Evaluation of the effect of exogenous melatonin on the placenta and inflammatory cytokines in rats submitted to chronic alcohol consumptionAbstract
Global alcohol consumption by pregnant women is approximately 9.8%. This consumption can hinder the interaction of the placenta with the fetus and increase levels of oxidative stress by altering and deregulating the expression of cytokines, leading to complications such as slow intrauterine development, such as low birth weight, premature babies and even stillbirths. It is known that melatonin is a natural antioxidant that acts mainly on the liver and also on various organs such as the ovary, placenta and the fetus itself. Melatonin can freely cross the placenta and exert effects on the mother and fetus. The present work aimed to analyze the effects of exogenous administration of melatonin during pregnancy on the placenta of rats subjected to chronic alcohol consumption. 30 albino rats were used, divided into 3 groups: Control – 10 pregnant rats that will not receive alcohol; Alcohol – 10 pregnant rats subjected to chronic alcohol consumption; Alcohol + Melatonin - 10 pregnant rats subjected to alcohol consumption associated with melatonin. Alcohol was administered at a dose of 3g/kg by gavage, while melatonin was administered at night at a dose of 0.8mg/kg intraperitoneally. The placentas were weighed and analyzed histopathologically, morphometrically and immunohistochemically. The results showed a significant reduction in placental weight in the alcohol group, disorganization in the labyrinthine zone and reduction of maternal and fetal vessels in the alcohol group. There was strong staining for TNF α and VEGF factors in the alcohol group. And as for PCNA, the control group and the alcohol + melatonin group showed the highest percentages of positive cells. And in relation to apoptosis, there was strong staining in the junctional zone of the placentas of females in the alcohol group. Therefore, it is concluded that melatonin has the potential to protect against the oxidative damage of ethanol in placental cells and can also regulate the generation of inflammatory cytokines.
Keywords:
placenta; inflammatory cytokines; alcohol; antioxidant; oxidative stress
Resumo
O consumo mundial de álcool por mulheres gestantes é de aproximadamente 9,8%. Esse consumo pode dificultar a interação da placenta com o feto, e aumentar os níveis de estresse oxidativo alterando e desregulando a expressão de citocinas, levando a complicações, como desenvolvimento intrauterino lento, como baixo peso ao nascer, bebês prematuros e até mesmo nascidos mortos. Sabe-se que a melatonina é um antioxidante natural que atua principalmente no fígado e também em diversos órgãos como o ovário, placenta e o próprio feto. A melatonina pode atravessar livremente a placenta e exercer efeitos na mãe e no feto. O presente trabalho teve como objetivo analisar os efeitos da administração exógena de melatonina durante a gestação sobre a placenta de ratas submetidas ao consumo crônico de álcool. Foram utilizadas 30 ratas albinas divididas em 3 grupos: Controle – 10 ratas prenhes que não receberão álcool; Álcool – 10 ratas prenhes submetidas ao consumo crônico de álcool; Álcool + Melatonina - 10 ratas gestantes submetidas ao consumo de álcool associado à melatonina. O álcool foi administrado na dose de 3g/kg por gavagem, enquanto a melatonina foi administrada à noite na dose de 0,8mg/kg por via intraperitoneal. As placentas foram pesadas e analisadas histopatologicamente, morfometricamente e imuno-histoquimicamente. Os resultados mostraram redução significativa do peso placentário no grupo álcool, desorganização na zona labiríntica e redução dos vasos maternos e fetais no grupo álcool. Ocorreu forte coloração para os fatores TNF α e VEGF no grupo álcool. E quanto ao PCNA, o grupo controle e o grupo álcool + melatonina apresentaram os maiores percentuais de células positivas. E em relação à apoptose, houve forte marcação na zona juncional das placentas das fêmeas do grupo álcool. Portanto, conclui-se que a melatonina tem potencial de ação protetora sobre o dano oxidativo do etanol nas células placentárias e também pode regular a geração de citocinas inflamatórias.
