Melatonin Receptor Expression Research Articles

6793 The Potential Of Melatonin As A Treatment Option For Cushing’s Syndrome In Humans Based On Observations In Cushingoid Dogs

Abstract Disclosure: K. Fecteau: None. It is known that the secretion of cortisol and melatonin is circadian, with the former increasing during morning hours and the latter increasing during the dark hours of night. Studies investigating melatonin concentrations in patients with Cushing’s syndrome have reported differing results, with some having low melatonin concentrations, and others normal or high concentrations. Interestingly, adrenal glands have been shown to express melatonin receptors, and in vitro studies with human adrenal cells have shown a decrease in cortisol, as well as other adrenocortical steroids, when treated with melatonin. In human adrenal gland explants, melatonin reportedly inhibited ACTH stimulated circadian clock genes, as well as cortisol and progesterone production. Melatonin is often used as a first-line of treatment in dogs with atypical Cushing’s, where cortisol is normal, other adrenocortical steroids are elevated, and typical clinical signs of Cushing’s are present. Retrospective data collected in our diagnostic laboratory from dogs that had at least two submissions (before & during treatment), showed 34 percent of dogs on melatonin improved clinically. Of the 34 percent, 26.4 percent experienced either similar or more aberrant steroid concentrations, and 7.6 percent experienced lower steroid concentrations. Approximately 40 percent of dogs had significantly lower androstenedione when retested on melatonin. In humans, Cushing’s disease is a rare cause of androgen excess, with one study reporting occurrence of less than 4 percent in women. Adrenocortical carcinoma is also rare, however, women with this type of tumor may exhibit clinical signs of androgen excess due to adrenal androgen hypersecretion, and up to 25 percent of women with clinical signs had normal testosterone and elevated androstenedione. Therefore, based on the effectiveness of melatonin on lowering androstenedione concentrations and improving clinical signs associated with increased androgens and glucocorticoids in dogs, it may be an inexpensive and conservative option, especially for patients presenting with clinical signs but diagnostic testing is either negative of equivocal for Cushing’s. Presentation: 6/2/2024

Melatonin Ameliorates Abnormal Sleep-Wake Behavior via Facilitating Lipid Metabolism in a Zebrafish Model of Parkinson's Disease.

Sleep-wake disorder is one of the most common nonmotor symptoms of Parkinson's disease (PD). Melatonin has the potential to improve sleep-wake disorder, but its mechanism of action is still unclear. Our data showed that melatonin only improved the motor and sleep-wake behavior of a zebrafish PD model when melatonin receptor 1 was present. Thus, we explored the underlying mechanisms by applying a rotenone model. After the PD zebrafish model was induced by 10 nmol/L rotenone, the motor and sleep-wake behavior were assessed. In situ hybridization and real-time quantitative PCR were used to detect the expression of melatonin receptors and lipid-metabolism-related genes. In the PD model, we found abnormal lipid metabolism, which was reversed by melatonin. This may be one of the main pathways for improving PD sleep-wake disorder.

Melatonin Receptor Expression in Primary Uveal Melanoma.

Melatonin, noted for its anti-cancer properties in various malignancies, including cutaneous melanoma, shows promise in Uveal melanoma (UM) treatment. This study aimed to evaluate melatonin receptor expression in primary UM and its association with UM-related mortality and prognostic factors. Immunohistochemical analysis of 47 primary UM tissues showed low expression of melatonin receptor 1A (MTNR1A) and melatonin receptor 1B (MTNR1B), with MTNR1A significantly higher in patients who succumbed to UM. Analysis of TCGA data from 80 UM patients revealed RNA expression for MTNR1A, retinoic acid-related orphan receptor alpha (RORα), and N-ribosyldihydronicotinamide:quinone oxidoreductase (NQO2), but not MTNR1B or G protein-coupled receptor 50 (GPR50). Higher MTNR1A RNA levels were observed in patients with a BRCA1 Associated Protein 1 (BAP1) mutation, and higher NQO2 RNA levels were noted in patients with the epithelioid tumor cell type. However, Kaplan-Meier analysis did not show distinct survival probabilities based on receptor expression. This study concludes that UM clinical samples express melatonin receptors, suggesting a potential mechanism for melatonin's anti-cancer effects. Despite finding higher MTNR1A expression in patients who died of UM, no survival differences were observed.

