Squid ink polysaccharide prevents autophagy and oxidative stress affected by cyclophosphamide in Leydig cells of mice: a pilot study

Document Type : Original Article


1 College of Food & Biological Engineering, Hezhou University, Hezhou 542899, China

2 College of Chemistry & Environment, Guangdong Ocean University, Zhanjiang 524088, China

3 School of Food Science & Technology, Guangdong Ocean University, Zhanjiang524088, China

4 Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China


Objective(s): The aim of this study was to explore the effects of Squid ink polysaccharide (SIP) on prevention of autophagy and oxidative stress induced by cyclophosphamide (CP) in Leydig cells of mice.
Materials and Methods: Examination of reproductive organ exponents, abnormal sperm rate, activities of superoxide dismutase (SOD), catalase (CAT), contents of malondialdehyde (MDA), and histological structure were performed to detect the optimal dose of SIP against oxidative stress damage in vivo, and autophagy-associated protein LC3 and Beclin-1 were examined by immunofluorescence, and their expression was detected by Western blot analysis. Leydig cells ultrastructural changes were observed by transmission fluorescent microscope.
Results: SIP significantly inhibited sperm aberration, histological structure and injury of seminiferous tubules caused by CP, as well as the antioxidant activity of SOD and CAT were increased; contents of MDA were decreased. The optimal dose of SIP for prevention of oxidative stress injury by CP was 80 mg/kg. In addition, LC3 and Beclin-1 fluorescent granules were much less in the Leydig cell layer after treatment via SIP compared with the CP-treated group, and the expression levels of LC3 and Beclin-1 were also decreased. Furthermore, characteristics of cell autophagy such as mitochondrial swelling, autophagic vacuoles, and chromatin pyknosis were observed in CP-treated Leydig cells, but SIP could effectively weaken injury of Leydig cell ultrastructure by CP.
Conclusion: SIP, as an antioxidant, prevents the cytoskeleton damage through up-regulation antioxidant capacity and inhibition autophagy caused by CP.


