Squid ink polysaccharide reduces cyclophosphamide-induced testicular damage via Nrf2/ARE activation pathway in mice


College of Science, Guangdong Ocean University, Zhanjiang, China


Objective(s):Cyclophosphamide (CP) toxicity on testis was hampered by squid ink polysaccharide (SIP) via restoration of antioxidant ability in our previous investigations. This study investigated roles of Nrf2/ARE signal pathway in testis of treated mice.
Materials and Methods: Male Kunming mice were employed to undergo treatment with SIP and/or CP. Protein levels of Nrf2, keap-1, histone deacetylase 2 (HDAC2), quinone oxidoreductase 1 (NQO-1), and heme oxygenase 1 (HO-1) and phosphorylation level of protein kinase C (PKC) in testis were evaluated by Western blotting.
Results: Data showed that SIP elevated expressions of NQO-1 and HO-1 genes, two downstream target molecules of Nrf2, via activating Nrf2 to play preventive roles on CP-treated testis, and further discovered that upstream regulators of Nrf2, keap-1, HDAC2, and PKC, were concerned with the regulation of Nrf2.
Conclusion: These results suggest that SIP could effectively weaken CP-associated testicular damage via Nrf2/ARE signal pathway.


1. Emadi A, Jones RJ, Brodsky RA. Cyclophosphamide and cancer: golden anniversary. Nat Rev Clin Oncol 2009; 6:638-647.

2. Pryzant RM, Meistrich ML, Wislon G, Brown B, McLaughlinP. Long-term reduction in sperm count after chemotherapy with and without radiation therapy for non-hodgkin’s lymphomas. J Clin Oncol 1993; 11:239–247.

3. Elangovan N, Chiou TJ, Tzeng WF, Chu ST. Cyclophosphamide treatment causes impairment of sperm and its fertilizing ability in mice. Toxicology 2006; 222:60-70.

4. Takaya Y, Uchisawa H, Narumi F, Matsue H. Illexins A, B, and C from squid ink shoule have a branched structure. Biochem Biophys Res Commun 1996; 226:335-338.

5. Chen S, Xu J, Xue C, Dong P, Sheng W, Yu G, Chai W. Sequence determination of a non-sulfated glycosaminoglycan-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.

6. Le XY, Luo P, Gu YP, Tao YX, Liu HZ. Interventional effects of squid ink polysaccharides on cyclophosphamide-associated testicular damage in mice. Bratisl Lek Listy 2015; 116: 334-339.

7. Tang Q, Zuo T, Lu S, Wu J, Wang J, Zheng R, Chen S, Xue C. Dietary squid ink polysaccharides ameliorated the intestinal microflora dysfunction in mice undergoing chemotherapy. Food Funct 2014; 5: 2529-2535.

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

9. Zuo T, Cao L, Li X, Zhang Q, Xue C, Tang QJ. The Squid Ink Polysaccharides Protect Tight Junctions and Adherens Junctions from Chemotherapeutic Injury in the Small Intestinal Epithelium of Mice. Nutr Cancer 2015; 67: 364–371.

10. Luo P, Liu HZ. 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.

11. Li Y, Paonessa JD, Zhang Y. Mechanism of chemical activation of Nrf2. Plos One 2012; 7: e35122.

12. Tripathi DN, Jena GB. Astaxanthin inhibits cytotoxic and genotoxic effects of cyclophosphamide. Toxicology 2008; 248:96-103.

13. Balogun E, Hoque M, Gong P, Killeen E, Green CJ, Foresti R, et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J 2003; 371:887-895.

14. Garg R, Gupta S, Maru GB. Dietary curcumin modulates transcriptional regulators of phaseⅠand phaseⅡenzymes in benzo[a]pyrene-treated mice: mechanism of its anti-initiating action. Carcinogenesis 2008; 29:1022-1032.

15. Liu F, Li XL, Lin T, He DW, Wei GH, Liu JH, et al. The cyclophosphamide metabolite, acrolein, induces cytoskeletal changes and oxidative stress in Sertoli cells. Mol Biol Rep 2012; 39: 493-500.

16. Miao W, Hu L, Scrivens PJ, Batist G. Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway direct cross talk between phaseⅠand phaseⅡ drug-metabolizing enzymes. J Biol Chem 2005; 280:20340-20348.

17. Stepkowski TM, Kruszewski MK. Molecular cross-talk between the Nrf2/KEAP1 signaling pathway, autophagy, and apoptosis. Free Radic Biol Med 2011; 50:1186-1195.

18. Tripathi DN, Jena GB. Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress, DNA damage and early hepatocarcinogenesis in
rat: role of Nrf2, p53, p38 and phase-Ⅱ enzymes. Mutat Res 2010; 696:69-80.

19. Huang HC, Nguyen T, Pickett CB. Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J Biol Chem 2002; 277:42769-42774.

20. Niture SK, Jain AK, Jaiswal AK. Antioxidant-induced modification of INrf2 cysteine 151 and PKC-δ-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance. J Cell Sci 2009; 122:4452-4464.

21. Keum YS, Yu S, Chang PP, Yuan X, Kim JH, Xu C, et al. Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element–mediated heme oxygenase-1 in human hepatoma hepG2 cells. Cancer Res 2006; 66:8804-8813.

22. Velichkova M,Hasson T. Keap1 regulates the oxidation-sensitive shuttling of Nrf2 into and out of the nucleus via a Crm1-dependent nuclear export mechanism. Mol Cell Biol 2005; 25:4501-4513.

23. Sun Z, Chin YE, Zhang DD. Acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 during the antioxidant response. Mol Cell Biol 2009; 29:2658-2672.

24. Mercado N, Thimmulappa R, Thomas CM, Fenwick PS, Chana KK, Donnelly LE, Biswal S, Ito K, Barnes PJ. Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress. Biochem Biophys Res Commun 2011; 406:292-298.

25. Forney GB, Morre DJ, Morre DM. Oxidative stress reduced by a green tea concentrate and capsicum combination: synergistic effects. J Diet Suppl 2013; 10:318-324.

26. Li B, Cui W, Tan Y, Luo P, Chen Q, Zhang C, et al. Zinc is essential for the transcription function of Nrf2 in human renal tubule cells in vitro and mouse kidney in vivo under the diabetic condition. J Cell Mol Med 2014; 18:895-906.

27. Kobayashi M, Yamamoto M. Nrf2–Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv Enzyme Regul 2006; 46:113-140.

28. Yang JH, Shin BY, Han JY, Kim MG, Wi JE, Kim YW, et al. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes. Toxicol Appl Pharm 2013; 274:293-301.

29. Zhang C, Su ZY, Khor TO, Shu L, Kong AN. Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation. Biochem Pharmacol 2013; 85: 1398-1404.