The role of melatonergic system in intervertebral disc degeneration and its association with low back pain: a clinical study.

The mechanisms of intervertebral disc degeneration (IVDD) in low back pain (LBP) patients are multiples. In this study, we attempt to investigate whether melatonergic system plays a potential role in IVDD patients with LBP by analyzing their clinical specimens. The fucus will be given to the correlation between the melatonin receptor expression and intervertebral disc tissue apoptosis. In this clinical study, 107 lumbar intervertebral disc nucleus pulposus (NP) specimens from patients with LBP were collected with patients' consents. The disc height (DH) discrepancy ratio, range of motion and sagittal parameters of the pathological plane were measured and Pfirrmann grade was used to classified the grades of IVDD level. Discs at grades 1-3 were served as normal control and grades 4-5 were considered as IVDD. The expression levels of melatonin receptor 1A (MT1) and 1B (MT2) were measured by immunohistochemistry. The apoptosis of NP was assessed using TUNEL staining. Their potential associations among MT1/2, DH, apoptosis, sagittal parameters with IVDD and LBP were evaluated with statistical analysis. The incidence of IVDD was positively associated with age and negatively related to VAS scores for LBP (p<0.001). Patients with higher degree of IVDD also have higher DH discrepancy ratio (p<0.001), higher prevalence of lumbar instability (p=0.003) and higher cell apoptosis compared to the control. Nevertheless, no statistically significant correlation was identified between Pfirrmann grade and lumbar sagittal parameters. MT1 and MT2 both were highly expressed in the NP tissues. Importantly, MT1 expression but not MT2 was significantly increased in the intervertebral disc tissue of patients with IVDD and its level correlated well with cell apoptosis level and the severity of IVDD as well as lower VAS scores for LBP. The highly elevated MT1 expression was found in NP tissues of patients with IVDD and LBP compared to the control. This phenomenon probably reflects the compensating response of the body to the pathological alteration of the IVDD and LBP. Therefore, these findings provide the novel information to use selective agonists of MT1 to target IVDD and LBP clinically.

The Role of Melatonin and Matrix Metalloproteinases in Breast Cancer Pathogenesis: A Comprehensive Review

Background: Breast cancer is a heterogeneous disease with molecular subtypes including Triple Negative Breast Cancer (TNBC), HER2/neu-positive, Luminal Type A, and Luminal Type B, each with unique clinical behaviors and therapeutic responses. This study investigates the role of melatonin, matrix metalloproteinases (MMPs), pro-inflammatory cytokines (IL6 and TNF-alpha), and lactate dehydrogenase (LDH) isoenzymes in breast cancer pathogenesis. Methods: Over three years, 171 breast cancer patients were studied. Blood and urine samples were collected to measure melatonin, MMPs, IL-6, TNF-alpha, LDH-1, and LDH-5 levels. Circulating tumor cells (CTCs) were isolated using density gradient centrifugation and immunomagnetic separation. Melatonin receptor expression on CTCs was analyzed by flow cytometry and immunocytochemistry. Plasma concentrations of melatonin were measured using high-performance liquid chromatography (HPLC), and MMPs, IL-6, TNF-alpha, LDH isoenzymes were quantified using enzyme-linked immunosorbent assays (ELISAs) and electrophoretic methods. Results: The expression of melatonin receptors was lowest in TNBC patients, higher in HER2/neu-positive and ER/PRnegative patients, and highest in Luminal Type A and B patients. TNBC patients exhibited elevated LDH-1 and inflammatory cytokines (IL-6, TNF-alpha), with significantly decreased melatonin levels. HER2/neu-positive patients showed elevated LDH5 and moderate decreases in melatonin. Luminal Type B and A patients had varying degrees of biomarker elevation and melatonin receptor expression. Discussion: The findings suggest a correlation between reduced melatonin signaling and aggressive breast cancer phenotypes, particularly TNBC. Elevated MMPs and pro-inflammatory cytokines underscore the role of inflammation and ECM degradation in cancer progression. Melatonin’s potential inhibitory effects on these pathways highlight its therapeutic promise. The combined assessment of melatonin, MMPs, cytokines, and LDH could guide personalized treatment strategies. Conclusion: This study underscores melatonin’s multifaceted role in breast cancer pathogenesis. Melatonin receptor expression, along with MMP, IL-6, TNF-alpha, and LDH levels, provides a detailed molecular landscape of breast cancer subtypes. Integrating melatonin supplementation into therapeutic regimens holds promise for improving outcomes, particularly inaggressive subtypes like TNBC. Further research and clinical trials are essential to validate these findings and develop effective melatonin-based therapies.

