Preventive Effects of a Novel Polysaccharide from Sepia esculenta Ink

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Preventive effects of a novelty polysaccharide from Sepia esculenta ink on ovarian failure and its action mechanisms in cyclophosphamide-treated mice Huazhong Liu, Yexing Tao, Ping Luo, Chunmei Deng, Yipeng Gu, Lei Yang, and Jieping Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01854 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 25

Journal of Agricultural and Food Chemistry

1

Preventive effects of a novel polysaccharide from Sepia esculenta ink on ovarian

2

failure and its action mechanisms in cyclophosphamide-treated mice

3 †

‡

†

4

Hua-Zhong Liu, ,© Ye-Xing Tao, ,© Ping Luo, Chun-Mei Deng,

5

Yi-Peng Gu,∮ Lei Yang, ,* and Jie-Ping Zhong ,*

†

†

†

6 7 8

†

College of Sciences, Guangdong Ocean University, Zhanjiang 524088, China;

‡

Science Experiment Center, Guilin Medical University, Guilin 541004, China;

∮

Institute of Food Science & Engineering Technology, Hezhou University, Hezhou 542899, China;

10

©

The authors contributed equally to this manuscript.

11

* Corresponding authors: Lei Yang, e-mail: [email protected]

9

12

Jie-Ping Zhong, e-mail: [email protected]

13 14 15 16 17 18 19 20 21 22 23 1

ACS Paragon Plus Environment


Page 2 of 25

24

ABSTRACT：：Based on our findings about chemo-preventive roles of squid ink polysaccharide and the

25

well-known toxicity of cyclophosphamide (CP) on female gonad, this research investigated the protective

26

effects of a novel polysaccharide from Sepia esculenta ink (SEP) on the ovarian failure resulting from CP,

27

as well as the action mechanisms underpinning this. The results indicated that CP destroyed the ovaries of

28

mice caused depletion of various follicles, and led to a reduction in estradiol content, increases in FSH-

29

and LH-contents in sera, decreases in ovary and uterus masses and their relative mass ratios, disruption of

30

the ultrastructure of granulosa cells, as well as induction of apoptosis and autophagy via p38 MAPK and

31

PI3K/Akt signalling pathways. The phenomenon resulted in ovarian failure. However, SEP exposure

32

altered the negative effects completely. The data indicated that SEP can effectively prevent ovarian failure

33

CP caused in mice by inhibiting the p38 MAPK signalling pathway and activating the PI3K/Akt

34

signalling pathway as regulated by CP. SEP was a novel polysaccharide from Sepia esculenta ink with a

35

unique primary structure mainly composed of GalN and Ara that accounted for almost half of all

36

monosaccharides: their ratio was nearly one-to-one. Besides, the polysaccharide contained a small

37

number of Fuc and tiny amounts of Man, GlcN, GlcA, and GalA.

38 39

KEYWORDS:

SEP; cyclophosphamide; autophagy; apoptosis; ovarian failure.

40 41

■ INTRODUCTION

42

Chemotherapy has been widely used in the treatment of various malignancies and autoimmune diseases,

43

however, female patients undergoing chemotherapy have to face some severe adverse effects, such as

44

premature ovarian failure.1-4 Among the widely used chemotherapeutic drugs, alkylating agents are shown

45

to be the most gonadotoxic. As an important alkylating agent, cyclophosphamide (CP)-induced ovarian

46

failure in treated female patients of child-bearing age has been already verified and characterised by 2


Page 3 of 25


47

irreversible amenorrhoea and infertility.5-7 Immature primordial follicles and primary follicles in

48

CP-treated mice are more sensitive to CP that can injure granulosa cells of preantral follicles in both rats

49

and mice.5 CP not only results in DNA cross-linking of granulosa cells and a reduction in their quantity,

50

but also decreases circulatory levels of both progesterone and oestrogen, which induces ovarian fibrosis.6

51

Squid ink polysaccharide (SIP) has been considered as a potential effective, non-toxic, broad-spectrum,

52

cytoprotective agent due to its positive physiological functions such as antioxidant,8 anti-tumour,9-11 and

53

anti-chemotherapy.8,12-15 Presently sourced from two different cuttlefishes, two types of SIP were isolated

54

and identified as glycosaminoglycan with diverse primary structures.16-18 Over the last ten years research

55

has shown that SIP could weaken the toxicity of CP on liver, lung, kidney, heart, and testis.8,12,19 The

56

chemo-preventive effects of marine polysaccharides were also observed in intestine.13-15 Up to now, no

57

evidence indicates SIP’s interventional effects against ovarian failure in CP-exposed mice, which could

58

be confirmed by reasonable deduction according to the existing published reports. This research not only

59

first reported that SIP prevented CP-caused ovarian failure by inducing autophagy and apoptosis of

60

ovarian cells through activating p38 MAPK and PI3K/Akt signalling pathways, but more importantly that

61

a novel polysaccharide, with a different primary structure from others reported, extracted from Sepia

62

esculenta ink was isolated and characterised.

