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-KA treatment 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.

Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

Mouse Models in Circadian Rhythm and Melatonin Research.

This contribution reviews the role of inbred and transgenic mouse strains for deciphering the mammalian melatoninergic and circadian system. It focusses on the pineal organ as melatonin factory and two major targets of the melatoninergic system, the suprachiasmatic nuclei (SCN) and the hypophysial pars tuberalis (PT). Mammalian pinealocytes sharing molecular characteristics with true pineal and retinal photoreceptors synthesize and secrete melatonin into the blood and cerebrospinal fluid night by night. Notably, neuron-like connections exist between the deep pinealocytes and the habenular/pretectal region suggesting direct pineal-brain communication. Control of melatonin biosynthesis in rodents involves transcriptional regulation including phosphorylation of CREB and upregulation of mPer1. In the SCN, melatonin acts upon MT1 and MT2 receptors. Melatonin is not necessary to maintain the rhythm of the SCN molecular clockwork, but it has distinct effects on the synchronization of the circadian rhythm by light, facilitates re-entrainment of the circadian system to phase advances in the level of the SCN molecular clockwork by acting upon MT2 receptors and plays a stabilizing role in the circadian system as evidenced from locomotor activity recordings. While the effects in the SCN are subtle, melatonin is essential for PT functions. Via the MT1 receptor it drives the PT-intrinsic molecular clockwork and the retrograde and anterograde output pathways controlling seasonal rhythmicity. Although inbred and transgenic mice do not show seasonal reproduction, the pathways from the PT are fully intact if the animals are melatonin proficient. Thus, only melatonin-proficient strains are suited to investigate the circadian and melatoninergic systems.

Melatonin receptor structure and signaling.

Melatonin (5-methoxy-N-acetyltryptamine) binds with high affinity and specificity to membrane receptors. Several receptor subtypes exist in different species, of which the mammalian MT1 and MT2 receptors are the best-characterized. They are members of the G protein-coupled receptor superfamily, preferentially coupling to Gi/o proteins but also to other G proteins in a cell-context-depending manner. In this review, experts on melatonin receptors will summarize the current state of the field. We briefly report on the discovery and classification of melatonin receptors, then focus on the molecular structure of human MT1 and MT2 receptors and highlight the importance of molecular simulations to identify new ligands and to understand the structural dynamics of these receptors. We then describe the state-of-the-art of the intracellular signaling pathways activated by melatonin receptors and their complexes. Brief statements on the molecular toolbox available for melatonin receptor studies and future perspectives will round-up this review.

Deregulation of Melatonin Receptors and Differential Modulation of After-Hyperpolarization and Ih Currents Using Melatonin Treatment Due to Amyloid-ß-Induced Neurotoxicity in the Hippocampus.

Treatment with melatonin is routinely prescribed for its potent antioxidant and cognitive-promoting effects, nevertheless, it has yet to find neuromodulatory effects in normal and disease conditions. Therefore, to investigate its neuromodulatory mechanisms, melatonin was systemically administered over 10 consecutive days to both intracortical normal saline- and amyloid-ß 1-42 (Aß) peptide-injected rats. At the behavioral level, treatment with melatonin was associated with reduced efficacy in restoring Aß-induced deficit in passive-avoidance memory. Whole-cell patch-clamp recordings from CA1 pyramidal neurons revealed that melatonin treatment reduced spontaneous and evoked intrinsic excitability in control rats while exerting a reduction of spontaneous, but not evoked activity, in the Aß-injected group. Interestingly, treatment with melatonin enhances after-hyperpolarization in control, but not Aß-injected rats. In contrast, our voltage-clamp study showed that Ih current is significantly enhanced by Aß injection, and this effect is further strengthened by treatment with melatonin in Aß-injected rats. Finally, we discovered that the transcription of melatonin receptors 1 (MT1) and 2 (MT2) is significantly upregulated in the hippocampi of Aß-injected rats. Collectively, our study demonstrates that systemic treatment with melatonin has differential neuromodulation on CA1 neuronal excitability, at least in part, via differential effects on after-hyperpolarization and Ih currents due to Aß-induced neurotoxicity.

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.

Association of rotating night shift work, CLOCK, MTNR1A, MTNR1B genes polymorphisms and their interactions with type 2 diabetes among steelworkers: a case-control study.

