Modular effect of melatonin on diabetic heart disease in ratsABSTRACT
The aim of this study was to analyze the effects of melatonin administration on the hearts of rats induced with diabetes. Twenty male Wistar albino rats, 70 days old, were divided into the following groups: Control - non-diabetic rats; Diabetic; Diabetic + insulin; Diabetic + melatonin. All treatments lasted 30 days. Diabetes was induced by streptozotocin (60mg/kg ip) and melatonin (10mg/kg, ip). Insulin was administered at a dose of 5 IU/day. In the histopathological analysis, the animals in groups II and III presented significant disorganization and vacuolization of cardiomyocytes with histopathological scores above 2 (moderate to extensive degeneration of cardiomyocytes with diffuse infiltration of inflammatory cells) and score 1 (mild; degeneration of cardiomyocytes with discrete infiltration of inflammatory cells), respectively. Morphometric analysis revealed a significant increase in cardiac wall thickness, reduction of the lumen and a higher lumen/wall thickness ratio in diabetic animals, in addition to presenting greater collagen staining and elevated levels of total creatine kinase, creatine kinase MB, lactate dehydrogenase, troponin, IL-6 and TNF-α, differing statistically from the other groups. These effects were prevented by melatonin. We conclude that melatonin has great potential as an adjuvant in preventing the development of diabetic cardiomyopathy in rats.
Keywords:
antioxidant; hyperglycemia; heart; cytokines; cardiac injury markers
RESUMO
O objetivo desta pesquisa foi analisar os efeitos da administração de melatonina no coração de ratos induzidos ao diabetes. Foram utilizados 20 ratos albinos Wistar, machos, com 70 dias de idade, divididos nos grupos: controle - ratos não diabéticos; diabéticos; diabéticos + insulina; diabéticos + melatonina. Todos os tratamentos duraram 30 dias. O diabetes foi induzido por estreptozotocina (60mg/kg ip) e melatonina (10mg/kg ip). A insulina foi administrada na dose de 5UI/dia. Na análise histopatológica, os animais dos grupos II e III apresentaram significativa desorganização e vacuolização dos cardiomiócitos com escore histopatológico acima de 2 (degeneração moderada a extensa dos cardiomiócitos com infiltração difusa de células inflamatórias) e escore 1 (leve; degeneração dos cardiomiócitos com infiltração discreta de células inflamatórias), respectivamente. A análise morfométrica revelou, nos animais diabéticos, aumento significativo da espessura da parede cardíaca, redução do lúmen e maior relação lúmen/espessura da parede, além de apresentar maior marcação para colágeno e níveis elevados de creatina quinase total, creatina quinase MB, lactato desidrogenase, troponina, IL-6 e TNF-α, diferindo estatisticamente dos demais grupos. Esses efeitos foram evitados pela melatonina. Conclui-se que a melatonina tem grande potencial coadjuvante na prevenção do desenvolvimento de cardiomiopatia diabética em ratos.
Palavras-chave:
antioxidante; hiperglicemia; coração; citocinas; marcadores de lesão cardíaca
INTRODUCTION
Diabetes mellitus is the most widespread metabolic disease worldwide, often accompanied by a wide range of cardiovascular complications or heart diseases, being the most prevalent cause of morbidity and mortality in diabetics, due to left ventricular hypertrophy, accelerated atherosclerosis, fibrillation atrial, diastolic and systolic dysfunction, heart failure, coronary artery disease and myocardial infarction (Zheng et al., 2018).
In addition to diabetic heart complications being more frequent in individuals with type II diabetes, we cannot ignore the fact that individuals with type I diabetes with poor glycemic control are at high risk of developing them, with possible progression to heart failure. Generally, individuals with type I diabetes are younger at the onset of the disease, resulting in the loss of more years of life due to cardiovascular disease than those with type II diabetes. In relation to the mortality rate due to cardiovascular diseases, young adults with type I diabetes are between eight and forty times more likely than the general population. Furthermore, both types of diabetes lead to serious cardiovascular complications, such as functional changes and structures of the heart that make it more susceptible to stress.