63 64

■ MATERIALS AND METHODS

65

Preparation of Sepia esculenta ink polysaccharide (SEP)

66

Fresh squid (Sepia esculenta) caught from Beibu Gulf were sacrificed to get ink sacs which were then

67

stored at –70 °C until use. SEP was prepared using a slightly modified method as described by Chen et

68

al.18 Briefly, the thawed frozen squid ink at 4 °C was diluted with an equal volume of PBS (0.01 mol/L,

69

pH 7.4) and then treated by sonication in an ice-bath. After storage at 4 °C for more than 8 h, the mixture 3



Page 4 of 25

70

was centrifuged (8000 rpm) at 4 °C for 50 min, and the supernatant was collected and hydrolyzed with

71

papain (1.5 ‰) at 50 °C for 90 min, and it was then heated in boiling water to denature the protease. The

72

proteins in the treated supernatant were removed by using the Sevag method.20 The aqueous phase was

73

mixed with four volumes of ethanol to precipitate the polysaccharides. Crude polysaccharides were

74

obtained from the precipitate and then separated by DEAE-52 cellulose column chromatography. The

75

major fraction was collected, dialysed, concentrated, and further purified in a Sephacryl S-300HR column.

76

The largest elution peak was obtained from the S-300HR column and dialysed, concentrated, freeze-dried,

77

and stored at -20 °C. The harvested polysaccharide was SEP.

78 79

Purity determination of SEP

80

For determining purity, SEP was prepared as a 1 mg/L solution with ultrapure water and assayed by

81

UV-vis absorption spectroscopy at a range of wavelengths between 190 and 400 nm using a

82

spectrophotometer, or made into a 3 mg/L solution with ultrapure water that was further used to identify

83

the purity of SEP with size exclusion high-performance liquid chromatography (Shimadzu LC-20AT,

84

RID-10A, Nakagyo-ku, Kyoto, Japan). As a mobile phase, ultrapure water flowed at a rate of 1 ml/min

85

past the chromatographic column (TSK-GEL G5000PWXL,7.8 × 300 mm, Shanghai, China), at a column

86

temperature of 50 °C.

87 88

Monosaccharide composition analysis

89

Nine kinds of monosaccharides, including: glucose (Glc), xylose (Xyl), L-arabinose (L-Ara), L-fucose

90

(L-Fuc), D-mannose (D-Man), glucosamine (GlcN), D-glucuronic acid (D-GlcA), D-galacturonic acid

91

(D-GalA), and D-galactosamine (D-GalN), were confected into 2 mmol/L solutions, 50 µl of each

92

solution was mixed with 450 µl of PMP derivatisation reagent (0.5 mol/L PMP methanol solution) and 4


Page 5 of 25


93

450 µl of 0.3 mol/L NaOH. The mixture was heated in a water bath for 30 min followed by being cooled

94

at room temperature for 10 min and being neutralised with 450 µl of 0.3 mol/L HCl, and then thrice

95

extracted with 1 ml chloroform. Ten microlitres of filtrate of water phase was passed through a 0.22 µm

96

aperture filter membrane for HPLC analysis.

97

SEP (2.0 mg) was hydrolysed with 1 ml TFA (2 mol/L) at 110 °C for 8 h in an ampoule that was

98

recharged with nitrogen. Unreacted TFA in reaction system cooled down to room temperature was

99

removed at 50 °C using pressured gas blowing concentrator. The reaction system was adjusted to neutral

100

with 2 mol/L, 0.3 mol/L NaOH solutions in turn, and was then diluted with water to 1 ml. The product

101

(450 µl) was derived with PMP by the method of aforementioned monosaccharide derivatisation.

102 103

Animal experimental scheme

104

Following habituation for one week, sexually mature female Kunming mice were allocated to four

105

groups, ten mice per group, a control group (CON), a CP-treated group (CP), a SEP-treated group (SEP),

106

and a co-treated group (SEP plus CP). The SEP was administered orally at 80 mg/kg body mass every day

107

for 21 consecutive days, and CP (dissolved in normal saline) was injected intraperitoneally at a dose of

108

120 mg/kg body mass two times on day 7 and day 14, respectively. The treatment procedure was

109

presented in Table 1. Animals were housed under standardised conditions in a room on a 12 h light/dark

110

cycle with food and water available ad libitum.

111 112

Detection of the relative organ mass and counts of bilateral ovarian follicles

113

After the last SEP treatment, animals were fasted, but water was available ad libitum for 24 h. Mouse

114

blood was sampled until death. Uterus and bilateral ovaries were collected and the fat around the organs

115

removed. Ice-cold normal saline cleaned organs were dried with filter paper and then weighed. The 5



Page 6 of 25

116

relative organ mass ratio was calculated as: womb or ovary mass (mg) / mouse body mass (g).