Palavras-chave:
placenta; citocinas inflamatórias; álcool; antioxidante; estresse oxidativo
1. Introduction
Alcohol consumption during pregnancy plays a strong and significant role in reducing offspring development (Disney et al., 2008). There are differences in the percentage of alcohol consumption by pregnant women around the world, in the United Kingdom 28.5%, in Russia 26.5%, in Switzerland 20.9%, in Serbia 15.4% and in Italy 18.2% % of women say they drank some percentage of alcohol during pregnancy (Mardby et al., 2017). The global average prevalence of alcohol consumption by pregnant women is approximately 9.8%. And in Brazil the percentage is higher than the world average at 15.2% (Popova et al., 2017).
This consumption can develop several problems that hinder the interaction of the placenta with the developing fetus, and increase levels of oxidative stress (Costa, 2017). Furthermore,
[…] in chronic alcohol users, levels of Interleukin 1 (IL-1), Interleukin 6 (IL-6) and Tumor Necrosis Factor Alpha (TNF α) increase significantly (Ahluwalia et al., 2000). And when cytokine expression is dysregulated, fetal and placental development pathways are disrupted (Yockey and Iwasaki, 2018), leading to complications during pregnancy, such as slow intrauterine development, and difficulty in childbirth, such as low birth weight, babies premature babies and even stillbirths (Popova et al., 2017).
It is known that melatonin (N-acetyl-5-methoxytryptamine) is a natural antioxidant that acts mainly in the liver, contributing to the reduction of oxidative stress and also in various organs such as the ovary, placenta and the fetus itself (Reiter et al., 2014a). Melatonin can freely cross the placenta and exert similar effects and levels in the mother and fetus (Reiter et al., 2014b). However, it is not yet possible to say whether exogenous melatonin administered during pregnancy can act on the cytokines produced by the placenta and, thus, prevent damage caused by chronic alcohol consumption. Therefore, this study aimed to evaluate morphometrically, histologically and immunohistochemically (IL6, TNF-ɑ, PCNA, VEGF and Apoptotic Index) the effect of exogenous melatonin administered during pregnancy for possible prevention of the harmful effects produced by alcohol on the placenta of rats.
2. Material and Methods
2.1. Animals and experimental groups
This experiment was carried out at the Laboratory of Morphological Studies of Vertebrates and Invertebrates (LABEMOVI) of the Department of Animal Morphology and Physiology (DMFA) of the Federal Rural University of Pernambuco (UFRPE) with approval from the Institutional Ethics Committee (CEUA) under number 5329121120. Thirty albino rats (Rattus norvegicus albinus), weighing approximately 200 ± 30 g, of the Wistar strain, with 90 days of age, coming from the vivarium of DMFA, UFRPE. The animals were kept in cages with food and water ad libitum, remaining under standard temperature conditions of 22 ± 1 °C and artificial lighting with a photoperiod of 12 hours light and 12 hours dark. Treatments with alcohol and melatonin were started on the first day of pregnancy confirmation and were administered until the twentieth day of pregnancy and the day of euthanasia. Females that presented three consecutive regular estrous cycles were selected for the experiment and divided into 10 rats per group as follows: Control Group: rats that received no alcohol and no treatment; Alcohol Group: rats submitted only to alcohol ingestion; Alcohol + Melatonin Group: rats submitted to alcohol consumption and treated with melatonin simultaneously.
2.2. Mating of animals
Females were submitted to mating always at the beginning of the night (18:00h), when they are more biologically active. They were placed in the cage at a ratio of one male for every three females. The following day, vaginal Pap smears were performed on the rats to confirm mating.
