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Laminaria japonica polysaccharide inhibits vascular calcification via preventing osteoblastic differentiation of vascular smooth muscle cells Xue-Ying Li, Qiang-Ming Li, Qing Fang, Xue-Qiang Zha, panlihua panlihua, and Jian-Ping Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06115 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Journal of Agricultural and Food Chemistry

1

Laminaria

japonica

polysaccharide

inhibits

vascular

2

calcification via preventing osteoblastic differentiation of

3

vascular smooth muscle cells

4 5

†‡ ‡ ‡ †‡ ‡ Xue-Ying Li#, , , Qiang-Ming Li#, , Qing Fang , Xue-Qiang Zha*, , , Li-Hua Pan ,

6

Jian-Ping Luo*,

‡

7 8 9 10 11

†

School of Biological and Medical Engineering, Hefei University of Technology,

Hefei 230009, People’s Republic of China ‡

School of Food Science and Engineering, Hefei University of Technology, Hefei

230009, People’s Republic of China

12 13

*Correspondence authors: Prof. Dr. Xue-Qiang Zha and Jian-Ping Luo, School of

14

Food Science and Engineering, Hefei University of Technology, Hefei 230009,

15

People’s Republic of China. E-mail: [email protected] (X.-Q. Zha);

16

[email protected] (J.-P. Luo)

17 18

#

Xue-Ying Li, Qiang-Ming Li and Qing Fang contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT: This study aimed to investigate the effect and underlying mechanism of

20

a purified Laminaria japonica polysaccharide (LJP61A) on preventing vascular

21

calcification (VC). In adenine-induced chronic renal failure (CRF) mice VC model

22

and β-glycerophosphate (β-GP)-induced vascular smooth muscle cells (VSMC)

23

calcification model, LJP61A was found to significantly inhibit VC phenotypes as

24

determined

25

immunohistochemical staining. Meanwhile, LJP61A remarkably up-regulated the

26

mRNA levels of VSMC related markers and down-regulated the mRNA levels of

27

sodium-dependent phosphate cotransporter Pit-1. In addition, LJP61A could

28

significantly decrease the protein levels of core-binding factor-1, osteocalcin, bone

29

morphogenetic protein 2 and receptor activator for nuclear factor-κB ligand, and

30

increase the protein levels of osteoprotegerin and matrix gla protein. These results

31

indicated that LJP61A ameliorated VC both in vivo and in vitro via preventing

32

osteoblastic differentiation of VSMC, suggesting LJP61A might be a potential

33

therapeutic agent for VC in CRF patients.

by

biochemical

analysis,

von

Kossa,

alizarin

red

and

34 35

KEYWORDS: Laminaria japonica, Polysaccharide, Vascular calcification, Chronic

36

renal failure, Osteoblastic differentiation

37


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INTRODUCTION

39

The vascular calcification (VC) is demonstrated as calcium over deposition in vessel

40

wall, which is an important risk marker in the pathogenesis of cardiovascular diseases,

41

such as myocardial infarction and coronary death.1 VC is an active and regulated

42

process involved with osteoblastic differentiation of vascular smooth muscle cells

43

(VSMC), which is one major mechanism in the development of VC.2-4 Under normal

44

conditions, VSMC produce endogenous calcification inhibitors, such as osteopontin

45

(OPN)5, osteoprotegerin (OPG)6, 7 and matrix gla protein (MGP)8, to suppress the

46

osteoblastic differentiation of VSMC. However, in the occurrence of VC, the

47

expression of these endogenous calcification inhibitors will be decreased. Additionally,

48

the intracellular phosphate concentration will be enhanced by sodium dependent

49

phosphate cotransporter Pit-1 to cause VSMC transforming to osteoblast-like cells.9

50

Under the control of core-binding factor alpha 1 (Cbfa-1), these osteoblast-like cells

51

will express alkaline phosphatase (ALP) and bone-associated proteins, such as

52

osteocalcin (OC) and bone morphogenetic protein 2 (BMP-2) to further induce the

53

osteoblastic differentiation and calcification of VSMC.9-12

54

Evidences exhibited that VC is highly prevalent in patients with chronic renal

55

failure (CRF), especially those treated by dialysis. The morbidity of VC in dialysis