117

Furthermore, ovary was made into routine paraffin sections with a thickness of 5 µm that was subjected to

118

measurement using H.E. staining and the middle section of every ovary was selected to count bilateral

119

ovarian follicles.

120 121

Detection of sex hormones in serum

122

Blood was stored at 4 °C and allowed to coagulate to prepare serum that was used to determine

123

follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) contents with detection

124

kits according to the manufacturer’s protocol.

125 126

Detection of ultrastructure of ovary with transmission electron microscopy

127

Ovary was fixed with 3% glutaraldehyde for 4 h, and thrice washed with 0.1 mol/L dimethyl sodium

128

arsenate. At 4 °C, the fixed ovary was treated with 1% osmic acid, and thrice washed with 0.1 mol/L

129

dimethyl sodium arsenate followed by dehydration of gradient ethanol and embedding of epoxypropane.

130

An ultramicrotome was used to made sections that were stained with 2% uranyl acetate and lead citrate

131

which were then observed by using transmission electron microscopy.

132 133

Western blotting analysis

134

The supernatant of ovary homogenate added to the protein sample buffer was denatured in boiling

135

water for 5 min. After SDS-PAGE, the protein was electro-transferred to a nitrocellulose membrane and

136

then probed with monoclonal antibody that would be captured as a secondary antibody conjugated with

137

horseradish peroxidase. The membrane was visualised using a SuperSignal West Pico chemiluminescence

138

detection system. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal 6


Page 7 of 25

139


reference.

140 141

Statistical analysis

142

All experimental data were presented as mean ± standard deviation. Data were analysed by JMP 7.0.2.

143

One-way analysis of variance and the post hoc Tukey HSD test were used to evaluate differences between

144

groups, lowercasep < 0.05 and capitalp < 0.01 were considered significant.

145 146

■ RESULTS

147

Isolation of a novel polysaccharide from Sepia esculenta and its monosaccharide composition

148

The crude polysaccharides were separated into three fractions, A, B, and C, by DEAE-52 cellulose

149

column chromatography with gradient NaCl solutions at 0, 0.3, and 0.5 mol/L. Thereinto the first fraction

150

content was considerably greater than the others (Figures 1A, 1B, 1C), which meant that the

151

polysaccharides content in fraction A should be the major ingredient of the crude polysaccharides. In the

152

next purification step, Sephacryl S-300HR failed to separate the product in fraction A into various

153

fractions, only one elution peak was observed (Figure 1D), so it could be deduced that the product may be

154

a purified polysaccharide, which was deemed to be SEP.

155

With UV-vis absorption spectra at a range of wavelengths between 190 and 400 nm, by

156

spectrophotometry, it was found that the SEP absorption peak occurred at 190 nm and was a

157

characteristic absorption peak of such saccharides, and that no obvious absorption peak could be found at

158

260 and 280 nm (Figure 1E). Meanwhile, results of HPLC analysis showed a single

159

chromatographic peak at 10.917 min that was symmetrical (Figure 1F). The data suggested that SEP was

160

a purified polysaccharide.

161

After being degraded and derived, it can be seen from Figures 1G and 1H that SEP was mainly 7



Page 8 of 25

162

composed of GalN and Ara (accounting for almost half of all monosaccharides) in the ratio one-to-one

163

(Figure 1I). Besides, the polysaccharide contained a small number of Fuc and tiny amounts of Man, GlcN,

164

GlcA, and GalA.

165 166

Attenuation of SEP on the negative effects of CP-induced on body mass, ovary mass, uterus mass,

167

and the relative mass ratios of ovary and uterus in mice

168

CP not only seriously reduced body mass, ovary mass and uterus mass, also markedly decreased the

169

relative mass ratios of two female reproductive organs in mice. However the data presented in Table 2

170

showed that in co-stimulated mice exposed to SEP and CP, although body mass failed to increase

171

significantly, ovary mass, uterus mass, and their two ratios all returned to normal, which suggested that

172

SEP successfully suppressed CP toxicity on ovary, uterus, and body of mice.

173 174

Inhibition of SEP on depletion of follicles in CP-exposed mice

175

As shown in Table 3, counts of every stage follicle and total follicle in ovary from the mice exposed to

176

CP were significantly diminished compared to vehicle-treated mice; but, under the protection of SEP, the

177

follicles were hardly affected by CP-induced toxicity, no obvious quantitative differences were found

178

between vehicle- and co-treated mice.

