Effect of Fucoidan on PCNA, INSL3, JNK, TGF-β1, IL-1β and Akt Expressions in Diabetic Rat Testicular Tissues

Abstract

The aim of our study is to evaluate the possible effects of fucoidan, which is an antioxidant with high potential to be effective in histopathological damages in diabetic testicular tissue, on the proliferation, Leydig cell function, apoptosis and inflammation. For this purpose, we planned to evaluate protein expression of proliferated cell nuclear antigen (PCNA), insulin-like peptide 3 (INSL3) phospho-c-Jun N-terminal kinase (p-JNK), transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) phospho-serine/threonine protein kinase (p-Akt) in testicular tissue. Although fucoidan has been presented to consumer use in tablet form for various purposes, there are insufficient data to make an assessment of infertility in male patients with diabetes. The animals were divided into four groups (n=6) using twenty-four Wistar albino male rats; K: control group, Group D: 40 mg/kg streptozotocin (STZ, 5 consecutive days, intraperitoneal (i.p.), EF group: 40 mg/kg STZ i.p (5 consecutive days)+50 mg/kg i.p. fucoidan (one time, every other day, during six weeks), GF group: 40 mg/kg STZ (5 consecutive days)+50 mg/kg i.p. fucoidan (after fifteen days induction diabetes, every other day, during six weeks). The shedding of immature cells and loss of the seminiferous tubule wall were seen as significant degenerative changes by diabetes induction. However diabetes decreased proliferation index, INSL3 and f-Akt immunoreactivity, whereas increased f-JNK, TGF-β1, IL-1β immunoreactivity. All parameters studied in EF and GF groups was observed reducing effects of diabetes. It was concluded that fucoidan may contribute to the prevention of some serious problems that may occur in male reproductive health, such as infertility.

Keywords

• Fucoidan
• Testes
• Diabetes
• JNK
• INSL3

Project Number

114s153

Fukoidanın Diyabetik Sıçan Testis Dokularındaki PCNA, INSL3, JNK, TGF-β1, IL-1β ve Akt İfadelerine Etkisi

Abstract

Çalışmamızın amacı, diyabetik testis dokularında gözlenen hasarlarda etkili olabilme potansiyeli yüksek bir antioksidan olan fukoidanın, spermatogenez seri hücrelerine proliferasyon, apoptozis ve inflamasyon yönünden olası etkilerini araştırmaktır. Fukoidan; çeşitli amaçlarla tablet formunda tüketici kullanımına sunulmuş olmasına rağmen, diyabetli erkek hastalarda infertiliteye yönelik bir değerlendirme yapabilmek için yeterli veriler bulunmamaktadır. Bu amaçla, testis dokusunda prolifere hücre nükleer antijeni (PCNA), insulin-benzeri peptid 3 (INSL3), fosfo (f)-c-Jun N-terminal kinaz (f-JNK), dönüştürücü büyüme faktörü-β1 (TGF-β1), fosfo-serin/treonin protein kinaz (f-Akt) ve interlökin-1β (IL-1β) gibi biyobelirteçlerin ifadelerinin değerlendirilmesi planlanmıştır. Çalışmamızda yirmi dört adet Wistar albino erkek sıçan kullanılarak 4 deney grubu (n=6); K: Kontrol grubu, D: diyabet grubu; 40 mg/kg streptozotosin (STZ, 5 ardışık gün, intraperitoneal (i.p.) verilen grup, EF grubu: 40 mg/kg STZ i.p (5 ardışık gün) + 50 mg/kg i.p. fukoidan (Diyabet oluşumunun ertesi günü başlanarak 6 hafta süresince, günaşırı bir kez) verilen grup, GF grubu: 40 mg/kg STZ i.p. (5 ardışık gün) + 50 mg/kg i.p. fukoidan (diyabet oluşturulduktan 15 gün sonra, 6 hafta süresince günaşırı bir kez) verilen grup oluşturulmuştur. Diyabet indüksiyonuyla; immatur hücrelerde dökülme ve seminifer tübül duvarındaki hücrelerde izlenen sitoplazma kayıpları, belirgin dejeneratif değişiklikler olarak görülmüştür. Bununla birlikte diyabet, proliferasyon indeksinin yanısıra INSL3 ve f-Akt immunoreaktivitelerinde azalmaya; f-JNK, TGF-β1, IL-1β immunreaktivitelerinde ise artışa neden olmuştur. EF ve GF gruplarında ise çalışılan tüm parametrelerde, diyabetin etkilerini azaltma yönünde etki gözlenmiştir. Yaptığımız çalışma sonucunda, moleküler düzeyde etkili olabildiği önceki çalışmalarla da saptanan fukoidanın; diyabetik testis dokusundaki histopatolojik hasarlara karşı koruyucu etkileriyle, infertilite gibi üreme sağlığında oluşabilecek ciddi problemlerin önlenmesine katkı yapacağı kanısındayız.

