Preventive Effects of a Novel Polysaccharide from Sepia esculenta

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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

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Journal of Agricultural and Food Chemistry

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

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failure and its action mechanisms in cyclophosphamide-treated mice

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‡

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

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

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†

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

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

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

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©

The authors contributed equally to this manuscript.

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

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

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ACS Paragon Plus Environment


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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KEYWORDS:

SEP; cyclophosphamide; autophagy; apoptosis; ovarian failure.

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■ INTRODUCTION

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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esculenta ink was isolated and characterised.

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■ MATERIALS AND METHODS

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Preparation of Sepia esculenta ink polysaccharide (SEP)

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

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

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

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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



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

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

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

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

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

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

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

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

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Purity determination of SEP

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

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

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

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

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

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

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temperature of 50 °C.

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Monosaccharide composition analysis

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

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

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

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


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

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

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

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aperture filter membrane for HPLC analysis.

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

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

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

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

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

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Animal experimental scheme

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

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

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

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

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

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

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cycle with food and water available ad libitum.

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

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

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

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



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

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

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

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ovarian follicles.

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Detection of sex hormones in serum

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

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

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kits according to the manufacturer’s protocol.

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Detection of ultrastructure of ovary with transmission electron microscopy

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

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

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

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

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which were then observed by using transmission electron microscopy.

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Western blotting analysis

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

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

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

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

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


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reference.

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Statistical analysis

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

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

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

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■ RESULTS

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

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

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

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

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

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

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

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a purified polysaccharide, which was deemed to be SEP.

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

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

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

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

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

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a purified polysaccharide.

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



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

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

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GlcA, and GalA.

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

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and the relative mass ratios of ovary and uterus in mice

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

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

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

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

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

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

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

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

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

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between vehicle- and co-treated mice.

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

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

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

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

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destruction of ovarian oestrogen production was prevented by SEP. 8


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

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

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

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

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

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

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

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

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

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that SEP prevented apoptosis of CP-treated granulosa cells.

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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■ DISCUSSION

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

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

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Takaya et al.

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

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

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

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

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

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

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

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and GalA were observed in SEP.

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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also impaired autophagy induced by using CP.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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and inhibiting apoptosis.

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■ AUTHOR INFORMATION

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Corresponding Author

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

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

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Funding

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

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

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Notes

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No potential conflicts of interest were disclosed.

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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


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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


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234x173mm (96 x 96 DPI)


Preventive Effects of a Novel Polysaccharide from Sepia esculenta

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