179 180

SEP impaired the negative effects of CP on serum sex hormone contents

181

Data were presented in Figure 2: in sera of CP treated mice, the E2 content was decreased significantly,

182

and the FSH and LH contents were increased remarkably. However the changes were almost reversed to

183

normal levels in the mice that were co-treated with SEP and CP, which suggested that the CP-induced

184

destruction of ovarian oestrogen production was prevented by SEP. 8


Page 9 of 25


185 186

Prevention of SEP on CP-induced ultrastructure disruption of ovarian granulosa cells

187

In Figure 3, using transmission electron microscopy, normal cellular morphology and ultrastructure of

188

granulosa cells in ovaries from control group mice were observed. Chromatin was evenly distributed and

189

cellular membranes were intact. Endoplasmic reticulum and mitochondria were rich and had normal

190

structure. Results in granulose cells of CP-exposed mice showed chromatin condensed into pieces,

191

nuclear membranes marginalised, and nuclei undergoing pycnosis and deformation. Organelles were

192

fewer and of abnormal shape. Mitochondria were swelling and underwent vacuolation, but in SEP

193

administered mice exposed to CP, the quantity and structure of organelles were reversed to some extent.

194

Cellular morphology and ultrastructure of granulosa cells almost returned to normal. These data implied

195

that SEP prevented apoptosis of CP-treated granulosa cells.

196 197

Impairment of SEP on CP-induced apoptosis and autophagy in ovary of mice

198

Ultrastructure characteristics of granulosa cells in Figure 3 and TUNEL assay data on granulosa cells in

199

Figure 4 indicated that apoptosis occurrence in ovary was mediated by CP and SEP impaired the

200

programmed cell death, which was shown to be true by detection data relating to expression or

201

phosphorylation levels of apoptosis- and autophagy-associated genes, as well as p38 and Akt proteins in

202

ovary (Figure 6). An increase of Bax content and decrease of Bcl-2 content in CP-treated ovary, compared

203

with vehicle-treated ovary, indicated induction of apoptosis in ovary cells by the antitumor drug that also

204

resulted in autophagy of the female gonad cells for promoting expression of two autophagy associated

205

genes, LC-3 and beclin-1, in CP administered ovary and its granulosa cells (Figure 5). The two cell

206

physiological processes were all suppressed simultaneously by SEP exposure. These data suggested that

207

CP-induced apoptosis and autophagy in ovary can be inhibited effectively by SEP, and that CP-mediated 9



Page 10 of 25

208

autophagy may be autophagic cell death, the secondary programmed cell death form. Since, under SEP

209

exposure, apoptosis and autophagy occurred simultaneously in ovary, then apoptosis inhibition may be

210

attributed to the suppression of autophagy. It was also found that the phosphorylation level of p38 or Akt

211

protein was elevated, or reduced, by using CP, respectively. SEP exposure converted the effects of CP on

212

the two important proteins of p38 MAPK and PI3K/Akt signalling pathways in ovary.

213 214

■ DISCUSSION

215

Squid ink polysaccharide (SIP), a mucopolysaccharide isolated from squid ink, has been proved to be a

216

multifunctional marine substance that is rich in biological activity, including chemoprophylaxis.8,12-15,19

217

Takaya et al.

218

further confirmed by Chen et al..17 Afterwards, another SIP with a new primary structure was reported by

219

Liu et al.18 In the last decade, research revealed that SIP could fully protect model animals from

220

damaging chemotherapeutic agent-induced effects in various tissues/organs,19 especially in testis of

221

mouse.8,12 Moreover a novel SIP originated from Sepia esculenta ink (named SEP in this paper) was

222

isolated and characterised. It was found that SEP was a novel SIP with a unique primary structure that

223

was different from the two reported SIP.16-18 SEP contained little else apart from two monosaccharides,

224

GalN and Ara, at almost 50% each. Also a small number of Fuc and tiny amounts of Man, GlcN, GlcA,

225

and GalA were observed in SEP.

16

firstly reported that SIP was a mucopolysaccharide with a unique structure that was

226

CP-associated ovarian damage has been recognised for several decades, and the damage characteristics

227

are clear and certain, such as reductions of various follicles, ovary mass and its relative mass ratio, as well

228

as a reduced E2 content in serum, with increases of FSH and LH contents in serum,21-25 and so on. These

229

adverse results were also observed in this study. Apart from these outcomes, this research also found that

230

womb mass and its relative mass ratio were all spared by using CP, suggesting chemotherapeutic damage 10


Page 11 of 25


231

to the uterus. Disruption of uterus must lead to negative effects for the ovary, for which uterine supply of

232

blood to the ovary should be part-responsible. However these pernicious effects of CP were all reversed

233

by SEP. Now it is widely believed that CP toxic roles are correlated with two important mechanisms –

234

oxidative stress and DNA damage – which are critical reasons for CP-mediated destruction of ovary. In

235

our previous work, we have proven that squid ink polysaccharide prevented DNA damage caused by

236

hydrogen peroxide and ultraviolet radiation in vitro,26 and attenuated CP-induced oxidative stress damage

237

in testis of mice via activating the Nrf2/ARE signalling pathway.8,12 Therefore we can reasonably deduce

238

that SEP potentially triggered an Nrf2/ARE signalling cascade to inhibit oxidative stress and CP-induced

239

DNA strand breakage in ovary, thus resulting in functional restoration of the gonad.