Keywords

• Fukoidan
• Testis
• Diyabet
• JNK
• INSL3

Supporting Institution

TÜBİTAK

Project Number

114s153

Thanks

Çalışmamız, TÜBİTAK 114S153 numaralı proje ile desteklenmiştir.

References

1. 1. International Diabetes Federation, Diabetes atlas 9th edition. https://www.diabetesatlas.org/en/sections/worldwide-toll-of-diabetes.html (24.03.2020) tarihinde erişildi). 2019.
2. 2. Alves MG, Martins AD, Rato L, Moreira PI, Socorro S, Oliveira PF. Molecular mechanisms beyond glucose transport in diabetes-related male infertility. Biochim Biophys Acta. 2013;1832(5):626-35.
3. 3. Yigitturk G, Acara AC, Erbas O, Oltulu F, Yavasoglu NUK, Uysal A, et al. The antioxidant role of agomelatine and gallic acid on oxidative stress in STZ induced type I diabetic rat testes. Biomed Pharmacother. 2017;87:240-6.
4. 4. Ersoy O, Kizilay G. Effects of fucoidan on diabetic rat testicular tissue. Biotech Histochem. 2018;93(4):277-85.
5. 5. Oroojan AA, Ahangarpour A, Paknejad B, Zareian P, Hami Z, Abtahi SR. Effects of Myricitrin and Solid Lipid Nanoparticle-Containing Myricitrin on Reproductive System Disorders Induced by Diabetes in Male Mouse. World J Mens Health. 2019;doi: 10.5534/wjmh.190010.
6. 6. Song J, Gao X, Tang Z, Li H, Ruan Y, Liu Z, et al. Protective effect of Berberine on reproductive function and spermatogenesis in diabetic rats via inhibition of ROS/JAK2/NFkappaB pathway. Andrology. 2020;8(3):793-806.
7. 7. La Vignera S, Calogero AE, Condorelli R, Lanzafame F, Giammusso B, Vicari E. Andrological characterization of the patient with diabetes mellitus. Minerva Endocrinol. 2009;34(1):1-9.
8. 8. Bener A, Al-Ansari AA, Zirie M, Al-Hamaq AO. Is male fertility associated with type 2 diabetes mellitus? Int Urol Nephrol. 2009;41(4):777-84.