It is also known that diabetes is a chronic inflammation and an important pathophysiological factor in increasing the levels of pro-inflammatory cytokines in the body such as: Tumor Necrosis Factor-α (TNF-α), Interleukin IL-1β and IL-6, demonstrated in patients with metabolic syndrome and insulin-resistant patients. In animal studies, it was observed that systemic inflammation may occur during DM, such as an increase in tumor necrosis factor-α (TNFα), interleukins (IL-1β, IL-6), transforming growth factor-β (TGF-β), interferon-γ (IFNγ), among others (Ritchie and Abel, 2020).
Regarding the treatment of heart disease with insulin, the literature reports that the administration of this hormone can cause frequent hypoglycemic events, which can be dangerous in patients with heart failure. The hypoglycemic effects of insulin cause adverse cardiovascular consequences through sympathetic activation, leading to increased heart rate and myocardial infarction. Although these data show a poor prognosis for insulin therapy, there are conflicting results about insulin therapy in individuals with heart failure (Jang et al., 2021).
Studies also report that the high glycemic index can induce oxidative stress in cardiomyocytes or fibroblasts, leading to deficits in contractile function and myocardial fibrosis in the left ventricle of diabetic rats, in addition to influencing the increase in apoptosis and myocardial inflammation and the development of heart disease. Since oxidative stress plays an important role in the pathogenesis of diabetes and can trigger diabetic heart disease, interest in the use of antioxidant agents as complementary therapy has been growing, aiming to improve redox homeostasis in the heart (Zhang et al., 2020).
Melatonin (5-methoxy-N-acetyltryptamine), an indoleamine produced and secreted mainly by the pineal gland, is a good option due to its physicochemical characteristics, as well as strong antioxidant properties, in addition to having robust potential as a treatment option, due to its low and infrequent toxicity (Abdulwahab et al., 2021). Thus, this work aimed to analyze the effects of melatonin administration on the histophysiology of the heart with emphasis on its morphometry, enzymatic analysis and inflammatory cytokines.
MATERIALS AND METHODS
The experiment was carried out at the Histology Laboratory of the Department of Morphology and Animal Physiology (DMFA) of the Federal Rural University of Pernambuco (UFRPE). Twenty male Wistar albino rats, aged 70 days, weighing approximately 250 ± 30 g, from the DMFA vivarium of UFRPE, were used. They were kept in an environment with a temperature of 22 ± 1 ºC, in a photoperiod of 12 hours of light and 12 hours of darkness, on an ad libitum diet and water intake. The experimental protocol was submitted and approved by the Institutional Ethics Committee nº: 130/2019.
The animals were divided equally and randomly into 4 groups: Control: rats without diabetes induction; Diabetic: rats induced diabetes; Diabetic + insulin: rats induced to diabetes and after confirmation of diabetes treated with insulin for 30 days: Diabetic + melatonin: rats induced to diabetes and after confirmation of diabetes treated with melatonin.
Diabetes was induced by intraperitoneal administration of streptozotocin solution (Sigma Chemical Co., USA) after a 14-hour food fast. Streptozotocin was diluted in 10 mM sodium citrate buffer, pH 4.5 at a single dosage of 60mg/kg of animal weight. Non-diabetic animals (control group) received equal doses of saline solution in the same way and after 30 minutes of administration all animals were fed normally (Labina® Purina, Brazil) (Dall'ago et al., 2002). After seven days of induction, diabetes was confirmed using the Glucometer (Accu-Chek Active Performa, Roche, Germany), where a drop of blood is placed on strips and taken to the device to measure blood glucose levels. Only animals with blood glucose above 200 mg/dL, except the control group, were considered diabetic and included in the study to start treatment with melatonin or insulin (Rehman et al., 2023).
The administration of melatonin, N-acetyl-5-methoxytryptamine (Sigma, St Louis, MO, USA) was carried out intraperitoneally always from 6:00 pm to 7:00 pm for 30 days at a dose of 10 mg/kg. For this purpose, melatonin was dissolved in ethanol (0.2 mL) and diluted in 0.9 mL 0.9% NaCl. This dose of melatonin was chosen because this dose of 10 mg is not associated with risks for the animal, whether human or rat (Xiong et al., 2018).
Insulin was administered subcutaneously for 30 days at a dose of 5IU (international units)/day, with two units of insulin at 10 am and the remaining three units at 7 pm (Pinheiro et al., 2011).