240

Mice are more sensitive to the effects of CP on the immature primordial and primary follicles, and

241

granulosa cells of more mature antral follicles.5 CP mediates granulosa cells to apoptosis through

242

mitochondrial pathway via inducing oxidative stress.27-29 In primordial and small primary follicles, CP

243

targets the oocytes for apoptotic destruction, whereas in larger follicles, CP induces granulosa cell

244

apoptosis followed by death of the oocyte.30,31 The above findings suggest that granulosa cell apoptosis

245

plays an important role in the process of CP-mediated ovary damage. To investigate the preventive

246

mechanism of SEP on CP toxicity in ovary, this study determined the ultrastructure and apoptosis of

247

ovarian granulosa cells. Results revealed that CP-induced apoptosis of ovarian granulosa cells, giving rise

248

to ovarian failure, was suppressed by SEP, which was ensured by the further detection on expression

249

levels of apoptosis-related genes, bcl-2 and bax. Treatment with CP resulted in significant Bcl-2 content

250

reduction and Bax content elevation in ovary when compared with vehicle treatment, but exposure to SEP

251

blocked these trends and reverted the expression levels of the two genes.

252

In addition, this study provided further evidence about SEP and CP actions on ovary based on

253

autophagy. In CP-treated mice, ovarian autophagy was promoted, which was supported by expressional 11



Page 12 of 25

254

upregulation of LC-3 and beclin-1, two autophagy-related pivotal genes that were inhibited in

255

SEP-administered mice exposed to CP. It is well known that autophagy is an important cell physiological

256

phenomenon that can mediate cells into two different processes, promoting cell survival via inhibiting

257

apoptosis and accelerating programmed cell death via inducing autophagic cell death.32,33 Although it

258

cannot be concluded whether CP-induced autophagy in ovarian cells enhanced cell survival or cell death:

259

the anticancer agent probably resulted in autophagic death on the basis of the comprehensive effects of

260

SEP and CP on ovary apoptosis and autophagy since SEP not only inhibited apoptosis of ovarian cells,

261

also impaired autophagy induced by using CP.

262

Our data revealed that LC-3 and Beclin-1 proteins were upregulated significantly and Akt was

263

dephosphorylated significantly, which implied that regulation of SEP and/or CP on ovary was correlated

264

with the PI3K/Akt/mTOR signalling pathway. PI3K/Akt/mTOR is a critical signalling pathway during

265

autophagy. Thereinto activation of PI3K-I should inhibit autophagy, an activated PI3K-III pathway can

266

promote autophagy.34 Our results showed that, in CP-treated mice, phosphorylation of Akt protein was

267

inhibited, but expression of LC-3 and becline-1 genes was promoted, which implied that CP could

268

probably not induce autophagy by activating the PI3K/Akt/mTOR signalling pathway, although it can be

269

supposed that CP possibly promoted autophagy via inhibiting the PI3K-I/Akt/mTOR signalling pathway

270

which was effectively relieved, in CP-stimulated mice, by SEP.

271

Except for the PI3K/Akt/mTOR signalling pathway, we also found that p38 MAPK was involved in the

272

induction of SEP and/or CP on ovary. Promotion of CP on activation of p38 observed in CP-mediated

273

ovary was impaired by SEP. Mediation of p38 MAPK on apoptosis and autophagy has been confirmed.35

274

Chemotherapeutic agents and ROS may activate p38 to induce apoptosis, but the activated p38 MAPK

275

signalling cascade can also induce tumour cells to autophagic cell death.35

276

In summary, we can suppose logically that SEP prevented chemotherapeutic damage to ovary through 12


Page 13 of 25


277

inhibiting CP induced autophagic cell death which contributed to impairment of programmed cell death,

278

and inhibiting apoptosis.

279 280

■ AUTHOR INFORMATION

281

Corresponding Author

282

*(L. Yang) Mailing address: East of the Huguang Lake, Zhanjiang/Guangdong, China 524088. E-mail:

283

[email protected]. Phone: +86 759 2383300.

284

Funding

285

This work was supported by the National Natural Science Foundation of China (Grant no. 31171667) and

286

the Natural Science Foundation of Guangdong Province, China (Grant no. 2016A030313753).

287

Notes

288

No potential conflicts of interest were disclosed.

289 290

■ REFERENCES

291

(1) Howell, S.; Shalet, S. Gonadal damage from chemotherapy and radiotherapy. Endocr. Metab. Clin. N.

292

Am. 1998, 27, 927-943.

293

(2) Manger, K.; Wildt, L.; Kalden, J.R.; Manger, B. Prevention of gonadal toxicity and preservation of

294

gonadal function and fertility in young women with systemic lupus erythematosus treated by

295

cyclophosphamide: the prego-study. Autoimmin. Rev. 2006, 5, 269-272.