56

patients was 2-5 times higher than those in age-matched normal subjects.13 Moreover,

57

the occurrence of VC was considered as the most important reason that the high

58

cardiovascular mortality was observed in end-stage CRF patients.3, 14 However, the

59

definite mechanisms of VC in CRF patient are not entirely understood and current



60

therapeutic agents are not efficient. Therefore, finding a target agent to delay the

61

occurrence and development of VC has great sense for CRF patients. In recent years,

62

to achieve this goal, there is considerable interest in developing these agents from

63

herbs or edible-medicinal materials.15-17

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Laminaria japonica, a famous edible-medicinal marine vegetable, has long been

65

recorded as a valuable therapeutic agent for detumescence, phlegm elimination, and

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weight loss for many centuries.18 In our previous works, a purified L. japonica

67

polysaccharide (LJP61A) was isolated and characterized as a repeating unit consisting

68

of →3,6)-α-D-Manp-(1→,→4)-α-D-Manp-(1→,→4)-2-O-acetyl-

69

β-D-Glcp-(1→,→4)-β-D-Glcp-(1→,→6)-4-O-SO3-β-D-Galp-(1→,→6)-β-D-Galp-(1

70

→,→3)-β-D-Galp-(1→, and a terminal residue of α-D-Glcp-(1→ (Figure 1).19 We

71

have demonstrated that LJP61A have good potential for suppressing atherosclerosis

72

via regulating cellular lipid metabolism, inhibiting cellular inflammation and

73

ameliorating insulin resistance.19, 20 Because atherosclerosis is a common pathological

74

basis of cardiovascular diseases and VC is a common pathological manifestation of

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atherosclerosis,1 we speculated that LJP61A might have the potential of preventing

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VC development. We examined the preventive effect of L. japonica polysaccharide

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LJP61A on vascular calcification using the adenine-induced CRF mice VC model and

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β-glycerophosphate (β-GP) induced VSMC calcification model. The underlying

79

mechanism was further clarified in vivo and in vitro. To our best knowledge, similar

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action about polysaccharides has not been reported.

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MATERIALS AND METHODS


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Chemicals

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LJP61A was extracted and purified as described previously.19 Adenine and β-GP were

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purchased from Biosharp (Hefei, China). Cinacalcet tablets were purchased from

85

Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Fetal bovine serum, DMEM,

86

streptomycin and penicillin were purchased from Hyclone Co. (UT, USA). The

87

fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH) and ALP ELISA

88

kits and radio immunoprecipitation assay lysis buffer were purchased from Nanjing

89

Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent was purchased

90

from Invitrogen (Darmstadt, Germany). BCA was purchased from Sangon Biotech

91

Co., Ltd. (Shanghai, China). The primary antibodies against calcium-sensing receptor

92

(CaSR), Cbfa-1, OC, BMP-2, receptor activator for nuclear factor-κB ligand

93

(RANKL), OPG, MGP and β-actin were purchased from Santa Cruz Biotechnology,

94

Inc. (TX, USA). The secondary antibody was purchased from Boster Co. (Wuhan,

95

China). All other reagents were analytical grade and purchased locally.

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Animal and treatment

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Eight-week-old male C57BL/6 mice (20 ± 2 g) were obtained from the Laboratory

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Animal Center of Anhui Medical University. After an acclimatization period of one

99

week, mice were randomly divided into six groups (20 mice per group): including

100

normal group, model group, positive group and three LJP61A treatment groups. The

101

normal group was fed a normal diet, while the others groups were fed the diet

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supplemented with adenine at the dose of 0.2% (w/w).21 The positive group was



103

administered with Cinacalcet tablet at the dosage of 150 mg/kg/day. Three LJP61A

104

treatment groups were administered with LJP61A by 50, 100 and 200 mg/kg/day,

105

respectively. The normal and model groups were administered with the same volume

106

of physiological saline. After thirty days feeding, all mice were euthanized with CO2.