On the 30th day of the experiment (day of euthanasia), blood was collected by cardiac puncture, centrifuged and the serum stored in a freezer at -20ºC for subsequent biochemical analyses.
To collect the heart, the rats were anesthetized with ketamine hydrochloride (80mg/kg) and xylazine (6.0 mg/kg) intramuscularly, associated with thiopental (100 mg/kg) intraperitoneally. After opening the abdominal cavity, the heart was collected and placed in a container with 10% buffered formaldehyde at pH 7.4 for fixation, remaining there for 48 h. For histological and histochemical analysis, heart fragments were dehydrated in ethyl alcohol in increasing concentrations, cleared with xylene, impregnated and embedded in paraffin. The paraffin blocks were cut with a Minot -type microtome (Leica RM 2035) adjusted to 6 µm. The sections thus obtained were placed on slides previously greased with Mayer's albumin and kept in an oven set at a temperature of 37 º C, for 24 hours, for drying and gluing. Subsequently, the sections were subjected to the hematoxylin and eosin (HE) staining technique for histopathological analysis and Mallory's Trichomic to identify collagen fibers and were then analyzed and photographed under a light microscope, brand OLYMPUS BX-49 and OLYMPUS BX-50 respectively. Quantification in pixels of collagen fibers was carried out using the GIMP 2.8 program.
To analyze the histopathology, the dilation and thickness of the left ventricular wall were evaluated, using three slides per group. The following histopathological score was applied: 0 (absence of histopathological anomalies); 1 (mild; degeneration of cardiomyocytes with slight infiltration of inflammatory cells); 2 (moderate to extensive degeneration of cardiomyocytes with diffuse infiltration of inflammatory cells); and 3 (severe; necrotic tissue with massive infiltration of inflammatory cells) (Saber et al., 2020). The degree of cardiac tissue damage was determined by examining 5 randomly chosen fields for each slide/group.
For morphometry, an eyepiece containing a micrometer reticle was used (Graticles Tonbridge, Kent, England). Three slides per group were used to estimate (I) lumen diameter, (II) thickness of the entire wall and (III) the wall/lumen ratio of the left ventricle. The lumen diameter of the sections (Di) was calculated from the equation Di = √ ab, where (a) is the major axis and (b) the minor axis, at right angles to the major axis of the lumen (Williams, 1977).
At the end of the experimental protocol, the following parameters were measured in serum: Lactate Dehydrogenase (LDH), Creatine Kinase - MB (CK-MB), Total Creatine Kinase (CK) by kinetic methods, and Troponin I (cTnI) by immunochromatographic method, all with Bioclin commercial kit (Pinto et al., 2010). The test was performed by adding 100 µL of serum to the center of the cassette application window. After this step, readings were taken between 15 and 30 minutes after application. The test was considered positive when the formation of two lines was observed, one in the control region (C) and the other in the test region (T), indicating the presence of cTnI in the samples. The test was considered negative when only one line was formed in the control region (C) and there was complete absence in the test region (T). The relative CK-MB index was also calculated, according to the formula recommended by Capellan et al. (2003).
For immunohistochemical analysis, the salinized slides with 6 µm heart sections were deparaffinized and dehydrated in xylene and alcohol respectively and subjected to antigen retrieval in citrate buffer (pH 8.0) in the microwave for 5 minutes. Endogenous peroxidase was inhibited using 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 one hour. They were then treated with primary antibodies IL-6 (sc-28343 - Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and TNFα (sc-33639 - Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in a ratio of 1:50, for one hour and then with N- Histofine as a secondary antibody for thirty minutes. All antibodies were diluted in 1% PBS/BSA. The antigen-antibody reaction was observed through a brown precipitate after application of DAB (3,3 diaminobenzidine) for four minutes and stained with hematoxylin. Two slides from each group were used, where four fields/slide were photographed to measure the markings.
Immunohistochemistry 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), which is based in luminescence intensity where the tones of the image pixels vary from 0 to 255, with tone 0 representing absolute darkness (lowest luminescence), while tone 255 represents absolute white (highest luminescence).