2.3. Colpocytological exam
In the early morning, to collect the vaginal material from the rats, cotton swabs moistened in saline were used and introduced into the animal's vagina with rotating movements. Only then, the collected material was transferred to the histological slide, also from rotating movements of the rod over the slides. These were submitted to staining with Toluidine Blue for 3 seconds and observed through a light microscope, brand OLYMPUS BX-49 for analysis of the presence or absence of sperm. This being, if confirmed, considered as the first day of pregnancy.
2.4. Ethanol administration
It was administered by gavage, at a dosage of 3 g/kg of ethyl alcohol in rats from the first day of pregnancy (Marco et al., 2017) diluted in distilled water.
2.5. Treatment with melatonin
Melatonin, N-acetyl-5-methoxytryptamine (Sigma Chemical Co., St. Louis, USA) was administered with daily injections of 0.8 mg/kg throughout pregnancy. This was dissolved in 0.2 mL of ethanol and diluted in 0.8 mL of 0.9% NaCl. The injection was applied intraperitoneally, always from 18:00h to 19:00h. (Abd-Allah et al., 2003; Paget and Barne, 1964). This dose is comparable to the human dose (9 mg/kg), which was converted based on body surface area.
2.6. Euthanasia
After 20 days of treatment, the animals were anesthetized with ketamine hydrochloride (80 mg/kg) and xylazine (6 mg/kg), intramuscularly and euthanized by deepening anesthetic. Placentas were removed from the opening of the abdominal cavity and fixed in 10% buffered formalin solution for 48 hours.
2.7. Histopathology
After euthanasia and fixation, the material was dehydrated in ethyl alcohol, diaphanized by xylol and impregnated in paraffin for inclusion and subsequent cuts in the microtome adjusted to 5 µm per section. The sections were placed on histological slides, and these underwent the Hematoxylin-Eosin (HE) staining procedure, and analyzed under a light microscope, OLYMPUS BX-49, and photographed under an OLYMPUS BX-50 microscope.
2.8. Morphometric analysis of the placenta
Ten slides from each of the three groups were used and the placental disc cells in the labyrinth, trophospongi, spongiotrophoblast and giant trophoblastic cells were analyzed. The morphometry of points was analyzed by the quantification, through the graticule of 110 points, of the structures and cells, which were classified in: (1) Maternal vascularization; (2) Small trophoblastic cells - undifferentiated (in contact with fetal vessels); (3) Intermediate cells (in contact with maternal vessels); (4) Giant trophoblastic (binucleated) cells; (5) Syncytial cells (near the spongioblast region) and (6) Mesenchyme, in the spongioblast region in the trophospongi region, and in the labyrinth region in: (1) Syncytial trophoblast; (2) Fetal vessel wall; (3) Lumen of fetal vessels and (4) Maternal blood space Labyrinthine vascular bed (maternal vessel), being analyzed in the 40X objective, randomly chosen 15 fields per placental region (Lemos et al., 2014).
2.9. Immunohistochemical analysis (TNF-α, VEGF, PCNA and apoptosis)
Antibodies TNFα (sc-33639, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF-A (MBS2540134, MyBioSource) and PCNA (Santa Cruz Biotechnology) were used, all at a dilution ratio of 1:100. were deparaffinized and dehydrated in xylene and alcohols, respectively. Antigen retrieval was performed using citrate buffer solution pH 8.0 at high temperature in the microwave for 5 minutes. Endogenous peroxidase was inhibited by a solution of hydrogen peroxide (3%) in methanol. The nonspecific antigen-antibody reaction was blocked by incubating the slides in PBS and 5% bovine serum albumin (BSA) for 1 hour and diluted in PBS/1% BSA. Subsequently, the sections were treated with Histofine® (Code 414191F, - Nichirei Biosciences, Tokyo, Japan) for 30 min. The antigen-antibody reaction (brown precipitate) occurred after application of 3,3 diaminobenzidine for four minutes and contrasted with hematoxylin. The images were captured using a Sony® video camera, coupled to the Olympus ® Bx50 microscope, which were submitted to the Gimp 2.0 application for quantification using RGB Histogram (Red-Green-Blue) (Oberholzer et al., 1996; Lee et al., 2001).For cell proliferation, counting was performed with a 25-point WEIBEL reticle, in a 10x eyepiece. Three slides per group were used, in which four fields in the placentas were analyzed, with a 40x objective. In each field, 300 PCNA-stained positive cells were counted and transformed into a percentage of positive cells (Weibel, 1963). For apoptosis, the TUNEL method (s7101 ApopTag Plus Peroxidation InSitu Apoptosis Detectin Kit) was used and the manufacturer's instructions were carefully followed. The apoptotic index was determined by counting the percentage of positive cells from at least 500 nuclei subdivided into 10 randomly chosen fields using a 40X objective (Wu et al., 2013).
