4 apoptosis, Guías, Proyectos, Investigaciones de Bioquímica

¡Descarga 4 apoptosis y más Guías, Proyectos, Investigaciones en PDF de Bioquímica solo en Docsity! ORIGINAL PAPER Does melatonin influence the apoptosis in rat uterus of animals exposed to continuous light? Cecı́lia S. Ferreira1,2 • Kátia C. Carvalho2 • Carla C. Maganhin2 • Ana P. R. Paiotti3 • Celina T. F. Oshima3 • Manuel J. Simões4 • Edmund C. Baracat2 • José M. Soares Jr.2 Published online: 5 November 2015  Springer Science+Business Media New York 2015 Abstract Melatonin has been described as a protective agent against cell death and oxidative stress in different tissues, including in the reproductive system. However, the information on the action of this hormone in rat uterine apoptosis is low. Our objective was to evaluate the effects of melatonin on mechanisms of cell death in uterus of rats exposed to continuous light stress. Twenty adult Wistar rats were divided into two groups: GContr (vehicle control) and GExp which were treated with melatonin (0.4 mg/mL), both were exposed to continuous light for 90 days. The uterus was removed and processed for quantitative real time PCR (qRT-PCR), using PCR-array plates of the apoptosis pathway; for immunohistochemistry and TUNEL. The results of qRT-PCR of GEXP group showed up-regulation of 13 and 7, pro-apoptotic and anti-apoptotic genes, respectively, compared to GContr group. No dif- ference in pro-apoptotic proteins (Bax, Fas and Faslg) expression was observed by immunohistochemistry, although the number of TUNEL-positive cells was lower in the group treated with melatonin compared to the group not treated with this hormone. Our data suggest that melatonin influences the mechanism and decreases the apoptosis in uterus of rats exposed to continuous light. Keywords Melatonin  Female rats  Continuous light  Apoptosis Introduction Melatonin is an indoleamine synthesized and secreted in a rhythm pattern by the pineal gland of vertebrates, it has high serum levels during the nocturnal phase of the circa- dian cycle [1–4]. This neurohormone is an important endogenous mediator of photoperiodic information and is involved in the control of circadian and seasonal processes in many species [5, 6]. In fact, melatonin is a highly pleiotropic molecule, present in many tissues and systems (nervous, cardiovascular, gastrointestinal, reproductive, etc.). It produces anti-inflammatory, antioxidant and immunomodulating activities and further, modulates pro- liferation, differentiation and apoptosis in many tissues with the interaction of hormones and growth factors [7– 11]. In the reproductive system the presence of specific receptors for melatonin in ovaries, mammary glands, testes and uterus was demonstrated [12–15]. In rodents, mela- tonin could be considered to directly influence ovarian function; increasing the secretion of estrogen and proges- terone by the granulosa cells and disrupt the progression of the estrous cycle [16, 17]. Research also demonstrated that melatonin associated with progesterone might also inhibit ovarian aromatase expression and increase uterine recep- tors in female mice [18]. Other studies show that pinealectomy and continuous light exposure (a method of & Cecı́lia S. Ferreira csferreira29@gmail.com 1 Departamento de Ginecologia, Universidade Federal de São Paulo, Avenida Doutor Arnaldo, 455. Sala 2113. Cerqueira César, CEP: 01246-923 São Paulo, Brazil 2 Laboratory of Structural and Molecular Gynecology (LIM- 58), Disciplina de Ginecologia - Departmento de Ginecologia e Obstetrı́cia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil 3 Laboratory of Pathology Molecular, Departamento de Patologia, Universidade Federal de São Paulo, São Paulo, Brazil 4 Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, Brazil 123 Apoptosis (2016) 21:155–162 DOI 10.1007/s10495-015-1195-0 decreasing the endogenous melatonin) [19] on rodents can decrease the levels of gonadotropins (LH and FSH), induce precocious puberty, ovarian atrophy, hyperprolactinemia and inhibition of uterine implantation [20–23], this indi- cates that the hormone has an important role to play in the maintenance of the homeostasis in the female reproductive tract. Studies