The data on histopathological scoring, morphometry, quantification of collagen fibers, quantification of cardiac injury markers and the expression of the inflammatory cytokines IL-6 and TNFα were subjected to Analysis of Variance, when significant this was complemented by the Multiple Comparisons test of Tukey and Kramer (p < 0.05).
RESULTS
Histopathological analysis of the left ventricle of animals in the control group showed a normal architecture with well-organized cardiomyocytes, long cylindrical cells with a centralized nucleus (Fig. 1A). In the diabetic group, cardiomyocytes showed significant disorganization, in addition to the presence of several cytoplasmic vacuoles (Fig. 1B). These same findings were verified in the ventricles of animals in the diabetic + insulin group (Fig. 1C). Melatonin treatment prevented such effects (Fig. 1D).
The diabetic group presented the highest histopathological score with a score above 2 (moderate to extensive degeneration of cardiomyocytes with diffuse infiltration of inflammatory cells), followed by the diabetic + insulin group with a score of 1 (mild; degeneration of cardiomyocytes with mild infiltration of inflammatory cells). The control group and the diabetic + melatonin group showed a score close to 0 (absence of histopathological abnormalities), not differing from each other (Fig. 2).
Morphometric analysis of the left ventricle of animals in the diabetic group showed increased wall thickness, reduced lumen and increased wall/lumen thickness ratio compared to the control group and the one treated with melatonin. Insulin treatment promoted intermediate values between these groups (Table 1).
The histochemical analysis for collagen fibers revealed greater staining in the ventricle of animals in the diabetic group in relation to the other experimental groups, which was confirmed by quantification in pixels (Fig. 3).
Left ventricle of rats in the experimental groups. A - Control; B - Diabetic; C - Diabetic + insulin; D - Diabetic + melatonin. Note in B and C cardiomyocytes presenting a disorganized structure and with cytoplasmic vacuoles (arrows), while in A and D the cardiac tissue is organized and preserved. HE.
Histopathological score of the left ventricle of rats in the experimental groups Control; Diabetes; Diabetic + insulin and Diabetic + melatonin. Note lower scores in the control group and those treated with melatonin, while the diabetic group showed higher scores, followed by the group treated with insulin. Means followed by the same letter do not differ significantly from each other using the Tukey and Kramer Multiple Comparisons test (p<0.05).
Histochemistry for total collagen in the left ventricle of rats in the experimental groups. A - Control; B - Diabetic; C - Diabetic + insulin; D - Diabetic + melatonin. Note in B greater staining between cardiomyocytes. Arrows - collagen fibers. Mallory's trichrome. E - Quantification in pixels of total collagen. Verify significant increase in the diabetic group. Means followed by the same letter do not differ significantly from each other using the Tukey and Kramer Multiple Comparisons test (p>0.05).
Diabetes produced significant increases in total CK, CK-MB, relative CK-MB index and LDH levels compared to the control, diabetic + insulin and diabetic + melatonin groups. Furthermore, in relation to cTnI, the test was reactive for the diabetic group, as two lines formed, one in the control region (C) and another more intense in the test region (T) between 15 and 30 minutes, indicating an increase in cTnI in the sample. For the control and diabetic + melatonin groups, the tests were not reactive, as there was only the formation of a line in the control region (C) and complete absence in the test region (T) between 15 and 30 minutes. In the diabetic + insulin group, the tests were not reactive, as a line formed in the control region (C) and another very clear line in the test region (T) in the interval of 15 to 30 minutes (Table 2).
Immunohistochemical analysis revealed greater staining of the cytokines IL-6 and TNFα in the ventricle of animals in the diabetic group compared to the other experimental groups, which was confirmed by quantification in pixels (Fig. 4 and 5).
Immunohistochemistry for IL-6 in the left ventricle of rats in the experimental groups. A - Control; B - Diabetic; C - Diabetic + insulin; D - Diabetic + melatonin. Note in B greater staining in cardiomyocytes. E - Quantification in pixels of IL-6. Verify significant increase in the diabetic group. Means followed by the same letter do not differ significantly from each other using the Tukey and Kramer Multiple Comparisons test (p<0.05).