2.10. Statistical analysis
For statistical analysis of placental weight, morphometry, immunohistochemistry with IL-6, TNF-α, VEGF, PCNA and Apoptosis, the non-parametric Kruskal -Wallis method with Dunn's post-hoc was used (P<0.05).
3. Results
The placentas with twenty days of development, from the experimental control and alcohol + melatonin groups, did not show significant histological changes, characterized by the observation of the decidua basalis region and the well-developed placental disc region, with the labyrinth zone, the most external and thicker, with the presence of maternal and fetal vessels, in addition to syncytial trophoblasts (Figures 1AC, 2AC and 3AC). In the junctional zone, also called spongioblasts or trophospongia, undifferentiated trophoblasts and giant trophoblastic cells (binucleated) were observed (Figures 3A and C).
Histology of junctional zone. (A) and (B) showing trophospongium (T) and trophoblastic giant cells (GC); (C) Trophospongia (T) and glycogen cells (Cg). HE staining.
The analysis of the placentas of females in the alcohol experimental group was characterized by presenting a poorly developed region of the decidua basalis and placental disc, with disorganization of the labyrinth zone, with a predominance of maternal vessels and syncytial trophoblasts, but rare fetal vessels (Figures 1B and 2B). In the junctional zone, undifferentiated trophoblasts, numerous clusters of glycogen cells, but absence of trophoblastic cells were observed. giants (Figure 3B).
Histology of placental disc. (A) Control; (B) Alcohol; (C) Alcohol + Mel. Note the well-developed placental disc (P) in A and C. In B poorly developed placental disc. HE staining.
Histology of the labyrinth zone. (A) Control; (B) Alcohol; (C) Alcohol + Mel. In A and C - Labyrinth zone showing maternal vessels (short arrows), presence of fetal vessels (long arrows) and syncytial trophoblast (arrowheads). In B disorganized labyrinth zone with maternal vessels and prevalence of syncytial trophoblasts. HE staining.
Regarding placental weight, there was a significant reduction in the group that received only alcohol (Figure 4). The morphometric analysis of the placentas showed alterations in the labyrinth and junctional zones. In the labyrinth, there was a reduction in maternal and fetal vessels and an increase in syncytial trophoblasts in females that received alcohol. In the junctional zone, there was a reduction in undifferentiated trophoblastic cells and giant trophoblastic cells, in addition to an increase in glycogen cells (Table 1).
Weight of the placentas. Means followed by the same letter did not differ significantly from each other by the Wilcoxon -Mann-Whitney test (p<0.05).
Percentages of the constituent elements of the Labyrinth Zones (LZ) and Junctional Zones (JZ) of the placentas of the females of the experimental groups.
Immunohistochemical analyzes revealed strong marking for the factors TNF α and VEGF in the placentas of females from the alcohol group, when compared to the placentas of females from the control and alcohol + melatonin groups, which was confirmed by the quantification of these factors (Figures 5 and 6). Regarding PCNA staining for cell proliferation, the females of the control group and the alcohol + melatonin group had the highest percentage of positive cells, significantly differing from the placentas of the females of the alcohol group, which had the lowest percentage (Figure 7). Regarding apoptosis, there was a strong marking in the junctional zone of the placentas of females in the alcohol group in relation to the others, resulting in a high apoptotic index (Figure 8).