296 297

(3) Mastro, L.D.; Catzeddu, T.; Venturini, M. Infertility and pregnancy after breast cancer: current knowledge and future perspectives. Cancer Treat. Rev. 2006, 32, 417-422.

298

(4) Stearns, V.; Schneider, B.; Henry, N.L.; Hayes, D.F.; Flockhart, D.A. Breast cancer treatment and

299

ovarian failure: risk factors and emerging genetic determinants. Nat. Rev. Cancer. 2006, 6, 886-893. 13



300 301 302 303 304 305 306 307

Page 14 of 25

(5) Plowchalk, D.R.; Mattison, D.R. Reproductive toxicity of cyclophosphamide in the C57BL/6N mouse: 1. Effects on ovarian structure and function. Reprod. Toxicol. 1992, 6, 411–421. (6) Slater, C.A.; Liang, M.H.; McCune, W.J.; Christman, G.M.; Laufer, M.R. Preserving ovarian function in patients receiving cyclophosphamide. Lupus 1999, 8, 3–10. (7) Emadi, A.; Jones, R.J.; Brodsky, R.A. Cyclophosphamide and cancer: golden anniversary. Nat. Rev. Clin. Onco. 2009, 6, 638-647. (8) Le, X.Y.; Luo, P.; Gu, Y.P.; Tao, Y.X.; Liu, H.Z. Interventional effects of squid ink polysaccharides on cyclophosphamide-associated testicular damage in mice. Bratisl. Lek. Listy. 2015, 116, 334-339.

308

(9) Zong, A.; Liu, Y.; Zhang, Y.; Song, X.; Shi, Y.; Cao, H.; Liu, C.; Cheng, Y.; Jiang, W.; Du, F.; Wang, F.

309

Anti-tumor activity and the mechanism of SIP-S: a sulfated polysaccharide with anti-metastatic effect.

310

Carbohyd. Polym. 2015, 129, 50-54.

311

(10) Zong, A.; Zhao, T.; Zhang, Y.; Song, X.; Shi, Y.; Cao, H.; Liu, C.; Cheng, Y.; Qu, X.; Cao, J.; Wang,

312

F. Anti-metastatic and anti-angiogenic activities of sulfated polysaccharide of Sepiella maindroni ink.

313

Carbohyd. Polym. 2013, 91, 403-409.

314

(11) Chen, S.G.; Wang, J.F.; Xue, C.H.; Li, H.; Sun, B.B.; Xue, Y.; Chai, W.G. Sulfation of a squid ink

315

polysaccharide and its inhibitory effect on tumor cell metastasis. Carbohyd, Polym. 2010, 81,

316

560-566.

317

(12) Le, X.Y.; Luo, P.; Gu, Y.P.; Tao, Y.X.; Liu, H.Z. Squid ink polysaccharides reduces

318

cyclophosphamide-induced testicular damage via Nrf2/ARE activation pathway in mice. Iran. J.

319

Basic Med. Sci. 2015, 18, 827-831.

320

(13) Zuo, T.; He, X.; Cao, L.; Xue, C.; Tang, Q.J. The dietary polysaccharide from Ommastrephes

321

bartrami prevents chemotherapeutic mucositis by promoting the gene expression of antimicrobial

322

peptides in Paneth cells. J. Funct. Foods 2015, 12, 530-539. 14


Page 15 of 25

323 324


(14) Zuo, T.; Cao, L.; Xue, C.; Tang, Q.J. Dietary squid ink polysaccharide induces goblet cells to protect small intestine from chemotherapy induced injury. Food Funct. 2015, 6, 981-986.

325

(15) Zuo, T.; Cao, L.; Li, X.; Zhang, Q.; Xue, C.; Tang, Q.J. The squid ink polysaccharides protect tight

326

junctions and adherens junctions from chemotherapeutic injury in the small intestinal epithelium of

327

mice. Nutr. Cancer 2015, 67, 364–371.

328 329 330

(16) Takaya, Y.; Uchisawa, H.; Narumi, F.; Matsue, H. Illexins A, B, and C from squid ink should have a branched structure. Biochem. Bioph. Res. Co. 1996, 226, 335-338. (17) Chen, S.; Xu, J.; Xue, C.; Dong, P.; Sheng, W.; Yu, G.; Chai, W. Sequence determination of a

331

non-sulfated glycosaminoglycan-like polysaccharide from

melanin-free ink of the

squid

332

Ommastrephes bartrami by negative-ion electrospray tandemmass spectrometry and NMR

333

spectroscopy. Glycoconj. J. 2008, 25, 481-492.

334

(18) Liu, C.H.; Li, X.D.; Li, Y.H.; Feng, Y.; Zhou, S.; Wang, F.S. Structural characterization and

335

antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink. Food Chem.

336

2008, 110, 807-813.

337

(19) Liu, H.Z.; Wang, G.; Wu, J.L.; Shi, L.S.; Zhong, J.P.; Pan, J.Q. Amelioratory effects of squid ink

338

polysaccharide on partial internal organs injured by cyclophosphamide. Chin. J. Mod. App. Pharm.

