Influence of calcium supplementation against fluoride-mediated

Subscriber access provided by Georgetown University | Lauinger and Blommer Libraries

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Influence of calcium supplementation against

2

fluoride-mediated osteoblasts impairment in vitro:

3

Involvement of canonical Wnt/β-catenin signaling pathway

4

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

5

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

6

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

7

8

&

9

University. Taigu 030801, Shanxi, PR China.

College of Animal Science and Veterinary Medicine, Shanxi Agricultural

10

#

11

University. Taigu 030801, Shanxi, PR China.

12

§

13

Shanxi, PR China.

14

※

15

PR China

16

* For correspondence:

17

Jinming Wang,

18

Associate Professor, Shanxi Key Laboratory of Environmental Veterinary Medicine,

19

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

20

[email protected]

21

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,

ACS Paragon Plus Environment


22

Full Professor, Shanxi Key Laboratory of Environmental Veterinary Medicine,

23

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

24

[email protected]

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Page 2 of 38

Page 3 of 38


43

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

44

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

45

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

46

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

47

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

48

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

49

mRNA

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

group.

levels

of

wingless/integrated

3a

(Wnt3a),

low-density

lipoprotein

65

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

66

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

67

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

68

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

69

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

70

pathway



71

Page 4 of 38

Introduction

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

canonical

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

Wnt/β-catenin

signaling

pathway

affects


the

development

and

Page 5 of 38


100

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

101

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

102

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

103

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

104

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

105

OC cells.14

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

signaling pathway or not.

126

MATERIALS AND METHODS

127

Animals



Page 6 of 38

128

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

129

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

130

experimentation was performed according to the regulations of laboratory animal care

131

approved by the Ethics Committee of Shanxi Agricultural University.

132

Chemicals and Instruments

133

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

134

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

135

(FCS)

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

purchased from Sangon (Shanghai, China).

149

Primary isolation and culture of OB cells

were

obtained

from

Gibico

(Grand

Island,

NY,

USA),

1

%

150

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

151

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

152

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

153

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

154

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

155

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


Page 7 of 38


156

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

157

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

158

Passage and identification of OB cells

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

Screening the F concentration

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

optimal NaF concentration for promoting OB cells proliferation with an EIA

178

Analyzer.

179

Screening the Ca2+ concentration

180

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

181

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

182

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



183

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

184

method was applied to screen the Ca2+ concentration.

185

Screening the optimal time for DKK1 treatment

186

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

187

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

188

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

189

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

190

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

191

screened.

192

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

193

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

194

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

195

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

196

Table 1.

197

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

198

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

199

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

200

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

201

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

202

the

203

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

204

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

205

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

206

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

207

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

208

evaluate the relative expression levels of genes by comparing sample expression

209

relative to a housekeeping gene (β-Actin).

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


Page 8 of 38

Page 9 of 38

210


Immunofluorescence staining for β-catenin nuclear translocation

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

Immunofluorescence staining for Wnt3a and Cyclin D1

224

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

225

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

226

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

227

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

228

observed under a fluorescence microscope (Japan, OLYMPUS).

229

Western blotting

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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



238

proteins were determined in the gel and transferred onto polyvinylidene fluoride

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

Statistical analyses

248

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

249

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

250

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

251

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

252

significance was set at P < 0.05.

253

Results

254

Establishment of OB cells culture

255

The subcultured mouse osteoblasts were observed under an inverted phase contrast

256

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

257

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

258

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

259

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

260

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

261

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

262

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

263

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

264

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


Page 10 of 38

Page 11 of 38


265

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

266

Determination of appropriate NaF concentration

267

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

268

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

269

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

270

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

271

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

272

Determination of appropriate Ca2+ concentration

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283

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

284

1.

285

Determination of duration for NaF and Ca2+ exposure

286

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

287

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

288

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

289

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

290

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

291

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



Page 12 of 38

292

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

293

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

294

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

295

induced by NaF, DKK-1, and Ca2+

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

significantly decreased compared with the F group.

group were

304

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

Influence of calcium supplementation against fluoride-mediated

Recommend Documents