have shown the action of melatonin in the cell death of different tissues and systems, including the reproductive system. It seems that this endoleamine pre- sents a modulating role in the apoptosis process and it can present as both pro-apoptotic and anti-apoptotic actions [24, 25]. Anti-apoptotic action of melatonin was demon- strated on the thymus, kidney, brain and liver and this action is attributed by some authors, to the antioxidant properties of indoleamine [26] and by the elimination of hydroxyl anion (–OH), peroxyl (ROO–) and superoxide [27–32]. The modulation of immunological elements is also another mechanism by which melatonin may inhibit apoptosis by inducing the release of cytokines, such as interleukin-4 [33]. Melatonin may also interact with nuclear receptors and change the gene expression of inducers or inhibitors of apoptosis [34, 35]. According to a well-established model of decreasing the endogenous melatonin (exposition to continuous light) and the action of this hormone on cell death in different tissues, our study aimed to evaluate the effects of melatonin deprivation on the uterus. It is known that this endoleamine causes a decrease in the levels of estradiol, an important protector of cell death, although, some studies also show that melatonin has a protective action on mammalian ovaries [36, 37]. Materials and methods Animals Twenty virgin adult female Wistar rats (Rattus norvegicus albinus), with regular estrous cycle, weighing between 200 and 300 g, from the Development of Experimental Model in Medicine and Biology Center (CEDEME) of the Federal University of São Paulo (UNIFESP) were used in the research. The animals were housed in plastic cages with food and water ad libitum, under the controlled tempera- ture of (22 ± 2 C) and randomly divided into two groups: GContr and GExp, both groups had ten animals and were kept under constant artificial light (400 lux) for 90 days [19, 21]. The Laboratory Animal Care Committee of Federal University of São Paulo approved this study (CEP 103/09) and all the animals used were handled in accor- dance with the Guiding Principles for The Care and Use of Animals. Experimental groups GExp group animals were treated with melatonin (Sigma Chemical Company, St. Louis, MO, USA) dissolved in 1 ml ethanol and diluted in the drinking water (0.4 mg/mL) for 90 consecutive days from 6:00 p.m. to 8:00 a.m. While GContr group animals received only water with the same concentration of ethanol (vehicle) for the same period of time [21]. After 90 days under treatment, vaginal smears were collected from the animals, those animals who were in the proestrous phase, from the GExp group, were euth- anized by decapitation, their blood was collected and their uterus’ were removed. The remaining animals, from the same group, continued under the same treatment and were euthanized once they reached the stage of the estrous cycle. All GContr group animals were euthanized after 90 days under treatment, as they were in persistent estrous. The uterus’ of the animals were removed and properly preserved according to the method which it had been subjected to. Right horn was conserved in liquid nitrogen and then frozen at -80 C, for gene expression analysis, and left horn fixed in 10 % buffered formaldehyde, for immunohistochemical, TUNEL techniques and hematox- ilin–eosin staining. Melatonin measurement The serum was obtained from the blood by centrifugation and melatonin was measured by ELISA method using IBL Kit (IBL International GmbH, Hamburg, Germany). All samples were measured in triplicate and the concentration of melatonin was given in pg/mL. Extraction and purification of total RNA Total RNA was extracted using QIAZOL Lysis Reagent (QIAGEN, Venlo, Netherlands) according to manufac- turer’s instructions and then treated with RNase-Free DNase Set (QIAGEN, USA). The total RNA obtained from each sample was quantified by spectrophotometric (ND100 NanoDrop —Thermo Fisher Scientific Inc. Co.) and its integrity profile assessed by electrophoresis on 1 % agarose gel. Synthesis of complementary strand DNA (cDNA) and quantitative real time PCR (qPCR) One microgram of total RNA purified from each sample was transcribed into