RETRACTION: Changes in the Expression of Melatonin Receptors Induced by Melatonin Treatment in Hepatocarcinoma HepG2 Cells.

Carbajo-Pescador S, Martín-Renedo J, García-Palomo A, Tuñón MJ, Mauriz JL, González-Gallego J. Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells. Journal of Pineal Research, 2009;47: 330-338. https://doi.org/10.1111/j.1600-079X.2009.00719.x The above article, published online on 14 October 2009 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Gianluca Tosini, and John Wiley and Sons Ltd. Following publication, concerns were raised by third parties regarding Figure 5. The authors could not provide the original data for this figure, and were unable to provide a satisfactory explanation to resolve the concerns. The retraction has been agreed because of concerns that portions of the figure were duplicated, affecting the interpretation of the data and results presented. The authors disagree with this decision.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Melatonin Alleviates BPA-Induced Testicular Apoptosis and Endoplasmic Reticulum Stress.

The impact of melatonin on bisphenol A (BPA)-induced testicular apoptosis and endoplasmic reticulum (ER) stress was explored. The mice received BPA (50 mg/kg) by gavage for 30 days while being injected with 20 mg/kg melatonin. Protein expressions were detected with western blotting. The Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay measured testicular cell apoptosis. Testosterone was quantified by employing enzyme-linked immunosorbent assay (ELISA). Melatonin promoted the development of seminiferous tubules, restored the orderly arrangement of the germ cells, and increased epithelial layers in the seminiferous tubules in BPA-treated mice. Moreover, in BPA-treated mouse testicular cells, melatonin markedly upregulated melatonin receptor 1A (MTNR1A) and melatonin Receptor 2 (MTNR2) expressions while downregulating ER molecular chaperones glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94). Furthermore, it decreased p-PERK, p-IRE1, and ATF6α, as well as the apoptotic proteins cysteine-containing aspartate-specific proteases-12 (caspase-12) and cleaved cysteine-containing aspartate-specific proteases-3 (cleaved caspase-3), causing the suppression of testicular cell apoptosis. Additionally, melatonin increased the levels of cytochrome P450 17α-hydroxylase/20-lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3), and 3β-hydroxysteroid dehydrogenase 4 (3β-HSD4), in the ER, and elevated testosterone levels in testicular tissue. Melatonin can significantly alleviate testicular apoptosis and ER stress induced by BPA, which is because of the upregulation of melatonin receptor expression in testicular cells, inhibition of ER stress-related pathways, and enhancement of testosterone synthesis.

Complex Hippocampal Response to Thermal Skin Injury and Protocols with Hyperbaric Oxygen Therapy and Filipendula ulmaria Extract in Rats.