107

Blood and aortas were collected for the following analysis. All the animal

108

experiments were carried out in accordance with the national guidelines for the care

109

and use of laboratory animals and were approved by the Animal Ethics Committee of

110

Hefei University of Technology.

111

Biochemical analysis

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The aortas were washed with distilled water. After drying and weighing, a small piece

113

of aorta was extracted by 0.6 M HCl at 4 °C overnight. The levels of calcium and

114

phosphorus in aortas were further quantified using calcium assay kit (Nanjing

115

Jiancheng Bioengineering Institute, Nanjing, China) and ammonium molybdate

116

spectrophotometric method,22 respectively. The levels of FGF23 and PTH in plasma

117

and the activity of ALP in aortas were quantified using ELISA kits according to the

118

manufacturer’s instructions.

119

Morphometry and immunohistochemical analysis

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Aortic tissues were fixed in 4% formalin at 4 °C overnight and embedded in paraffin

121

wax. Paraffin sections were cut and stained for calcification using the von Kossa

122

method.23 For immunohistochemistry, the sections were submerged in boiling citrate

123

buffer for ten minutes to retrieve the antigenicity. Endogenous peroxidase was


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quenched with 0.3% H2O2 for 30 minutes. After washing with PBS, the sections were

125

incubated with primary antibodies against CaSR overnight at 4 ℃. After washing, the

126

sections were subsequently incubated with biotinylated secondary antibody for 2 h at

127

room temperature, followed by further incubation with streptavidin-horseradish

128

peroxidase complex. Finally, the sections were stained using diaminobenzidine and

129

imaged with an optical microscope.

130

Cell culture and treatment

131

The MOVAS cells were purchased from the cell bank of China Center for Type

132

Culture Collection and cultured in DMEM containing high glucose, 10% fetal bovine

133

serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 ℃ in a humidified

134

incubator with 5% CO2 .The effect of LJP61A on the cell viability was determined by

135

MTT assay as described.15 To evaluate the inhibitory effect of LJP61A on VC in vitro,

136

MOVAS cells were treated with or without LJP61A (5 or 25 mg/L) in DMEM in the

137

absence or presence of β-GP (10 mmol/L) for 10 days. After washing, the cells were

138

fixed in 4% formalin at room temperature, stained with 1% alizarin red S and imaged

139

with an optical microscope. For calcium quantification, calcium was extracted with

140

0.6 M HCl at 37 ℃ overnight and analyzed with a calcium assay kit. The calcium

141

content was normalized to protein content as previously described.24 In addition, the

142

activity of ALP was quantified using ELISA kit according to the manufacturer’s

143

instruction.

144

Quantitative real-time PCR



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Total RNA was isolated from aortas and MOVAS cells using TRIzol Reagent

146

according to the manufacturer’s instruction. Quantitative real-time PCR (qRT-PCR)

147

was performed as described previously.25 All primer sequences were listed as follows:

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smooth muscle 22α (SM22α), 5′-CCAGGAGCATAAGAGGGACTT-3′ (upper

149

primer) and 5′-CATTTGTGAGGCCTAAG-3′ (lower primer); α-smooth muscle

150

actin (α-SMA), 5′-AGGGAGTAATGGTTGGAATGG-3′ (upper primer) and

151

5′-GGTGATGATGGCGTGTTCTAT-3′

152

5′-GCCAAAGTGAGCGAAACCATCC-3′

(upper

primer)

and

153

5′-CCACACAGAAGAACCAAACATAGC-3′

(lower

primer);

OPN,

154

5′-ATGCTATCGACAGTCCAGGCG-3′

155

5′-GCTCAGGGCCCAAAACACTA-3′

(lower

156

5′-AAGCCCAGGAAAGAGTCCG-3′

(upper

157

5′-TCTTATTTGGCTCCTCGGG-3′

158

5′-GAAGGGTGGAGGCAAAAG-3′

159

5′-ACCAGTGGTTGCAGGGAT-3′ (lower primer).