BACKGROUND: The purpose of this study is to investigate the association of rotating night shift work, CLOCK, MTNR1A, MTNR1B genes polymorphisms and their interactions with type 2 diabetes among steelworkers. METHODS: A case-control study was conducted in the Tangsteel company in Tangshan, China. The sample sizes of the case group and control group were 251 and 451, respectively. The logistic regression, log-linear model and generalized multifactor dimensionality (GMDR) method were used to investigate the interaction between circadian clock gene, melatonin receptor genes and rotating night shift work on type 2 diabetes among steelworkers. Relative excess risk due to interaction (RERI) and attributable proportions (AP) were used to evaluate additive interactions. RESULTS: Rotating night shift work, current shift status, duration of night shifts, and average frequency of night shifts were associated with an increased risk of type 2 diabetes after adjustment for confounders. Rs1387153 variants in MTNR1B was found to be associated with an increased risk of type 2 diabetes, which was not found between MTNR1A gene rs2119882 locus, CLOCK gene rs1801260 locus and the risk of type 2 diabetes. The association between rotating night shift work and risk of type 2 diabetes appeared to be modified by MTNR1B gene rs1387153 locus (RERI = 0.98, (95% CI, 0.40-1.55); AP = 0.60, (95% CI, 0.07-1.12)). The interaction between MTNR1A gene rs2119882 locus and CLOCK gene rs1801260 locus was associated with the risk of type 2 diabetes (RERI = 1.07, (95% CI, 0.23-1.91); AP = 0.77, (95% CI, 0.36-1.17)). The complex interaction of the MTNR1A-MTNR1B-CLOCK-rotating night shift work model based on the GMDR methods may increase the risk of type 2 diabetes (P = 0.011). CONCLUSIONS: Rotating night shift work and rs1387153 variants in MTNR1B were associated with an increased risk of type 2 diabetes among steelworkers. The complex interaction of MTNR1A-MTNR1B-CLOCK-rotating night shift work may increase the risk of type 2 diabetes.

Differentiation of T cells producing interleukin-17 (Th17) against the background of exogenous melatonin during pregnancy.

The population of T lymphocytes producing IL-17 (Th17) plays a dual role during pregnancy and its activity is tightly controlled during this period. One of the factors involved in this process may be the pineal hormone melatonin, which can effectively regulate this T cell population. Here we have shown that exogenous melatonin in pharmacological concentrations is able to enhance the differentiation of Th17 cells of pregnant women in vitro. The stimulatory effects of melatonin were limited to in the first trimester of pregnancy and were apparently mediated by both membrane and nuclear melatonin receptors. Since exogenous melatonin is currently considered as a promising drug in solving various problems associated with reproduction, it is necessary to take into account its immunoregulatory effects.

Therapeutic potential of a single-dose melatonin in the attenuation of cardiac ischemia/reperfusion injury in prediabetic obese rats.

Although acute melatonin treatment effectively reduces cardiac ischemia/reperfusion (I/R) injury in lean rats by modulating melatonin receptor 2 (MT2), there is no information regarding the temporal effects of melatonin administration during cardiac I/R injury in prediabetic obese rats. Prediabetic obese rats induced by chronic consumption of a high-fat diet (HFD) were used. The rats underwent a cardiac I/R surgical procedure (30-min of ischemia, followed by 120-min of reperfusion) and were randomly assigned to receive either vehicle or melatonin treatment. In the melatonin group, rats were divided into 3 different subgroups: (1) pretreatment, (2) treatment during ischemic period, (3) treatment at the reperfusion onset. In the pretreatment subgroup either a nonspecific MT blocker (Luzindole) or specific MT2 blocker (4-PPDOT) was also given to the rats prior to melatonin treatment. Pretreatment with melatonin (10 mg/kg) effectively reduced cardiac I/R injury by reducing infarct size, arrhythmia, and LV dysfunction. Reduction in impaired mitochondrial function, mitochondrial dynamic balance, oxidative stress, defective autophagy, and apoptosis were observed in rats pretreated with melatonin. Unfortunately, the cardioprotective benefits were not observed when 10-mg/kg of melatonin was acutely administered to the rats after cardiac ischemia. Thus, we increased the dose of melatonin to 20 mg/kg, and it was administered to the rats during ischemia or at the onset of reperfusion. The results showed that 20-mg/kg of melatonin effectively reduced cardiac I/R injury to a similar extent to the 10-mg/kg pretreatment regimen. The MT2 blocker inhibited the protective effects of melatonin. Acute melatonin treatment during cardiac I/R injury exerted protective effects in prediabetic obese rats. However, a higher dose of melatonin is required when given after the onset of cardiac ischemia. These effects of melatonin were mainly mediated through activation of MT2.

Melatonin Protects Against Hyperoxia-Induced Apoptosis in Alveolar Epithelial type II Cells by Activating the MT2/PI3K/AKT/ETS1 Signaling Pathway.