339

2012, 2, 89-93.

340

(20) Staub, A.M. Removal of protein-Sevag method. Method carbohyd. Chem. 1965, 5, 5-6.

341

(21) Ataya, K.; Rao, L.V.; Lawrence, E.; Kimmel, R. Luteinizing Hormone-releasing hormone agonist

342

inhibits cyclophosphamide-induced ovarian follicular depletion in rhesus monkeys. Bio. Reprod. 1995,

343

52, 365-372.

344 345

(22) Ataya, K.M.; Ramahi-Ataya, A. Reproductive performance of female rats treated with cyclophosphamide and/or LHRH agonist. Reprod. Toxicol. 1993, 7, 229-235. 15



Page 16 of 25

346

(23) Sanders, J.E.; Buckner, C.D.; Leonard, J.M.; Sullivan, K.M.; Witherspoon, R.P.; Deeg, H.J.; Storb,

347

R.; Thomas, E.D. Late effects on gonadal function of cyclophosphamide, total-body irradiation, and

348

marriw transplantation. Transplantation 1983, 36, 252-255.

349

(24) Bokser, L.; Szende, B.; Schally, A. Protective effects of D-Trp 6-luteinizing hormone-releasing

350

hormone microcapsules against cyclophosphamide-induced gonadotoxicity in female rats. Br. J.

351

Cancer Res. 1990, 61, 861-865.

352 353 354 355

(25) Montz, F.J.; Wolff, A.J.; Gambone, J.C. Gonadal protection and fecundity rates in cyclophosphamide-treated rats. Cancer Res. 1991, 51, 2124-2126. (26) Luo, P.; Liu, H.Z. Antioxidant ability of squid ink polysaccharides as well as their protective effects on DNA damage in vitro. Afr. J. Pharm. Pharmacol. 2013, 7, 1382-1388.

356

(27) Tsai-Turton, M.; Luderer, U. Gonadotropin regulation of glutamate cysteine ligase catalytic and

357

modifier subunit expression in the rat ovary is subunit and follicle stage-specific. Am. J.

358

Physiol-Endoc. M. 2005, 289, E 391-402.

359

(28) Tsai-Turton, M.; Luderer, U. Opposing effects of glutathione depletion and FSH on reactive oxygen

360

species and apoptosis in cultured preovulatory rat follicles. Endocrinology 2006, 147, 1224-1236.

361

(29) Tsai-Turton, M.; Luong, B.T.; Tan, Y.; Luderer, U. Cyclophosphamide-induced apoptosis in COV434

362

human Granulosa cells involves oxidative stress and glutathione depletion. Toxicol. Sci. 2007, 98,

363

216-230.

364 365 366 367 368

(30) Desmeules, P.; Devine, P.J. Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries. Toxicol. Sci. 2006, 90, 500-509. (31) Lopez, S.G.; Luderer, U. Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis. Free Radic. Biol. Med. 2004, 36, 1366-1377. (32) Jia, G.; Sowers, J.R. Autophagy: A housekeeper in cardiorenal metabolic health and disease. Biochim. 16


Page 17 of 25

369


Biophys. Acta. 2015, 1852, 219-224.

370

(33) Eskelinen, E.L. The dual role of autophagy in cancer. Curr. Opin. Pharmacol. 2011, 11, 294-300.

371

(34) Kondo, Y.; Kondo, S. Autophagy and cancer therapy. Autophagy 2006, 2, 85-90.

372

(35) Nagelkerke, A.; Sweep, F.C.G.J.; Geurte-Moespot, A.; Bussink, J.; Span, P.N. Therapeutic targeting

373

of autophagy in cancer. Part I: Molecular pathways controlling autophagy. Semin. Cancer Biol. 2015,

374

31, 89-98.

375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 17



Page 18 of 25

392 393

Table 1. Animal experimental treatment procedure.

Groups

SEP

Normal saline

Normal saline

Experimental

(vehicle of SEP)

(vehicle of CP)

period (Day)

CP

CON

-

-

+

+

21

CP

+

-

-

+

21

SEP

-

+

+

-

21

CP+SEP

+

+

-

-

21

394 395 396 397 398

Table 2. Effects of SEP on the relative mass ratios of ovary and uterus in mice exposed to CP. Body mass

Ovary mass

Ovary relative

Uterus mass

Uterus relative

(g)

(mg)

mass (mg/g)

(mg)

mass (mg/g)

CON

24.68±1.29Aa

13.67±0.82Aa

0.56±0.05Aa

58.33±3.51 Aa

2.36±0.14 Aa

CP

23.75±0.79Ab

10.25±0.96Bb

0.43±0.04Bb

44.60±6.66 Bb

1.74±0.04 Bc

SEP

25.50±1.76Aa

12.80±1.30Aa

0.55±0.06 Aa

58.50±3.54 Aab

2.17±0.02 Ab

CP+SEP

24.55±2.15Aab

13.25±0.95Aa

0.53±0.04 Aab

57.67±4.73 Aa

2.19±0.04Ab

Groups

399

Note: Different letters indicate significant differences, abp < 0.05, ABp < 0.01.