Immunohistochemistry for TNF-α in the left ventricle of rats in the experimental groups. A - Control; B - Diabetic; C - Diabetic + insulin; D - Diabetic + melatonin. Note in B greater staining in cardiomyocytes. E - Quantification in pixels of IL-6. Verify significant increase in the diabetic group. Means followed by the same letter do not differ significantly from each other using the Tukey and Kramer Multiple Comparisons test (p>0.05).
DISCUSSION
According to the literature, heart damage observed in diabetes is strongly linked to oxidative stress resulting from hyperglycemia, which increases the production of free radicals such as reactive oxygen species, promoting cellular degeneration and fibrosis that leads to stiffness and reduces the elasticity of the wall (Tong et al., 2019 ).
Histopathological changes in the left ventricle of diabetic rats probably caused an increase in wall thickness, a reduction in the lumen and an increase in the wall/lumen thickness ratio. In fact, ventricular hypertrophy is directly linked to oxidative stress which, through molecular mechanisms, leads to the transient activation of genes that encode transcription factors such as c-jun and c-fos, which are upregulated in type I diabetes (Huynh et al., 2014).
Treatment with melatonin prevented the effects observed in the histopathology and morphometry of the diabetic group. The literature reports that melatonin receptors MTR1 and MTR2 are present in the human cardiovascular system and in rats (Sung et al., 2018). Studies have demonstrated the ability of melatonin to reduce damage to cardiac tissue resulting from oxidative stress, by reestablishing antioxidant enzymes, leading to cardioprotective effects on left ventricular remodeling in patients after acute myocardial infarction. It is assumed that the effect of melatonin on the heart is directly in the regulation of mitochondrial activity, as in diabetes, it not only generates reactive oxygen and nitrogen species, but is also the main target of this pathology (Wang et al., 2021).
Biochemical analysis in animals in the diabetic group revealed a significant increase in the levels of total CK, CK-MB, the CK-MB/total CK ratio, LDH and cTnI. Therefore, we can infer that these increased levels suggest heart muscle damage and ventricular dysfunction. On the other hand, melatonin treatment prevented the elevation of these biomarkers, demonstrating the ability of this indoleamine to protect cardiomyocytes. Nasseh et al. (2022) found that melatonin treatment regulated the levels of total CK, CK-MB, LDH and cTnI in rats with ischemia-induced myocardial injuries. Therefore, the administration of exogenous melatonin may be an alternative to maintain the integrity of the cell membrane, restricting the leakage of cardiac injury marker enzymes.
Elevated levels of TNF-α and IL-6 were observed in our study in animals in the diabetic group. These mediators are responsible for the rapid progression of diabetic cardiomyopathy (Ali et al., 2020) as they impair the capacity of cardiomyocytes through reduced the production of Ca++ ATPase of the sarcoplasmic/endoplasmic reticulum, leading to fibrosis, myocardial stiffness and diastolic dysfunction. However, we were able to verify the therapeutic potential of melatonin in inhibiting changes in the levels of the cytokines IL-6 and TNF-α in the left ventricle of the heart. According to Sadeghi et al. (2020) this effect of melatonin is related to the inhibition of NF- kB activity in patients with heart failure and comorbidities induced by diabetes.
In our study, insulin administration was not able to attenuate the damage observed in the histopathological analysis, although it was effective in the other parameters analyzed. Thus, glycemic control by insulin helps to reduce the progression of diabetic complications, however, it is not sufficient to reduce mortality from diabetic cardiomyopathy (Dunlay et al., 2019), as complications in the heart are mainly caused by oxidative stress.
CONCLUSION
Therefore, we can conclude that melatonin has great therapeutic potential in preventing the development of diabetic heart disease in rats. This hormone showed superior effects to insulin in protecting against cardiac structural changes and was equivalent to it in terms of effects on collagen deposition, markers of cardiac injury and expression of the pro-inflammatory cytokines IL-6 and TNF-α in affected cardiac tissue. by diabetes. However, it is necessary to carry out more research to better understand and elucidate the effects of this hormone on diabetic cardiomyopathy.
ACKNOWLEDGMENT
I would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financing this research through the granting of my master's scholarship.
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Publication Dates
-
Publication in this collection
28 Apr 2025 -
Date of issue
May-Jun 2025
History
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Received
14 May 2024 -
Accepted
04 Nov 2024