cDNA by reverse transcription reac- tion, using the First Strand kit RT2 (QIAGEN), according to manufacturer’s instructions. The cDNA was subjected to reactions in qPCR, using PCR-array plates which present a large panel of the apoptosis pathway gene expression 156 Apoptosis (2016) 21:155–162 123 for endometrial cell protection [22, 23, 46–48]. Also, melatonin increases the ovulation and formation of luteal bodies, which increase the progesterone levels that are responsible for endometrial apoptosis [49, 50]. This is the central mechanism through which melatonin may influence the reproductive system. However, the presence of the specific receptors MT1 and MT2 of melatonin in the uterus, confirms that there is also a local action of this hormone in this tissue [12, 16]. In addition, melatonin is able to up- regulate the expression of its receptors and decrease the estrogen and progesterone receptors in rat endometrium, which may influence the tissue homeostasis [51, 52]. This indoleamine therefore, may protect a cell from the oxida- tive stress and controls apoptosis [10, 31, 35]. In the uterus, apoptosis is involved mainly in main- taining homeostasis in the human menstrual cycle and eliminating senescent cells from the functional layer of the endometrium during the late secretory phase and during the menstrual phase [53, 54]. The cell death in the endometrium and other tissue from female reproductive system, for example ovaries and breasts, are regulated by the sexual hormones estradiol and progesterone, as well as cytokine action [54, 55], and it can be down-regulated by the action of prostaglandins locally produced [56, 57]. This is the limitations of our experiment and thus, further study in cell culture may be necessary to understand which mechanisms involved in apoptosis is really influenced by melatonin. In this present study we evaluated the expression of the important genes and proteins involved in the apoptosis process. We could observe the same pattern of expression of Bax protein in both groups of animals, in proestrous or estrous phases, corroborating studies that show that Bax does not have a cyclic pattern of expression, and is not hormonally regulated [58, 59]. On the other hand, there are studies which have shown that Bax and Bak reach higher expressions in the endometrium during the secretory phase, when the number of apoptotic cells increase [53–57]. The Fig. 2 Immunohistochemical analysis of adults rat uterus for Bax labeling (a, b), Fas (d, e) and Faslg (g, h). a, d and g GContr group. b, e and h: GExp group. In a, b and d, e note the positive and cytoplasmic staining in the glandular endometrial cells in both groups. No label is present for Falslg in both groups. In c, f, and i, negative control slides Apoptosis (2016) 21:155–162 159 123 changes in the expression of genes and proteins in endo- metrium, is related to the menstrual phases. The increase in the expression of an anti-apoptotic protein, for example Bcl-2, is important for maintaining the integrity of tissue and avoiding the premature cell death and endometrial sloughing [60–62]. In fact, we found seven up-regulated anti-apoptotic genes, included the BcL-2 family. Similar to that which occurs with the Bcl-2 family members-mediated apoptosis, the presence of estrogen and progesterone inhibits the Fas-mediated apoptosis in the human glandular epithelium [63–65]. Our data show that Fas and Falsg genes were up-regulated in animals which received melatonin, although the first, with a value beneath the cut off (data not shown), coincides with the probable decrease of the estradiol, this corroborates with previous studies. However, no labeling for the Faslg protein was detected in the uterus of the animals in both groups, whilst Fas protein was present in the tissue of all animals, with the same labeling profile. The main uterine source of Faslg comes from the embryo during pregnancy [66]. Therefore the amount of this ligand produced in the uterus is low and difficult to detect [66]. The difference in expression observed between other genes and proteins, may be due to the post-transcriptional and/or post-translational modifica- tions which can modulate the concentration of the