Immunohistochemistry of TNFα in placentas. Note in A (Control) and C (Alcohol + Melatonin) weak marking. In B (Alcohol) strong marking. D) Quantification in pixels of the expression of this factor. Note a significant increase in pixels in the alcohol group in relation to the other experimental groups. Means followed by the same letter did not differ significantly from each other by the Wilcoxon-Mann-Whitney test (p<0.05).
Immunohistochemistry of VEGF in placentas. Note in A (Control) and C (Alcohol + Melatonin) weak marking. In B (Alcohol) strong marking. D) Quantification in pixels of the expression of this factor. Note a significant increase in pixels in the alcohol group in relation to the other experimental groups. Means followed by the same letter did not differ significantly from each other by the Wilcoxon-Mann-Whitney test (p<0.05).
Immunohistochemistry for PCNA in placentas. Observe in A (Control) and C (Alcohol + Melatonin) several positive cells. In B (Alcohol) few positive cells. D) Quantification in pixels of the expression of this factor. Note a significant increase in pixels in the alcohol group in relation to the other experimental groups. Means followed by the same letter did not differ significantly from each other by the Wilcoxon-Mann-Whitney test (p<0.05). JZ - Junctional Zone
Immunohistochemistry for apoptosis (TUNEL) in placentas. Note in A (Control) and C (Alcohol + Melatonin) few positive cells (arrows). In B (Alcohol) several positive cells (arrows). D) Apoptotic index. Note a significant increase in the alcohol group in relation to the other experimental groups. Means followed by the same letter do not differ significantly from each other by the Wilcoxon-Mann-Whitney test (p<0.05). JZ - Junctional Zone.
4. Discussion
Morphometric analysis revealed alterations in the labyrinthine and junctional zones in the placentas of the alcohol group, characterized by the reduction of maternal and fetal vessels, in addition to trophoblasts and giant trophoblastic cells, suggesting that these alterations may cause changes in the placental structure promoting low nutrition to the fetus (Kalisch-Smith et al., 2016). However, the administration of melatonin was able to prevent such effects, since according to Lanoix et al. (2008), this indolamine is able to promote the survival of trophoblasts, in addition to being able to upregulate the mRNA synthesis of genes for the expression of antioxidant enzymes, thus eliminating the free radicals generated by alcohol (Hannan et al., 2018).
To provide possible nutritional compensation for the fetus arising from the effect of alcohol on the placenta, there is an increase in glycogen cells, as placental glycogen stores are known to provide a source of glucose to complete fetal growth in late pregnancy (Gårdebjer et al., 2014). In our experiments, the presence of melatonin prevented the increase of these cells, probably because it allowed an adequate nutritional flow, since there was no change in the quantification of maternal and fetal vessels.
Furthermore, alcohol ingested during pregnancy can modify specific uterine vascular adaptations and modulate estrogen-induced uterine angiogenesis. Such adaptations can cause the development of tortuous vessels and ineffective for the healthy development and maintenance of pregnancy (Woods et al., 2018). In our results, there was an increase in the expression of placental VEGF, which may have had a negative effect on maternal and fetal vessels, causing disorganization and lower placental development in the alcohol group. This drop in angiogenesis is a result of the direct and indirect effects that ethanol causes in the elevation of cytokines, possibly causing placental dysfunction (Holbrook et al. 2019). However, the administration of melatonin improves blood flow, as it can normalize the expression of inflammatory cytokines, which are the main mediators of coagulation activation (Yawno et al., 2012). Therefore, melatonin induces the expression and activity of catalase and superoxide dismutase, which can prevent oxidative stress and inhibit VEGF expression, contributing to placental regulation (Valenzuela et al., 2015). We can observe such a reduction in the group treated simultaneously with alcohol + melatonin, where the levels of VEGF demonstrated similar marking to the marking of the control group.
