| Squid ink polysaccharide(SIP), a marine bioactive substance which has various biological functions, separated and extracted from waste squid ink sac. Preliminary studies had found that SIP had a protective effect on kidney, lung, liver, testis and ovary damaged by cyclophosphamide(CP).Investigating the structure and the protective mechanism of ovarian cell apoptosis and autophagy induced by CP of SIP-1 which was purified from SIP is the primary purpose of this study.SIP was obtained through enzyme extraction and ethanol precipitation technique, and then purified by DEAE-52 cellulose column chromatography and sephacryl S-300 HR gel permeation chromatography. Three relative single sugar components namely SIP-1, SIP-2and SIP-3 were gained and the purity of SIP-1 was determined. UV scanning result showed that there are no absorption peak at 260 nm and 280 nm which explained that SIP-1 free from nucleic acid and protein. High performance liquid chromatography(HPLC) analysis had a symmetrical peak and narrow peak width which implied that SIP-1 was pure.The primary structure of SIP-1 was detected. Infrared spectrum showed that, SIP-1had strong O-H and N-H stretching vibration at 3385.96 cm-1,C-H stretching vibration at2931.23 cm-1,amino N-H angular vibration at 1637.74 cm-1,C-O of polysaccharide carboxyl stretching vibration at 1077 cm-1 and 1034.21 cm-1,and β-D-pyran glucose ring at901.95 cm-1.The monosaccharide composition of SIP-1 was analyzed by HPLC after completely acid hydrolysis which showed that SIP-1 contains galactosamine(GalN),arabinose(Ara) and the molar ratio was 1:1.Periodate oxidation and Smith degradation analysis showed that SIP-1 may have 1'3、1'2,3、1'3,4、1'3,6、1'2,3,4 glycosidic bond.1HNMR and13 CNMR showed that the possible SIP-1 monosaccharide glycosidic bond were α-L-Arap existing 1'5 glycosidic bond and α-D-GalNp existing 1'6glycosidic bond.40 healthy female Kunming mice were randomly divided into 4 groups: control group,model group, SIP-1 control group and treated group. The mice body weight, reproductive organ weight and index, ovarian tissue pathological change and serum hormone levels were detected after 15 d. The results showed that, CP had toxic damage effect on female mice ovary which was eliminated by SIP-1.Compared with control group, CP significantly(P<0.01,P<0.05) decreased the ovary weight and index, uterus weight and index and mice body weight, and the light microscope observation showed that total follicles and various follicle numbers significantly reduced and atretic follicle increased. In addition,follicle-stimulating hormone(FSH), serum luteinizing hormone(LH) level increased and estradiol level(E2) decreased. Compared with model group, SIP-1 could improve the changeable indicators caused by CP(P<0.01,P<0.05).40 healthy female Kunming mice were grouped as above. The mice were executed after 15 d and the bilateral ovaries were taken to analysis the ovarian ultrastructure through transmission electron microscope, the ovarian cell apoptosis rate through TUNEL staining,cell apoptosis and autophagy related protein levels in ovary through western blotting. The results showed that, compared with control group, model group could observe least organelles such as mitochondria and endoplasmic reticulum and apoptotic globule under TEM. Also, the ovarian cell apoptosis rate significantly decreased(P<0.01), Bax, p38 phosphorylation, LC3 and Beclin1 expression level significantly enhanced(P<0.01,P<0.05), Bcl-2 and p-Akt expression level significantly reduced(P<0.01,P<0.05).When compared with model group, SIP-1 could remarkably improve the ovarian injury induced by CP and the ovarian cell apoptosis rate of SIP-1 treated group decreased(P<0.05). At the same time, SIP-1 could relieve the reduction of Bcl-2, p-Akt expression level(P<0.01,P<0.05) and increase of p38 phosphorylation, LC3, Beclin1 expression level induced by CP(P<0.05). In total, SIP-1 could reduce CP-induced ovarian cell apoptosis and autophagy through regulating the expression level of cell apoptosis and autophagy related-proteins.SIP-1, a novel structure and purified squid ink polysaccharide, had an effect on eliminating reproductive damage caused by CP. The possible mechanism was to regulate the expression level of cell apoptosis and autophagy related-proteins. |
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