protein into the cells. But it also may be due to the technical limitations of detecting difference in protein expression in the tissue. Although the analysis of gene expression showed up regulation of 13 pro-apoptotic genes, we could also observe up regulation of important anti-apoptotic genes like Api5, Aven and Bcl2 [67–70] after melatonin treatment. These genes may justify the results of DNA fragmentation through the TUNEL analysis. Consequently, our data suggest that melatonin influences the mechanism of apop- tosis. This hormone also decreases the number of cells with DNA fragmentation in the uterus’ of rats exposed to con- tinuous light. However, further cell culture studies are necessary to investigate the exact melatonin action mech- anisms on the uterus, specially through MT1 and MT2 receptors. Acknowledgments We would like to thank Laboratory of Struc- tural and Molecular Gynecology (LIM - 58), Departamento de Ginecologia e Obstetrı́cia - Faculdade de Medicina da Universidade de São Paulo and Laboratory of Molecular Pathology, Universidade Federal de São Paulo. This work was supported by São Paulo Research Foundation (FAPESP - Process number: 09/51754-0) and National Council of Technological and Scientific Development (CNPq - Process Number: 137442/2009-2). We also thank Priscilla C. Addios for technical support. Fig. 3 Photomicrograph of rat uterus from GContr (a) and GExp (b) groups. Hemmatoxilin–eosin staining. Note zones of pseudostrar- tification with the simple cylindrical epithelium of the entometrium (arrows). c (Gcontr) and d (GExp): TUNEL-positive cells stained in dark brown (arrows) (Color figure online) 160 Apoptosis (2016) 21:155–162 123 Compliance with ethical standards Conflict of interest The authors declare no conflict of interest. References 1. Reiter RJ (1993) The melatonin rhythm: both a clock and a calendar. Experientia 49:654–664 2. Ganguly S, Coon SL, Klein DC (2002) Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation. Cell Tissue Res 309:127–137 3. Macchi MM, Bruce JN (2004) Human pineal physiology and functional significance of melatonin. Front Neuroendocrinol 25:177–195 4. Zawilska JB, Skene DJ, Arendt J (2009) Physiology and phar- macology of melatonin in relation to biological rhythms. Phar- macol Rep 61:383–410 5. Arendt J, Skene DJ (2005) Melatonin as a chronobiotic. Sleep Med Rev 9:25–39 6. Revel FG, Masson-Pevet M, Pevet P, Mikkelsen JD, Simonneaux V (2009) Melatonin controls seasonal breeding by a network of hypothalamic targets. Neuroendocrinology 90:1–14 7. Carrillo-Vico A, Lardone PJ, Naji L et al (2005) Beneficial pleiotropic actions of melatonin in an experimental model of septic shock in mice: regulation of pro-/anti-inflammatory cyto- kine network, protection against oxidative damage and anti- apoptotic effects. J Pineal Res 39:400–408 8. Claustrat B, Brun J, Chazot G (2005) The basic physiology and pathophysiology of melatonin. Sleep Med Rev 9:11–24 9. Hardeland R, Pandi-Perumal SR, Cardinali DP (2006) Melatonin. Int J Biochem Cell Biol 38:313–316 10. Radogna F, Paternoster L, Albertini MC et al (2006) Melatonin as an apoptosis antagonist. Ann NY Acad Sci 1090:226–233 11. Sarlak G, Jenwitheesuk A, Chetsawang B, Govitrapong P (2013) Effects of melatonin on nervous system aging: neurogenesis and neurodegeneration. J Pharmacol Sci 123:9–24 12. Vanecek J (1998) Cellular mechanisms of melatonin action. Physiol Rev 78:687–721 13. Clemens JW, Jarzynka MJ, Witt-Enderby PA (2001) Down-reg- ulation of mt1 melatonin receptors in rat ovary following estro- gen exposure. Life Sci 69:27–35 14. Ekmekcioglu C (2006) Melatonin receptors in humans: biological role and clinical relevance. Biomed Pharmacother 60:97–108 15. Tamura H, Takasaki A, Taketani T et al (2014) Melatonin and female reproduction. J Obstet Gynaecol Res 40:1–11 16. Soares JM Jr, Masana MI, Ersahin C, Dubocovich ML (2003) Functional melatonin receptors in rat ovaries at various stages of the estrous cycle. J Pharmacol Exp Ther 306:694–702 17. Itoh MT, Hosaka T, Takahashi N, Ishizuka B (2006) Expression of luteinizing hormone/chorionic gonadotropin receptor in the rat pineal gland. J Pineal Res 41:35–41 18. Bondi CD, Alonso-Gonzalez C, Clafshenkel