160

Western blot analysis

161

Total proteins were extracted from aortas and MOVAS cells using radio

162

immunoprecipitation assay lysis buffer. Then, the proteins were quantified by BCA kit

163

and denatured at 100 °C for 10 min and stored at -80 °C. Western blot was employed

164

to analyze the levels of Cbfa1, OC, RANKL, BMP-2, OPG, MGP and β-actin as

165

described previously.19

(lower

(upper

(lower (upper


primer);

primer) primer); primer) primer); primer)

Pit-1,

and MGP, and GAPDH, and

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166

Statistical analysis

167

All experiments were carried out independently in triplicates and the data were

168

expressed as mean ± SD values. All data were analyzed statistically using one-way

169

analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons tests.

170

Significant differences were set at p < 0.05.

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RESULTS

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LJP61A ameliorates VC of adenine-induced CRF mice

173

VC of adenine-induced CRF mice is a well-established in vivo model.21 Aortas

174

calcification was examined by von Kossa staining to identify the effect of LJP61A on

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VC. As shown in Figure 2A, no sign of calcification could be found in the aortas of

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normal group, while the aortas of model group showed several sign of calcification.

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Additionally, it could be found that the tissue structures of aortas in normal group

178

were tight and glossy, but those of model group were incompact and dimmish.

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However, these alterations of aortas in CRF mice could be attenuated by the treatment

180

of LJP61A in a dose-dependent manner. When LJP61A reached the dose of 200

181

mg/kg/day, the aortas appeared a small change compared to that of normal group. The

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expression of CaSR in aortas was also detected by immunohistochemical stainning.

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As shown in Figure 2B, abundant CaSR expression could be found in the aortas of

184

normal group, while few was found in the aortas of model group. However, the

185

down-regulated CaSR expression of CRF mice could be significantly reversed by

186

LJP61A in a dose-dependent manner. The results indicated LJP61A could remarkably



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improve the VC of CRF mice induced by adenine.

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To confirm the deduction above, the aortas and serum biochemical indexes of

189

CRF mice were investigated. As shown in Figure 2C-F, comparing with the normal

190

group, adenine induced significant increase in the levels of aortic calcium, aortic

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phosphate and serum PTH. It is different that the level of serum FGF-23 was

192

decreased by adenine. However, the alteration of these biochemical indexes could be

193

significantly reversed by LJP61A. When LJP61A reached the dose of 200 mg/kg/day,

194

comparing to those of model group, the levels of calcium, phosphate and PTH were

195

decreased by 64.5% (Figure 2C), 22.6% (Figure 2D) and 63.2% (Figure 2F),

196

respectively, and the level of FGF-23 was increased by 20.2% (Figure 2E). These

197

results further supported the conclusion that the VC of adenine-induced CRF mice

198

could be ameliorated by LJP61A.

199

LJP61A prevents osteoblastic differentiation of VSMC in adenine-induced CRF

200

mice

201

Previous studies showed that osteoblastic differentiation of VSMC is an important

202

event in the pathogenesis of VC.2-4 As shown in Figure 3A and 3B, comparing with

203

those of normal group, the mRNA levels of α-SMA and SM22α in aortas of model

204

group were significantly decreased by the administration of adenine, and the mRNA

205

levels of Pit-1 and the activity of ALP were remarkably increased. In addition,

206

compared to the normal group, the protein levels of Cbfa-1, OC, BMP-2 and RANKL

207

were also significantly increased in aortas of model group, and OPG and MGP were

208

remarkably increased (Figure 3C). However, these alterations seemed to be blocked


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by LJP61A in a dose-dependent manner, which indicated the inhibitory action of

210

LJP61A on VC is associated with the event of preventing osteoblastic differentiation

211

of VSMC.

212

LJP61A inhibits β-GP induced MOVAS cells calcification via preventing

213

osteoblastic differentiation

214

The β-GP induced MOVAS cells calcification model was used to evaluate the

215

inhibitory effect of LJP61A on VC in vitro. As expected, the data showed that

216

LJP61A could significantly decrease calcium deposition and ALP activity of MOVAS

217

cells induced by β-GP, and was non-cytotoxic to MOVAS cells (Figure 4A-D).