The aim of this study was to evaluate the alterations of the hippocampal function that may be related to anxiogenic response to thermal skin injury, including the morpho-functional alterations, and the effects of hyperbaric oxygen (HBO) and Filipendula ulmaria (FU) extract in the treatment of anxiety-like behavior that coincides with thermal skin injury. A rat thermal skin injury experimental model was performed on 2-month-old male Wistar albino rats. The evaluated therapeutic protocols included HBO and/or antioxidant supplementation. HBO was applied for 7 days in the hyperbaric chamber (100% O2, 2.5 ATA, 60 min). Oral administration of FU extract (final concentration of 100 mg/kg b.w.) to achieve antioxidant supplementation was also applied for 7 days. Anxiety level was estimated in the open field and elevated plus-maze test, which was followed by anesthesia, sacrifice, and collection of hippocampal tissue samples. HBO treatment and FU supplementation significantly abolished anxiogenic response to thermal skin injury. This beneficial effect was accompanied by the reduction in hippocampal pro-inflammatory and pro-apoptotic indicators, and enhanced BDNF and GABA-ARα2S gene expression, previously observed in untreated burns. The hippocampal relative gene expression of melatonin receptors and NPY positively responded to the applied protocols, in the same manner as µ and δ opioid receptors, while the opposite response was observed for κ receptors. The results of this study provide some confirmations that adjuvant strategies, such as HBO and antioxidant supplementation, may be simultaneously applied in the treatment of the anxiety-like behavior that coincides with thermal skin injury.

Melatonin promotes progesterone secretion in sheep luteal cells by regulating autophagy via the AMPK/mTOR pathway

The corpus luteum is primarily responsible for the production and secretion of progesterone. Melatonin has been established to regulate autophagy and induce progesterone secretion in luteal cell. However, whether melatonin affects progesterone secretion by interfering with autophagy is yet to be reported. In the present study, the expression levels of melatonin receptors (MT1 and MT2), autophagy-related protein Beclin1 (Bec1), microtubule-associated protein light chain 3 B (LC3B), progesterone and steroidogenic acute regulatory protein (StAR), and cytochrome P450scc (CYP11A1) were analyzed in the corpus luteum of sheep at different stages (early, middle, and late); specifically, enzyme-linked immunosorbent assays, immunohistochemical staining, and western blotting were utilized for this expression analysis. In addition, to determine whether melatonin regulated progesterone secretion via the regulation of autophagy, luteal cells were cultured before being exposed to different concentrations of melatonin (0.01–100 nM) and the autophagy inhibitor chloroquine (50 μM). Next, luteal cells were treated with the melatonin receptor inhibitors 4-phenyl-2-propionamidotetralin (1 μM) and luzindole (1 μM) before detecting Bec1, LC3B2, AMPK/mTOR, and progesterone secretion levels to ascertain whether the effect of melatonin on autophagy and progesterone secretion is mediated by its corresponding receptors in luteal cells. Finally, to determine the significance of the AMPK/mTOR pathway in this process, an AMPK inhibitor, Compound C (10 μM), was added to luteal cells. Overall, the highest expression of melatonin receptors, autophagy and progesterone secretion was observed in the middle-phase corpus luteum; additionally, melatonin promoted autophagy, at least partially, through its receptor-mediated AMPK/mTOR pathway, which thereby promoting progesterone secretion in luteal cells in vitro. Ultimately, this study is the first to clarify the important role of autophagy in the melatonin-mediated regulation of progesterone secretion in the corpus luteum of sheep; it also lays a foundation for further exploration into the role of melatonin in regulating sheep's ovarian function.

Ramelteon attenuates hippocampal neuronal loss and memory impairment following kainate-induced seizures.

Evidence suggests that the neuroprotective effects of melatonin involve both receptor-dependent and -independent actions. However, little is known about the effects of melatonin receptor activation on the kainate (KA)neurotoxicity. This study examined the effects of repeated post-KAtreatment with ramelteon, a selective agonist of melatonin receptors, on neuronal loss, cognitive impairment, and depression-like behaviors following KA-induced seizures. The expression of melatonin receptors decreased in neurons, whereas it was induced in astrocytes 3 and 7 days after seizures elicited by KA (0.12 μg/μL) in the hippocampus of mice. Ramelteon (3 or 10 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) mitigated KA-induced oxidative stress and impairment of glutathione homeostasis and promoted the nuclear translocation and DNA binding activity of Nrf2 in the hippocampus after KA treatment. Ramelteon and melatonin also attenuated microglial activation but did not significantly affect astroglial activation induced by KA, despite the astroglial induction of melatonin receptors after KA treatment. However, ramelteon attenuated KA-induced proinflammatory phenotypic changes in astrocytes. Considering the reciprocal regulation of astroglial and microglial activation, these results suggest ramelteon inhibits microglial activation by regulating astrocyte phenotypic changes. These effects were accompanied by the attenuation of the nuclear translocation and DNA binding activity of nuclear factor κB (NFκB) induced by KA. Consequently, ramelteon attenuated the KA-induced hippocampal neuronal loss, memory impairment, and depression-like behaviors; the effects were comparable to those of melatonin. These results suggest that ramelteon-mediated activation of melatonin receptors provides neuroprotection against KA-induced neurotoxicity in the mouse hippocampus by activating Nrf2 signaling to attenuate oxidative stress and restore glutathione homeostasis and by inhibiting NFκB signaling to attenuate neuroinflammatory changes.