9. 9. Shoorei H, Khaki A, Shokoohi M, Khaki AA, Alihemmati A, Moghimian M, et al. Evaluation of carvacrol on pituitary and sexual hormones and their receptors in the testicle of male diabetic rats. Hum Exp Toxicol. 2020;39(8):1019-30.
10. 10. Rezaei N, Mardanshahi T, Shafaroudi MM, Abedian S, Mohammadi H, Zare Z. Effects of l-Carnitine on the Follicle-Stimulating Hormone, Luteinizing Hormone, Testosterone, and Testicular Tissue Oxidative Stress Levels in Streptozotocin-Induced Diabetic Rats. J Evid-Based Integr. 2018;23:2515690X18796053.
11. 11. Shi GJ, Zheng J, Wu J, Qiao HQ, Chang Q, Niu Y, et al. Protective effects of Lycium barbarum polysaccharide on male sexual dysfunction and fertility impairments by activating hypothalamic pituitary gonadal axis in streptozotocin-induced type-1 diabetic male mice. Endocr J. 2017;64(9):907-22.
12. 12. Zhu X, Guo F, Tang H, Huang C, Xie G, Huang T, et al. Islet Transplantation Attenuating Testicular Injury in Type 1 Diabetic Rats Is Associated with Suppression of Oxidative Stress and Inflammation via Nrf-2/HO-1 and NF-kappaB Pathways. J Diabetes Res. 2019;2019:8712492.
13. 13. Wang Y, Xing M, Cao Q, Ji A, Liang H, Song S. Biological Activities of Fucoidan and the Factors Mediating Its Therapeutic Effects: A Review of Recent Studies. Mar Drugs. 2019; 20;17(3):183.
14. 14. Luthuli S, Wu S, Cheng Y, Zheng X, Wu M, Tong H. Therapeutic Effects of Fucoidan: A Review on Recent Studies. Mar Drugs. 2019;21;17(9):487.
15. 15. Kim KJ, Yoon KY, Lee BY. Fucoidan regulate blood glucose homeostasis in C57BL/KSJ m+/+db and C57BL/KSJ db/db mice. Fitoterapia. 2012;83(6):1105-9.
16. 16. Chen J, Wang W, Zhang Q, Li F, Lei T, Luo D, et al. Low molecular weight fucoidan against renal ischemia-reperfusion injury via inhibition of the MAPK signaling pathway. PLoS One. 2013;8(2):e56224.
17. 17. Lee H, Kim JS, Kim E. Fucoidan from seaweed Fucus vesiculosus inhibits migration and invasion of human lung cancer cell via PI3K-Akt-mTOR pathways. PLoS One. 2012;7(11):e50624.
18. 18. Kim TH, Lee EK, Lee MJ, Kim JH, Yang WS. Fucoidan inhibits activation and receptor binding of transforming growth factor-beta1. Biochem Biophys Res Commun. 2013;432(1):163-8.
19. 19. Zhu H, Zhang Y, Hu X, Yi C, Zhong S, Wang Y, et al. The effects of high-dose qinggan huoxue recipe on acute liver failure induced by d-galactosamine in rats. Evid Based Complement Alternat Med. 2013;2013:905715.
20. 20. Yuksel B, Kilic S, Lortlar N, Tasdemir N, Sertyel S, Bardakci Y, et al. Environmental Tobacco Smoke Exposure during Intrauterine Period, Promotes Caspase Dependent and Independent DNA Fragmentation in Sertoli-Germ Cells. ISRN Obstet Gynecol. 2014;2014:170124.
21. 21. Minagawa I, Sagata D, Pitia AM, Kohriki H, Shibata M, Sasada H, et al. Dynamics of insulin-like factor 3 and its receptor expression in boar testes. J Endocrinol. 2014;220(3):247-61.
22. 22. Kim KJ, Yoon KY, Lee BY. Low molecular weight fucoidan from the sporophyll of Undaria pinnatifida suppresses inflammation by promoting the inhibition of mitogen-activated protein kinases and oxidative stress in RAW264.7 cells. Fitoterapia. 2012;83(8):1628-35.
23. 23. Roy S, Metya SK, Rahaman N, Sannigrahi S, Ahmed F. Ferulic acid in the treatment of post-diabetes testicular damage: relevance to the down regulation of apoptosis correlates with antioxidant status via modulation of TGF-beta1, IL-1beta and Akt signalling. Cell Biochem Funct. 2014;32(1):115-24.
24. 24. Lee MW, Kwon JE, Lee YJ, Jeong YJ, Kim I, Cho YM, et al. Prunus mume leaf extract lowers blood glucose level in diabetic mice. Pharm Biol. 2016;54(10):2135-40.