WP et al (2014) The effect of estradiol, progesterone, and melatonin on estrous cycling and ovarian aromatase expression in intact female mice. Eur J Obstet Gynecol Reprod Biol 174:80–85 19. Wideman CH, Murphy HM (2009) Constant light induces alter- ations in melatonin levels, food intake, feed efficiency, visceral adiposity, and circadian rhythms in rats. Nutr Neurosci 12:233–240 20. Acuna-Castroviejo D, Fernandez B, Castillo JL, del Aguila CM (1993) Similarity between the effects of suprachiasmatic nuclei lesions and of pinealectomy on gonadotropin release in ovariec- tomized, sulpiride-treated and melatonin-replaced rats. Experi- entia 49:797–801 21. Prata Lima MF, Baracat EC, Simoes MJ (2004) Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: similarity with polycystic ovary syndrome. Braz J Med Biol Res 37:987–995 22. Dair EL, Simoes RS, Simoes MJ et al (2008) Effects of melatonin on the endometrial morphology and embryo implantation in rats. Fertil Steril 89:1299–1305 23. Dardes RC, Baracat EC, Simões MJ (2000) Modulation of estrous cycle and LH, FSH and melatonin levels by pinealectomy and sham-pinealectomy in female rats. Prog Neuropsychopharmacol Biol Psychiatry 24:441–453 24. Rodriguez C, Martin V, Herrera F et al (2013) Mechanisms involved in the pro-apoptotic effect of melatonin in cancer cells. Int J Mol Sci 14:6597–6613 25. Bizzarri M, Proietti S, Cucina A, Reiter RJ (2013) Molecular mechanisms of the pro-apoptotic actions of melatonin in cancer: a review. Expert Opin Ther Targets 17:1483–1496 26. Sainz RM, Mayo JC, Rodriguez C, Tan DX, Lopez-Burillo S, Reiter RJ (2003)Melatonin and cell death: differential actions on apoptosis in normal and cancer cells. Cell Mol Life Sci 60:1407–1426 27. Pieri C, Marra M, Moroni F, Recchioni R, Marcheselli F (1994) Melatonin: a peroxyl radical scavenger more effective than vitamin E. Life Sci 55:Pl271–Pl276 28. Barlow-Walden LR, Reiter RJ, Abe M et al (1995) Melatonin stimulates brain glutathione peroxidase activity. Neurochem Int 26:497–502 29. Nava M, Romero F, Quiroz Y, Parra G, Bonet L, Rodriguez- Iturbe B (2000) Melatonin attenuates acute renal failure and oxidative stress induced by mercuric chloride in rats. Am J Physiol Renal Physiol 279:F910–918 30. Hoijman E, Rocha Viegas L, Keller Sarmiento MI, Rosenstein RE, Pecci A (2004) Involvement of Bax protein in the prevention of glucocorticoid-induced thymocytes apoptosis by melatonin. Endocrinology 145:418–425 31. Baydas G, Koz ST, Tuzcu M, Etem E, Nedzvetsky VS (2007) Melatonin inhibits oxidative stress and apoptosis in fetal brains of hyperhomocysteinemic rat dams. J Pineal Res 43:225–231 32. Molpeceres V, Mauriz JL, Garcia-Mediavilla MV, Gonzalez P, Barrio JP, Gonzalez-Gallego J (2007) Melatonin is able to reduce the apoptotic liver changes induced by aging via inhibition of the intrinsic pathway of apoptosis. J Gerontol A Biol Sci Med Sci 62:687–695 33. Maestroni GJ (1993) The immunoneuroendocrine role of mela- tonin. J Pineal Res 14:1–10 34. Mediavilla MD, Cos S, Sanchez-Barcelo EJ (1999) Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro. Life Sci 65:415–420 35. Pedreanez A, Rincon J, Romero M, Viera N, Mosquera J (2004) Melatonin decreases apoptosis and expression of apoptosis-as- sociated proteins in acute puromycin aminonucleoside nephrosis. Nephrol Dial Transpl 19:1098–1105 36. Voznesenskaya T, Makogon N, Bryzgina T, Sukhina V, Grushka N, Alexeyeva I (2007) Melatonin protects against experimental immune ovarian failure in mice. Reprod Biol 7:207–220 37. Wang SJ, Liu WJ, Wu CJ et al (2012) Melatonin suppresses apoptosis and stimulates progesterone production by bovine granulosa cells via its receptors (MT1 and MT2). Theriogenology 78:1517–1526 38. Barrezueta LF, Oshima CT, Lima FO et al (2010) The intrinsic apoptotic signaling pathway in gastric adenocarcinomas of Brazilian patients: immunoexpression of the Bcl-2 family (Bcl-2, Bcl-x, Bak, Bax, Bad) determined by tissue microarray analysis. Mol Med Rep 3:261–267 39. Paiotti AP, Ribeiro DA, Silva RM et al (2012) Effect of COX-2 inhibitor lumiracoxib and the TNF-alpha antagonist etanercept on TNBS-induced colitis in Wistar rats. J Mol Histol 43:307–317 Apoptosis (2016) 21:155–162 161 123