Melatonin regulates the circadian rhythm to ameliorate postoperative sleep disorder and neurobehavioral abnormalities in aged mice.

Postoperative sleep disorder (PSD) and delirium, which may be associated with surgery and inhalational anesthetics, induce adverse effects in old adults. Emerging evidence indicates that circadian rhythm contributes to various neuropathological diseases, including Alzheimer's disease. Thus, we analyzed the potential role of circadian rhythm in PSD and delirium-like behavior in aged mice and determined whether exogenous melatonin could facilitate entrainment of the circadian rhythm after laparotomy under sevoflurane anesthesia. We selected old C57BL/6J mice which receiving laparotomy/sevoflurane anesthesia as model animals. We employed buried food, open field, and Y maze test to assess delirium-like behavior, and electroencephalography/electromyography (EEG/EMG) were used to investigate sleep changes. We analyzed the transcription rhythm of clock genes in superchiasmatic nucleus (SCN) to explore the effects of surgery and melatonin pretreatment on the circadian rhythm. Then, we measured melatonin receptor levels in SCN and ERK/CREB pathway-related proteins in hippocampus and prefrontal cortex to assess their role in PSDs and delirium-like behavior. Laparotomy under sevoflurane anesthesia had a greater influence than sevoflurane alone, leading to sleep disorder, a shift in sleep-wake rhythm, and delirium-like behavior. Bmal1, Clock, and Cry1 mRNA expression showed a peak shift, MT1 melatonin receptor expression level was increased in the SCN, and p-ERK/ERK and p-CREB/CREB were decreased in hippocampus and prefrontal cortex of aged mice 1 day after laparotomy. Melatonin showed significant efficacy in ameliorating PSD and delirium-like behavior and restoring the circadian rhythm, reversing melatonin receptor and ERK/CREB pathway expression abnormalities. In addition, most of the beneficial effect of melatonin was antagonized by luzindole, a melatonin receptor antagonist. Melatonin receptors in SCN, circadian rhythm, and ERK/CREB signaling pathway participate in the pathophysiological processes of PSD and delirium-like behavior. Melatonin intervention could be a potential preventative approach for PSD and delirium.