Regarding placental weights, the alcohol group showed a reduction when compared to the other groups. This result is similar to the one presented by Gårdebjer et al. (2014), where, points to a reduction in placental weight in addition to changes in its morphology. Due to the generation of oxidative stress after alcohol ingestion, there is a change in placental blood flow, which leads to deficiencies in placental circulation (Sebastiani et al., 2018), causing a loss in obtaining nutrients and releasing excreta by the fetus (Burd et al., 2007).
Our results evidenced the elevation of TNF-α in the placental tissue of the rats of the alcohol group. TNF-α is necessary for successful implantation, and in cases of increased concentrations, abortion may occur (Zhang et al., 2016). According to Holbrook et al. (2019), there is a high and significant pattern in the levels of some cytokines, such as TNF -α, in rats that were exposed to alcohol during the prenatal period. Thus, as the findings of Ahluwalia et al. (2000), where the amount of TNF-α increased almost 20 times during pregnancy subjected to alcohol consumption. According to our results, exogenous melatonin administered to rats in the alcohol + melatonin group prevented the increase in TNF-α. According to Berbets et al. (2021), melatonin can affect the activity of inflammatory mediators involved in pregnancy. This hormone exerts its anti-inflammatory effects by regulating pro- and anti-inflammatory cytokines in different situations (Zhang et al., 2020), as already demonstrated by Haddadi and Fardid (2015), where the administration of exogenous melatonin caused a decrease in the exacerbated secretion of TNF-α.
In this work, the placentas of the alcohol group showed lower PCNA markings in relation to the other groups studied. A balance between cellular control, apoptosis and cellular differentiation is necessary for the correct development of the placenta. Furthermore, human cytotrophoblasts undergo apoptosis when exposed to ethanol (Kalisch-Smith et al., 2016). In studies, Shanmugam et al. (2019), found that exposure to alcohol caused a reduction in the expression pattern of PCNA, and thus, a reduction in the growth of human trophoblasts. This may also explain the reduction of undifferentiated trophoblasts and trophoblastic giant cells in the junctional zone of the alcohol group placentas observed in our work. While our results of PCNA levels of the alcohol + melatonin group do not differ statistically from the results of the control group, as melatonin acts broadly as a potent antioxidant and has anti-inflammatory properties (Paradies et al., 2010).
It is known that cytotrophoblastic cells exhibit an increase in apoptosis levels after alcohol exposure (Bolnick et al., 2014). Therefore, oxidative stress can result in the phosphorylation and accumulation of the p53 protein, which can result in cellular apoptosis and impairment in embryonic invasion and placental proliferation (Fortis et al., 2018). As we can see in our results from the alcohol group, where there was strong marking in the junctional zone of the placentas, resulting in a high apoptotic index when compared to the control group and alcohol + melatonin group. Melatonin treatment may have prevented cell apoptosis after alcohol exposure. Lanoix et al. (2013) state that melatonin can reduce apoptosis because it acts on mitochondria through pathways activated by oxidative stress, being able to reduce the loss of villous cytotrophoblasts. Thus, it can act in the maintenance and/or restoration of placental activity.
5. Conclusion
In view of the above, we conclude that melatonin has a high protective potential on placental cells, regulating the expression of inflammatory cytokines. Furthermore, unlike the results observed in the alcohol-treated group, melatonin was able to ensure placental structure, attenuate fetal weight loss, conserve vascular endothelial growth and apoptotic indices, and also maintain cell proliferation. Being an important antioxidant for the body and for a range of pathologies caused by the high generation of free radicals.
Acknowledgements
The authors would like to thank UFRPE for the link, the laboratory of morphological studies of vertebrates and invertebrates and the vivarium of the Department of Animal Morphology and Physiology for providing the space to carry out the work And I would like to thank FACEPE for the financial support, which was essential for the accomplishment of this work.
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