218

Quantitative RT-PCR results showed that LJP61A could significantly up-regulate the

219

mRNA expression of α-SMA, SM22α, OPN and MPG, and down-regulate that of

220

Pit-1 in β-GP-induced MOVAS cells (Figure 4E). The western blot analysis exhibited

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that LJP61A significantly inhibited the protein expression of Cbfa-1, OC, BMP-2 and

222

RANKL, and enhanced those of OPG and MGP in β-GP induced MOVAS cells

223

(Figure 4F). These results were highly in consistent with the finding in vivo,

224

suggesting LJP61A prevents VC development possibly via inhibiting VSMC

225

osteoblast differentiation.

226

DISCUSSION

227

VC is a key risk marker of cardiovascular diseases, which are the most common cause

228

of death in CRF patients.3, 14, 26 However, the current therapeutic agents for VC in

229

CRF patients are not efficient. In the present work, we found that LJP61A

230

significantly inhibited VC both in vivo and in vitro as determined by calcium and



231

phosphate amounts, von Kossa and alizarin red staining (Figures 2A, 2C, 2D, 4A and

232

4B). In addition, serum biochemistry analysis showed the serum FGF-23 level of CRF

233

mice was significantly enhanced by LJP61A, and the serum PTH level was

234

remarkably decreased (Figure 2E and 2F). FGF-23 was reported to promote phosphate

235

excretion by reducing renal phosphate re-absorption,27 and decrease parathyroid gland

236

secreting PTH, which could induce VC.28 Moreover, the immunohistochemical

237

staining results demonstrated LJP61A could remarkably up-regulate the CaSR

238

expression of aortas in CRF mice to inhibit calcification (Figure 2B). It has been

239

demonstrated that the decrease of CaSR expression in the vasculature is directly

240

involved in the development of VC.29 These results suggested that LJP61A might be a

241

new compound to prevent the development of VC.

242

Evidences showed that the osteoblastic differentiation of VSMC is an important

243

mechanism for the development of VC.2-4 This event always accompany with the

244

down-regulation of VSMC related markers, such as α-SMA and SM22α.30 Phosphate

245

plays a key role in the osteoblastic differentiation of VSMC.2, 3 As we know, one

246

major clinical symptom of CRF patients is mineral imbalances, which will enhance

247

some serum mineral levels, particularly phosphate. Jono et al. reported that the high

248

extracellular phosphate would cause an increase of intracellular phosphate

249

concentration to induce the osteoblastic differentiation of VSMC, which was proved

250

to be mediated by the sodium dependent phosphate cotransporter Pit-1.9 Thus, Pit-1

251

was considered as one essential role in VSMC osteogenic differentiation and

252

calcification.23 In the process of osteoblastic differentiation of VSMC, Cbfa-1 was


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253

considered as a master and indispensable transcriptional regulator. The expression of

254

Cbfa-1 could also be enhanced by high extracellular phosphate.9

255

under the control of Cbfa-1, the osteoblast-like cells will express a vital marker of

256

ALP and bone-associated proteins (e.g. OC and BMP-2).9, 10

257

that both OC and BMP-2 could induces VSMC osteoblast differentiation and

258

calcification.11, 12 Besides phosphate, the endogenous calcification inhibitors, such as

259

OPN, OPG and MGP, produced by VSMC also play an important role in the process

260

of osteoblastic differentiation. OPN is a phosphor protein which could inhibit

261

mineralization via blocking the formation of hydroxyapatite and activating the

262

function of osteoclast.5 OPG, a member of the tumor necrosis factor receptor family,

263

plays an important role in bone resorption by inhibiting osteoclast differentiation and