EFFECT OF MELATONIN ON THE LEVEL OF CYTOKINES IN PREECLAMPSIA

Introduction. The literature reports a decrease in melatonin levels in preeclampsia, when the degree of decrease correlates with the severity of the process, as well as a decrease in the expression of melatonin receptors in the placental tissue in case of fetal growth restriction.Regarding changes in the cytokine profile in PE: there is evidence that the levels of proinflammatory cytokines, namely TNF-α and IL-6, increase in preeclampsia, while the concentrations of anti-inflammatory cytokines, namely IL-4 and IL-10, decrease. Melatonin, on the contrary, reduces the secretion of proinflammatory cytokines, in particular TNF-α, and increases the production of anti-inflammatory cytokines, namely IL-10. Therefore, normalization of melatonin levels may be a promising direction in the treatment of PE in pregnant women.Objective of the study. Assess the pathogenetic mechanisms of complications arising from a decrease in melatonin, and, in particular, find out more about the changes in melatonin, IL-6 and IL-10, determine the correlation between them.Material and research methodsThe study conducted at the Department of Pregnancy Pathology of the Chernivtsi Regional Perinatal Center, involved 32 women whose pregnancy was complicated by preeclampsia. The control group consisted of 33 women with uncomplicated pregnancies observed in the antenatal clinic of the Chernivtsi Regional Perinatal Center.The patient's informed consent form and the patient's examination card were approved by the Biomedical Ethics Committee of Bukovinian State Medical University (BSMU) of the Ministry of Health of Ukraine (Chernivtsi). The work was guided by the general provisions of the Declaration of Helsinki "Recommendations for Physicians for Biomedical Research Involving Human Subjects" (1964), the World Medical Association on Ethical Principles for Scientific Medical Research Involving Human Subjects (1964-2000), considering the requirements of Directive 2001/20/EC of the European Parliament and of the Council, ICH GCP, the Council of Europe Convention on Human Rights and Biomedicine (04.04.1997), Order of the Ministry of Health of Ukraine No. 690 of 23.09.2009.Statistical data were calculated using MedCalc software developed by MedCalc Software (Ostend, Belgium). The results were evaluated using the Mann-Whitney U-test for small groups. The P value of &lt;0.05 was considered valid. Correlation and regression statistical evaluations were also performed using the aforementioned software.R&amp;D «Preservation and restoration of reproductive health of women and girls in obstetric and gynecological pathology». State registration number: 0121U110020. Terms of execution: 01.2021–12.2025 yy.Research results. The level of melatonin in venous blood taken from women with diagnosed PE was significantly lower (p=0.029), as well as levels of placental growth factor PlGF (p&lt;0.0001), compared to healthy women. In our study we also found that the concentrations of both proinflammatory IL-6 and anti-inflammatory IL-10 were elevated in women with PE compared to women with uncomplicated pregnancies. We performed a prognostic assessment of the impact of the biochemical parameters we studied on the outcome of pregnancies in the examined patients. Melatonin, if present in the blood of a pregnant woman in a concentration higher than the threshold values we established (6.71 pg/ml), reduces the chances of clinical manifestations of severe preeclampsia with a high degree of reliability (p=0.00173). We assume that placental dysfunction in particular leads to impaired synthesis of the antioxidant melatonin, which causes disorders in the tissues of the mother, placenta and fetus and, as a result, provokes clinical manifestations of preeclampsia. The condition of the placenta also worsens the subclinical inflammatory process, as evidenced by an increase in IL-6 levels in patients with diagnosed preeclampsia; an increase in IL-10 in the study group, in our opinion, is a kind of compensatory reaction that prevents preterm labour. In women with pregnancies complicated by preeclampsia, melatonin is a prognostic criterion for delivery before 38 weeks of gestation, and this delivery is mainly associated with the progression of preeclampsia to its severe stage. In the group of patients with uncomplicated pregnancy, proinflammatory interleukin-6 is predictive of spontaneous onset of labour before 38 weeks of gestation. Placental growth factor PlGF was a predictor of the birth of children with a body weight of less than 3000 g in both study groups.Conclusions. Concentrations of melatonin and of placental growth factor in venous blood in women whose pregnancy was complicated by preeclampsia in the third trimester of pregnancy are significantly lower compared to uncomplicated pregnancies. On the other hand, the levels of proinflammatory IL-6 and anti-inflammatory IL-10 were significantly increased in the group of patients with diagnosed preeclampsia compared with normal pregnancy.

Melatonin-related dysfunction in chronic restraint stress triggers sleep disorders in mice.

Stress may trigger sleep disorders and are also risk factors for depression. The study explored the melatonin-related mechanisms of stress-associated sleep disorders on a mouse model of chronic stress by exploring the alteration in sleep architecture, melatonin, and related small molecule levels, transcription and expression of melatonin-related genes as well as proteins. Mice undergoing chronic restraint stress modeling for 28days showed body weight loss and reduced locomotor activity. Sleep fragmentation, circadian rhythm disorders, and insomnia exhibited in CRS-treated mice formed sleep disorders. Tryptophan and 5-hydroxytryptamine levels were increased in the hypothalamus, while melatonin level was decreased. The transcription and expression of melatonin receptors were reduced, and circadian rhythm related genes were altered. Expression of downstream effectors to melatonin receptors was also affected. These results identified sleep disorders in a mice model of chronic stress. The alteration of melatonin-related pathways was shown to trigger sleep disorders.