25. 25. Guneli E, Tugyan K, Ozturk H, Gumustekin M, Cilaker S, Uysal N. Effect of melatonin on testicular damage in streptozotocin-induced diabetes rats. Eur Surg Res. 2008;40(4):354-60.
26. 26. Singh R, Kaur N, Kishore L, Gupta GK. Management of diabetic complications: a chemical constituents based approach. J Ethnopharmacol. 2013;150(1):51-70.
27. 27. Sebai H, Selmi S, Rtibi K, Gharbi N, Sakly M. Protective Effect of Lavandula stoechas and Rosmarinus officinalis essential oils against reproductive damage and oxidative stress in alloxan-induced diabetic rats. J Med Food. 2015;18(2):241-9.
28. 28. Altay B, Cetinkalp S, Doganavsargil B, Hekimgil M, Semerci B. Streptozotocin-induced diabetic effects on spermatogenesis with proliferative cell nuclear antigen immunostaining of adult rat testis. Fertil Steril. 2003;80 Suppl 2:828-31.
29. 29. Cai L, Chen S, Evans T, Deng DX, Mukherjee K, Chakrabarti S. Apoptotic germ-cell death and testicular damage in experimental diabetes: prevention by endothelin antagonism. Urol Res. 2000;28(5):342-7.
30. 30. Sadik NA, El-Seweidy MM, Shaker OG. The antiapoptotic effects of sulphurous mineral water and sodium hydrosulphide on diabetic rat testes. Cell Physiol Biochem. 2011;28(5):887-98.
31. 31. Amaral S, Moreno AJ, Santos MS, Seica R, Ramalho-Santos J. Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes. Theriogenology. 2006;66(9):2056-67.
32. 32. Donmez YB, Kizilay G, Topcu-Tarladacalisir Y. MAPK immunoreactivity in streptozotocin-induced diabetic rat testis. Acta Cir Bras. 2014;29(10):644-50.
33. 33. Kizilay G, Bayram S, Ersoy O, Donmez Bozdemir Y. Jnk İnhibisyonunun Diyabetik Testis Dokusundaki Fas/Fasl Sinyal Yolağına Etkileri. Batı Karadeniz Tıp Dergisi 2017;1 (3): 112-118.
34. 34. Öztürk F, Gül M, Ağkadir M, Yağmurca M. Deneysel Diyabetin Sıçan Testislerinde Meydana Getirdiği Histolojik Değişiklikler. Turkiye Klinikleri Journal of Medical Sciences. 2002;22(2):173-8.
35. 35. Jiang X, Bai Y, Zhang Z, Xin Y, Cai L. Protection by sulforaphane from type 1 diabetes-induced testicular apoptosis is associated with the up-regulation of Nrf2 expression and function. Toxicol Appl Pharmacol. 2014;279(2):198-210.
36. 36. Khamis T, Abdelalim AF, Abdallah SH, Saeed AA, Edress NM, Arisha AH. Early intervention with breast milk mesenchymal stem cells attenuates the development of diabetic-induced testicular dysfunction via hypothalamic Kisspeptin/Kiss1r-GnRH/GnIH system in male rats. Biochim Biophys Acta Mol Basis Dis. 2020;1866(1):165577.
37. 37. Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003;79(4):829-43.
38. 38. Shrilatha B, Muralidhara. Early oxidative stress in testis and epididymal sperm in streptozotocin-induced diabetic mice: its progression and genotoxic consequences. Reprod Toxicol. 2007;23(4):578-87.
39. 39. Koh PO. Streptozotocin-induced diabetes increases apoptosis through JNK phosphorylation and Bax activation in rat testes. J Vet Med Sci. 2007;69(9):969-71.
40. 40. Koh PO. Streptozotocin-induced diabetes increases the interaction of Bad/Bcl-XL and decreases the binding of pBad/14-3-3 in rat testis. Life Sci. 2007;81(13):1079-84.
41. 41. Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W, et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci U S A. 2001;98(24):13681-6.
42. 42. Hu C, Su Q, Li F, Zhang G, Sun D, Han H, et al. Duodenal-Jejunal bypass improves glucose homeostasis in association with decreased proinflammatory response and activation of JNK in the liver and adipose tissue in a T2DM rat model. Obes Surg. 2014;24(9):1453-62.