1. Mythili Y, Sudharsan PT, Selvakumar E, Varalakshmi P. Protective effect of DL-α-lipoic acid on cyclophosphamide induced oxidative cardiac injury. Chem-Biol Interact 2004; 151: 13-19.
2. Zhang J, Ma K, Wang H. Cyclophosphamide suppresses thioredoxin reductase in bladder tissue and its adaptive response via inductions of thioredoxin reductase and gluththione peroxidase. Chem-Biol Interact 2006; 162: 24-30.
3. Aguilar-Mahecha A, Hales BF, Robaire B. Effects of acute and chronic cyclophosphamide treatment on meiotic progression and the induction of DNA double-strand breaks in rat spermatocytes. Biol Reprod 2005; 72: 1297-1304.
4. Le XY, Luo P, Gu YP, Tao YX, Liu HZ. Interventional effects of squid ink polysaccharides on cyclophosphamide-associated testicular damage in mice. Bratisl Med J 2015; 116: 334-339.
5. Le XY, Luo P, Gu Y P, Tao YX, Liu HZ. Squid ink polysaccha-rides reduces cyclophosphamide-induced testicular damage via Nrf2/ARE activation pathway in mice. Iran J Basic Med Sci 2015; 18: 827-831.
6. Tan C, Lai S, Wu S, Hu S, Zhou L, Chen Y, et al. Nuclear permeable ruthenium (II) β-carboline complexes induce autophagy to antagonize mitochondrial-mediated apoptosis. J Med Chem 2010; 53: 7613-7624.
7. Faleiro L, Lazebnik Y. Caspases disrupt the nuclear-cytoplasmic barrier. J Cell Biol 2000; 151: 951-960.
8. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012; 8:445-544.
9. Mizushima, N, Levine, B, Cuervo, AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069-1075.
10. Carchman EH, Rao J, Loughran PA, Rosengart MR, Zuckerbraun BS. Heme oxygenase-1-mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice. Hepatology 2011; 53: 2053-2062.
11. Serrone L, Hersey P. The chemoresistance of human malignant melanoma: an update. Melanoma Res 1999; 9: 51-58.
12. Kourtis N, Tavernarakis N. Autophagy and cell death in model organisms. Cell Death Differ 2009; 16: 21-30.
13. Ito H, Daido S, Kanzawa T, Kondo S, Kondo Y. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 2005; 26: 1401-1410.
14. Tang D, Kang R, Cheh CW, Livesey KM, Liang X, Schapiro N E, et al. HMGB1 release and redox regulates autophagy and apoptosis in cancer cells. Oncogene 2010; 29: 5299-5310.
15. Dalby K, Tekedereli I, Lopez-Berestein G, Ozpolat B. Targeting the pro-death and pro-survival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 2010; 6: 322-329.
16. Luo P, Liu H. Antioxidant ability of squid ink polysaccharides as well as their protective effects on
deoxyribonucleic acid DNA damage in vitro. Afr J Pharm Pharmacol 2013; 7: 1382-1388.
17. Zhong JP, Guang W, Shang JH, Pan JQ, Li K, Huang Y, et al. Protective effects of squid ink extract towards hemopoietic injuries induced by cyclophosphamide. Mar Drugs 2009; 7:9-18.
18. Chen S, Xu J, Xue C, Dong P, Sheng W, Yu G, et al. Sequence determination of a non-sulfated glycos- aminoglycan-like polysaccharide from melanin-free ink of the squid Ommastrephes bartrami by negative-ion electrospray tandemmass spectrometry and NMR spectroscopy. Glycoconjugate J 2008; 25: 481-492.
19. Liu HZ, Tao YX, Luo P, Deng CM, Gu YP, Yang L, et al. Preventive effects of a novel polysaccharide from Sepia esculenta ink on ovarian failure and its action mechanisms in cyclophosphamide-treated mice. J Agr Food Chem 2016; 64: 5759-5766.
20. Staub AM. Removal of protein-Sevag method. Methods Carbohyd Chem 1965; 5: 5-6.
21. Holdcraft RW, Braun RE. Hormonal regulation of spermatogenesis. Int J Androl 2004; 6: 335-342.
22. Handelsman DJ. Hormonal regulation of spermato- genesis: insights from constructing genetic models. Reprod Fert Develop 2011; 4: 507-19.
23. Iuchi Y, Kaneko T, Matsuki S, Ishii T, Ikeda Y, Uchida K, et al. Carbonyl stress and detoxification ability in the male genital tract and testis of rats. Histochem Cell Biol 2004; 2: 123-130.
24. Selvakumar E, Prahalathan C, Sudharsan PT, Varalakshmi P. Protective effect of lipoic acid on cyclophosphamide-induced testicular toxicity. Clin Chim Acta 2006; 1-2: 114-119.
25. Motawi TMK, Sadik NAH, Refaat A. Cytoprotective effects of DL-alpha-lipoic acid or squalene on cyclophos- phamide-induced oxidative injury: An experimental study on rat myocardium, testicles and urinary bladder. Food Chem Toxicol 2010; 48: 2326-2336.
26. Tinari A, Garofalo T, Sorice M, Esposti M D, Malorni W. Mitoptosis: Different Pathways for Mitochondrial Execution. Autophagy 2007; 3: 282-284.
27. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, et al. The mitochondrial permeability transition in cell death: A common mechanism in necrosis, apoptosis and autophagy. BBA-Bioenergetics 1998 1366: 177-196.
28. Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclin-phosphatidylinositol 3-kinase complex functions at the trans -Golgi network. EMBO Rep 2001; 2:330-335.
29. Karim MR, Kanazawa T, Daigaku Y, Fujimura S, Miotto G, Kadowaki M. Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells. Autophagy 2007; 3: 553-560.