Protective effect of melatonin against oxygen-induced retinopathy: a study based on the HMGB1/NF-κB/NLRP3 axis

To study the protective effect of melatonin (Mel) against oxygen-induced retinopathy (OIR) in neonatal mice and the role of the HMGB1/NF-κB/NLRP3 axis. Neonatal C57BL/6J mice, aged 7 days, were randomly divided into a control group, a model group (OIR group), and a Mel treatment group (OIR+Mel group), with 9 mice in each group. The hyperoxia induction method was used to establish a model of OIR. Hematoxylin and eosin staining and retinal flat-mount preparation were used to observe retinal structure and neovascularization. Immunofluorescent staining was used to measure the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis and lymphocyte antigen 6G. Colorimetry was used to measure the activity of myeloperoxidase. The OIR group had destruction of retinal structure with a large perfusion-free area and neovascularization, while the OIR+Mel group had improvement in destruction of retinal structure with reductions in neovascularization and perfusion-free area. Compared with the control group, the OIR group had significant increases in the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis, the expression of lymphocyte antigen 6G, and the activity of myeloperoxidase (P<0.05). Compared with the OIR group, the OIR+Mel group had significant reductions in the above indices (P<0.05). Compared with the control group, the OIR group had significant reductions in the expression of melatonin receptors in the retina (P<0.05). Compared with the OIR group, the OIR+Mel group had significant increases in the expression of melatonin receptors (P<0.05). Mel can alleviate OIR-induced retinal damage in neonatal mice by inhibiting the HMGB1/NF-κB/NLRP3 axis and may exert an effect through the melatonin receptor pathway.

Seasonal Variation of Melatonin Concentration and mRNA Expression of Melatonin-Related Genes in Developing Ovarian Follicles of Mares Kept under Natural Photoperiods in the Southern Hemisphere.

This study investigated the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr) receptors, melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods. For one year, ultrasonographic follicular aspiration procedures were performed monthly, and small (<20 mm), medium (20 to 35 mm) and large (>35 mm) follicles were recovered from five mares. One day before monthly sample collections, an exploratory ultrasonography conducted to record the number and the size of all follicles larger than 15 mm. The total number of large follicles were higher during the spring/summer (8.2 ± 1.9) than during autumn/winter (3.0 ± 0.5). Compared to autumn/winter seasons, there was an increase of Fshr and Aanat mRNA expressions in small, medium and large follicles, an increase of Lhr and Asmt mRNA expressions in medium and large follicles and an increase of Mt1 and Mt2 mRNA expressions in small and large follicles during spring/summer. The melatonin levels in follicular fluid were also higher during the spring/summer seasons. The present data show that melatonin locally upregulates the mRNA expression of Mt1 and Mt2 receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.

Photoperiod-driven concurrent changes in hypothalamic and brainstem transcription of sleep and immune genes in migratory redheaded bunting.

The molecular regulation of sleep in avian migrants is still obscure. We thus investigated this in migratory redheaded buntings, where four life-history states (LHS; i.e. non-migratory, pre-migratory, migratory and refractory states) were induced. There was increased night-time activity (i.e. Zugunruhe) during the migratory state with reduced daytime activity. The recordings of the sleep-wake cycle in buntings showed increased night-time active wakefulness coupled with drastically reduced front and back sleep during migratory phase. Interestingly, we found the buntings to feed and drink even after lights-off during migration. Gene expression studies revealed increased hypothalamic expression of glucocorticoid receptor (nr3c1), and pro-inflammatory cytokines (il1b and il6) in pre-migratory and migratory states, respectively, whereas in brainstem Ca2+/calmodulin-dependent protein kinase 2 (camk2) was upregulated during the migratory state. This suggested a heightened pro-inflammatory state during migration which is a feature of chronic sleep loss, and a possible role of Ca2+ signalling in promoting wakefulness. In both the hypothalamus and brainstem, the expression of melatonin receptors (mel1a and mel1b) was increased in the pre-migratory state, and growth hormone-releasing hormone (ghrh, known to induce sleep) was reduced during the migratory state. The current results demonstrate key molecules involved in the regulation of sleep-wake cycle across LHS in migratory songbirds.