43. 43. Bogoyevitch MA, Arthur PG. Inhibitors of c-Jun N-terminal kinases: JuNK no more? Biochim Biophys Acta. 2008;1784(1):76-93.
44. 44. Berdichevsky A, Guarente L, Bose A. Acute oxidative stress can reverse insulin resistance by inactivation of cytoplasmic JNK. J Biol Chem. 2010;285(28):21581-9.
45. 45. Bennett BL, Satoh Y, Lewis AJ. JNK: a new therapeutic target for diabetes. Curr Opin Pharmacol. 2003;3(4):420-5.
46. 46. Dönmez YB. Deneysel diyabet oluşturulmuş sıçanların testis dokularında JNK ve IL-6 ilişkisinin incelenmesi. (Doktora Tezi). Edirne: Trakya Üniversitesi; 2015.
47. 47. Hyun JH, Kim SC, Kang JI, Kim MK, Boo HJ, Kwon JM, et al. Apoptosis inducing activity of fucoidan in HCT-15 colon carcinoma cells. Biol Pharm Bull. 2009;32(10):1760-4.
48. 48. Park HY, Han MH, Park C, Jin CY, Kim GY, Choi IW, et al. Anti-inflammatory effects of fucoidan through inhibition of NF-kappaB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells. Food Chem Toxicol. 2011;49(8):1745-52.
49. 49. Chen J, Cui W, Zhang Q, Jia Y, Sun Y, Weng L, et al. Low molecular weight fucoidan ameliorates diabetic nephropathy via inhibiting epithelial-mesenchymal transition and fibrotic processes. Am J Transl Res. 2015;7(9):1553-63.
50. 50. Salama N, Tsuji M, Tamura M, Kagawa S. Transforming growth factor (beta1) in testes of aged and diabetic rats: correlation with testicular function. Arch Androl. 2001;47(3):217-26.
51. 51. Hong SW, Jung KH, Lee HS, Zheng HM, Choi MJ, Lee C, et al. Suppression by fucoidan of liver fibrogenesis via the TGF-beta/Smad pathway in protecting against oxidative stress. Biosci Biotechnol Biochem. 2011;75(5):833-40.
52. 52. Guo Z, Yan X, Wang L, Wu J, Jing X, Liu J. Effect of Telmisartan or Insulin on the Expression of Adiponectin and its Receptors in the Testis of Streptozotocin-Induced Diabetic Rats. Horm Metab Res. 2016;48(6):404-12.
53. 53. Shirneshan K, Binder S, Bohm D, Wolf S, Sancken U, Meinhardt A, et al. Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells. Mol Cell Endocrinol. 2008;295(1-2):79-86.
54. 54. Ermetici F, Donadio F, Iorio L, Malavazos AE, Dolci A, Peverelli E, et al. Peripheral insulin-like factor 3 concentrations are reduced in men with type 2 diabetes mellitus: effect of glycemic control and visceral adiposity on Leydig cell function. Eur J Endocrinol. 2009;161(6):853-9.
55. 55. Ivell R, Anand-Ivell R. Biological role and clinical significance of insulin-like peptide 3. Curr Opin Endocrinol Diabetes Obes. 2011;18(3):210-6.
56. 56. Valeri C, Schteingart HF, Rey RA. The prepubertal testis: biomarkers and functions. Curr Opin Endocrinol Diabetes Obes. 2013;20(3):224-33.
57. 57. Overvad S, Bay K, Bojesen A, Gravholt CH. Low INSL3 in Klinefelter syndrome is related to osteocalcin, testosterone treatment and body composition, as well as measures of the hypothalamic-pituitary-gonadal axis. Andrology. 2014;2(3):421-7.
58. 58. Burkhardt E, Adham IM, Hobohm U, Murphy D, Sander C, Engel W. A human cDNA coding for the Leydig insulin-like peptide (Ley I-L). Hum Genet. 1994;94(1):91-4.
59. 59. Foresta C, Bettella A, Vinanzi C, Dabrilli P, Meriggiola MC, Garolla A, et al. A novel circulating hormone of testis origin in humans. J Clin Endocrinol Metab. 2004;89(12):5952-8.
60. 60. Bay K, Hartung S, Ivell R, Schumacher M, Jurgensen D, Jorgensen N, et al. Insulin-like factor 3 serum levels in 135 normal men and 85 men with testicular disorders: relationship to the luteinizing hormone-testosterone axis. J Clin Endocrinol Metab. 2005;90(6):3410-8.