (p < 0.05),

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compared to the F group. Nevertheless, the proliferation rate of OB cells in the F+Ca

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Ⅰand F+CaⅡgroups were down-regulated significantly compared with the F group

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at 24 h (p < 0.05) and significantly down-regulated at 48 h only in the F+CaⅡgroup

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(p < 0.05). Hence, the 24 h was elected as a valid duration for further experiments.

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Changes of the expression of Wnt/β-catenin-signaling-pathway-related mRNA

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induced by NaF, DKK-1, and Ca2+

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Fig. 4 showed that the mRNA expression levels of Wnt3a, LRP5, Dv1, β-catenin,

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LEF1, cMYC and Cyclin D1 of mouse OB cells in the F group were markedly

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up-regulated compared to the C group. Nevertheless, the mRNA expression levels of

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the above genes in FY group were obviously down-regulated compared to the F group.

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In addition, the relative expression of Wnt3a, Dv1, β-catenin and cMYC in the F+Ca

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Ⅰ group were notably down-regulated, compared to the F group. Further, the relative

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expression of Wnt3a, LRP5, β-catenin and cMYC in F+Ca Ⅱ

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significantly decreased compared with the F group.

group were

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The F group was related to an obvious decrease in the expression of GSK3β (p