Photoperiodic modulation of melatonin receptor and immune genes in migratory redheaded bunting

The transcriptional regulation of innate immune function across annual life history states (LHS) remains obscure in avian migrants. We, therefore, investigated this in a migratory passerine songbird, redheaded bunting (Emberiza bruniceps), which exhibits long-distance vernal migration from India to Central Asia. We exposed the birds (N = 10) to differential photoperiodic conditions to induce a non-migratory (NM), pre-migratory (PM), migratory (MIG), and refractory (REF) state, and performed gene expression assays of melatonin receptors (MEL1A and MEL1B), and innate immunity-linked genes (IL1B, IL6, TLR4, and NFKB) in spleen and blood. We found a significant reduction in splenic mass and volume, and a parallel increase in fat accumulation, and testicular growth in birds under migratory state. The gene expression assay revealed an upregulation of MEL1A and MEL1B mRNA levels in both the tissues in MIG. Additionally, we found a nocturnal increase of splenic IL1B expression, and IL1B, IL6, and TLR4 expression in the blood. The mRNA expression of melatonin receptors and proinflammatory cytokine showed a positive correlation. These results suggest that melatonin relays the photoperiodic signal to peripheral immune organs, which shows LHS-dependent changes in mRNA expression of immune genes.

Anti-drug resistance, anti-inflammation, and anti-proliferation activities mediated by melatonin in doxorubicin-resistant hepatocellular carcinoma: in vitro investigations

Hepatocellular carcinoma (HCC) is the major life-threatening primary liver malignancy in both sexes all over the world. Unfortunately, the majority of patients are diagnosed at later stages because HCC does not elicit obvious symptoms during its early incidence. Consequently, most individuals escape the first-line HCC treatments and are treated with chemotherapy. Regrettably, the therapeutic outcomes for those patients are usually poor because of the development of multidrug resistance phenomena. Furthermore, most anti-HCC therapies cause severe undesired side effects that notably interfere with the life quality of such patients. Accordingly, there is an important need to search for an alternative therapeutic drug or adjuvant which is more efficient with safe or even minimal side effects for HCC treatment. Melatonin was recently reported to exert intrinsic antitumor activity in different cancers. However, the regulatory pathways underlying the antitumor activity of melatonin are poorly understood in resistant liver cells. Furthermore, a limited number of studies have addressed the therapeutic role of melatonin in HCC cells resistant to doxorubicin chemotherapy. In this study, we investigated the antitumor effects of melatonin in doxorubicin-resistant HepG2 cells and explored the regulatory pivotal targets underlying these effects. To achieve our aim, an MTT assay was used to calculate the 50% inhibitory concentration of melatonin and evaluate its antiproliferative effect on resistant cells. Additionally, qRT-PCR was used to quantify genes having a role in drug resistance phenotype (ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2); apoptosis (caspases-3, and -7, Bcl2, Bax, and p53); anti-oxidation (NRF2); expression of melatonin receptors (MT1, MT2, and MT3); besides, programmed death receptor PD-1 gene. The active form of the caspase-3 enzyme was estimated by ELISA. A human inflammatory antibody membrane array was employed to quantify forty inflammatory factors expressed in treated cells. We observed that melatonin inhibited the proliferation of doxorubicin-resistant HepG2 cells in a dose-dependent manner after 24-h incubation time with a calculated IC50 greater than 10 mM (13.4 mM), the expression levels of genes involved in drug resistance response (ABCB1, ABCC1, ABCC5, and ABCG2) were downregulated. Also, the expression of caspase-3, Caspase-7, NRF2, and p53 genes were expressed at higher levels as compared to control (DMSO-treated cells). An active form of caspase-3 was confirmed by ELISA. Moreover, the anti-inflammatory effect of melatonin was detected through the calculated fold change to control which was reduced for various mediators that have a role in the inflammation pathway. The current findings introduce melatonin as a promising anti-cancer treatment for human-resistant HCC which could be used in combination with current chemotherapeutic regimens to improve the outcome and reduce the developed multidrug resistance.