264

acting like a decoy receptor for RANKL.6 Thus, OPG is usually considered as a

265

protective factor against VC, whereas RANKL plays a negative role.7

266

well-known inhibitor of calcification and prevents osteoblast differentiation by

267

inhibiting hydroxyapatite formation.8 In the present study, we found that LJP61A

268

could significantly up-regulate the mRNA levels of α-SMA and SM22α, and

269

down-regulate the mRNA levels of Pit-1 and the activity of ALP in aortas of CRF

270

mice (Figure 3A and 3B). Moreover, in β-GP induced MOVAS cells, the mRNA

271

levels of α-SMA, SM22α, OPN and MPG were also enhanced by the treatment of

272

LJP61A, and the mRNA levels of Pit-1 and the activity of ALP were also decreased

273

(Figure 4C and 4E). In addition, western blot results revealed that LJP61A could

274

significantly decrease the protein levels of Cbfa-1, OC, BMP-2 and RANKL, and


Subsequently,

Evidences exhibited

MGP is a


Page 14 of 27

275

increase the protein levels of OPG and MGP both in adenine-induced CRF mice and

276

in β-GP induced MOVAS cells (Figures 3C and 4F). These results indicated that

277

LJP61A could regulate the expression of genes and proteins involved in the process of

278

VSMC osteoblast differentiation to inhibit the progression of VC both in vivo and in

279

vitro (Figure 5). Although some kinds of natural products, such as phenolic acid15 and

280

steroidal saponin16, have been proved to show this function, the preventive effect and

281

underlying mechanism of polysaccharide on the development of VC was reported for

282

the first time in this work.

283

In summary, the present work demonstrated that the purified L. japonica

284

polysaccharide LJP61A has an ability to inhibit VC via preventing VSMC osteoblast

285

differentiation, indicating LJP61A might be a new therapeutic or preventive agent to

286

delay the development of VC in CRF patients.

287

ACKNOWLEDGMENTS

288

This study was financially supported by the National Natural Science Foundation of

289

China (Grant No. 31271814, 21702040), the Fundamental Research Funds for the

290

Central Universities (Grant No. JZ2017HGPB0169) and the Science and Technology

291

Major Project of Anhui Province (Grant No. 17030701030).

292

NOTES

293

The authors declare no competing financial interest.

294

ABBREVIATIONS USED

295 296

ALP,

alkaline

phosphatase;

α-SMA,

α-smooth

muscle

actin;

β-GP

β-glycerophosphate; BMP-2, bone morphogenetic protein 2; CRF, chronic renal


Page 15 of 27


297

failure; CaSR, calcium-sensing receptor; Cbfa1, core-binding factor alpha 1; FGF23,

298

fibroblast growth factor 23; MGP, matrix gla protein; OC, osteocalcin; OPG,

299

osteoprotegerin; OPN, osteopontin; PTH, parathyroid hormone; RANKL, receptor

300

activator for nuclear factor-κB ligand; SM22α, smooth muscle 22α; VC, vascular

301

calcification; VSMC, vascular smooth muscle cells.

302



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Figure captions

304

Figure 1. The chemical structure of LJP61A.

305

Figure 2. The effect of LJP61A on VC of adenine-induced CRF mice. (A) Aortic von

306

Kossa staining; (B) aortic CaSR immunohistochemical staining; (C) aortic calcium

307

level; (D) aortic phosphate level; (E) serum FGF23 level; (F) serum PTH level.

308

0.01 (vs. normal group); *p< 0.05, **p< 0.01 (vs. model group).

309

Figure 3. LJP61A prevents osteoblastic differentiation of VSMC in adenine-induced

310

CRF mice. (A) Aortic mRNA levels of α-SMA, SM22α and Pit-1; (B) aortic ALP

311

activity; (C) aortic protein levels of Cbfa-1, OC, BMP-2, RANKL, OPG and MGP.

312

##

313

Figure 4. LJP61A inhibits β-GP induced MOVAS cells calcification via preventing

314

osteoblastic differentiation. (A) Cells alizarin red S staining; (B) cells calcium content;

315

(C) cells ALP activity; (D) cells viability; (E) cells mRNA levels of α-SMA, SM22α,

316

Pit-1, OPN and MGP; (F) cells protein levels of Cbfa-1, OC, BMP-2, RANKL, OPG

317

and MGP. ##p< 0.01 (vs. normal group); **p< 0.01 (vs. model group).

318

Figure 5. Possible mechanism of LJP61A inhibits the progression of VC.

##

p

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