400 401 402 18


Page 19 of 25

403


Table 3. Inhibition of SEP on depletion of follicles in CP-exposed mice Primordial

Primary

Secondary

Mature

Total follicl

follicle

follicle

follicle

follicle

e

CON

13.92±1.72Aa

12.23±1.70Aa

7.02±1.61Aa

3.10±0.75 Aa

36.19±2.44 Aa

CP

9.80±2.19Bb

7.55±1.46Bb

3.50±0.96Bb

1.08±0.44 Bb

21.92±2.77 Bb

SEP

12.57±2.52Aa

12.44±1.31Aa

7.90±2.22 Aa

2.92±0.53 Aa

35.88±2.23 Aa

CP+SEP

13.01±1.53Aa

11.53±1.78Aa

7.82±1.09 Aa

3.11±0.91 Aa

35.44±2.48Aa

Groups

404

Note: Different letters indicate significant differences, abp < 0.05, ABp < 0.01.

405

19



Page 20 of 25

5

4.60

4.48

4.5 4

Mole ratios

3.5 3 2.5 2 1.5 1.0 1 0.48 0.5 0.01

0.15

0.22

0.17

0 Man GlcN GlcA GalA GalN

Xyl

Ara

Fuc

Figure 1. Preparation and monosaccharide composition of SEP. A, B, and C represented elution peaks that were eluted with distilled water, and 0.3 mol/L and 0.5 mol/L NaCl solutions, respectively by column chromatography using cellulose DEAE-52. D represented the eluent of peak A which product was eluted with distilled water by column chromatography using Sephacryl S-300HR. E shows the ultraviolet spectrum of SEP produced by column chromatgraphy using Sephacryl S-300HR with spectrophotometry. F expressed the profile of SEP as determined using size exclusion high-performance liquid chromatography. G, H, and I show monosaccharide compositions of SEP as determined by high-performance liquid chromatography. 406 407 408 20


Page 21 of 25


409

Figure 2. SEP impaired the negative effects of CP on serum sex hormone contents. FSH, LH, and E2 contents in sera were detected with enzyme-linked immunosorbent assay detection kits, the primary antibodies were monoclonal antibodies produced in rabbit, the secondary antibodies were goat-originated polyclonal antibodies conjugated with peroxidase. Different letters indicate significant differences, abp < 0.05, ABp < 0.01. 410 411

412 413

CON

CP

SEP

CP+SEP

Figure 3. Prevention of SEP on ultrastructure disruption of ovarian granulosa cells caused by CP. Ultramicro-section of ovary showing the ultrastructure of granulose cells under transmission electron microscopy (15000×). Arrow, triangle or rectangle represents mitochondrion, nucleus or autophagic vacuole respectively.

21



CON

Page 22 of 25

CP 30

Bb

SEP

Apoptosis rate (%)

25

CP+SEP

20 15 10

Aa

Aa

SEP

SEP+CP

Aa

5 0 CON

414 415 416 417

CP

Figure 4. Apoptosis of granulosa cells CP induced was suppressed by SEP in mice ovaries. Ovaries from mice exposed to CP and/or SEP were subjected to make paraffin sections with routine methods and were used to determine apoptosis with TUNEL staining assay. Different letters indicate significant differences, abp < 0.05, ABp < 0.01.

CON

CP

SEP

CP+SEP

LC 3

Beclin 1

418 419

Figure 5. Autophagy of granulosa cells CP mediated was reduced by SEP in mice. Ovaries from mice exposed to CP and/or SEP were subjected to make paraffin sections with routine methods. LC 3 or Beclin 1 protein was probed with the corresponding first antibody and the fluorescein labelled secondary antibody, and then was observed under laser scanning confocal microscope and taken photos.

22


Page 23 of 25


Figure 6. Impairment of SEP on CP-induced apoptosis and autophagy in ovary of mice. Proteins were prepared from ovaries to detect expression levels of bax, bcl-2, beclin-1, and LC-3 genes and phosphorylation levels of p38 and Akt proteins by western blotting analysis. Protein levels were all normalised with glyceraldehyde-3-phosphate dehydrogenase protein content. Antibodies were purchased from Santa Cruz and Proteintech. Different letters indicate significant differences, abp < 0.05, ABp < 0.01. 420 421 422 423 424 425 426 427 428 429 430 431

23



432 433

TOC graphic

434

435

24


Page 24 of 25

Page 25 of 25


234x173mm (96 x 96 DPI)


Preventive Effects of a Novel Polysaccharide from Sepia esculenta Ink

Recommend Documents