PURPOSE: Hyperoxia-induced apoptosis in alveolar epithelial type II cells (AECIIs) plays a critical role in the development of bronchopulmonary dysplasia (BPD). Melatonin has been shown to improve BPD. However, the protective effect of melatonin on hyperoxia-induced apoptosis in AECIIs and the precise mechanisms involved remain unclear. METHODS: Human alveolar epithelial type II A549 cells were treated with hyperoxia as an in vitro model to investigate the antiapoptotic mechanism of melatonin. CCK-8 assays were performed to investigate the viability of A549 cells. Hoechst 33,258 staining was carried out to quantify apoptosis in A549 cells. The protein expression levels of E26 oncogene homolog 1 (ETS1), Bcl-2, Bax, Bim, Wnt, ß-catenin, AKT and phosphorylated AKT were measured by western blotting. LY294002, SC79 and the downregulation of ETS1, melatonin receptor 1 (MT1) and MT2 with specific siRNAs were used to investigate the role of the PI3K/AKT pathway, ETS1, MT1 and MT2 in hyperoxia-induced apoptosis in A549 cells. RESULTS: Melatonin prevented hyperoxia-induced apoptosis in A549 cells, and the upregulation of E26 oncogene homolog 1 (ETS1) contributed to the antiapoptotic effect of melatonin. Melatonin activated the PI3K/AKT axis, which led to ETS1 upregulation and inhibited apoptosis in hyperoxia-exposed A549 cells. Furthermore, melatonin-induced activation of the PI3K/AKT axis, upregulation of ETS1 and inhibition of apoptosis were reversed by melatonin receptor 2 (MT2) siRNA in hyperoxia-exposed A549 cells. CONCLUSION: Melatonin prevents hyperoxia-induced apoptosis by activating the MT2/PI3K/AKT/ETS1 axis in alveolar epithelial cells.

Artifical light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species.

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

Structural Basis for Agonistic Activity and Selectivity toward Melatonin Receptors hMT1 and hMT2.

Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results.

[Age-related features of expression of melatonin and its receptors in myocardial tissues in patients with dilated cardiomyopathy.]

In recent years, more and more attention of researchers has been paid to the study of dilated cardiomyopathy (DCMP). The prevalence of this disease in older age groups is higher than previously thought, and the course of the disease is associated with a worse prognosis and treatment difficulties. Researchers are considering various signaling molecules whose expression changes are associated with myocardial damage and the development of DCMP; evaluation of changes in the expression of melatonin and its receptors in DCMP requires further study. The aim of the study was to study the age-related features of the expression of melatonin and its receptors (MT1, MT2) in the myocardium and their changes depending on the presence of dilated cardiomyopathy. Immunocytochemical and immunohistochemical methods were used to evaluate the expression of melatonin and its MT1, MT2 receptors in myocardial autopsy material and cardiomyocyte cultures of people of different ages with and without cardiovascular pathology. The study revealed age-associated changes in the form of a decrease in the expression of melatonin and its MT1 and MT2 receptors in the myocardium. In individuals with DCMP of all age groups, a more significant decrease in expression was noted: melatonin by 1,6-1,7 times in old age and 3,2 times in old age; MT1 by 1,8 and 2 times, respectively; MT2 by 1,4 and 4 times, respectively. The relationship between the decrease in the expression of melatonin and its receptors in myocardial tissues with age and the presence of DCMP was revealed. The data obtained allow us to clarify age-dependent changes in melatonin and its receptors, as well as to assume their important role in the development of DCMP, which requires further study.

Exploring the mechanisms and impacts of melatonin on fish colouration.

The pineal hormone melatonin is a multi-functional molecule with a recognized role in pigment aggregation in chromatophores, mediating its actions through binding to subtypes of its specific receptors. Since its discovery, melatonin has been known to be responsible for pigment aggregation towards the cell centre in fishes, including their embryos, as an adaptation to reduced light and thus results in pale body colouration. Diversity exists in the sensitivity of melanophores towards melatonin at interspecies, intraspecific levels, seasons, and amongst chromatophores at different regions of the animal body. In most of the fishes, melatonin leads to their skin paling at night. It is indicated that the melatonin receptors have characteristically maintained to show the same aggregating effects in fishes and other vertebrates in the evolutionary hierarchy. However, besides this aggregatory effect, melatonin is also responsible for pigment dispersion in certain fishes. Here is the demand in our review to explore further the nature of the dispersive behaviour of melatonin through the so-called ß-melatonin receptors. It is clear that the pigment translocations in lower vertebrates under the effect of melatonin are mediated through the melatonin receptors coupled with other hormonal receptors as well. Therefore, being richly supplied with a variety of receptors, chromatophores and melanocytes can be used as in vitro test models for pharmacological applications of known and novel drugs. In this review, we present diverse effects of melatonin on chromatophores of fishes in particular with appropriate implications on most of the recent findings.

MTNR1A and MTNR1B Gene Variants of the Melatonin Receptor and Arterial Stiffness in Persons without Arterial Hypertension.

A comparative analysis of vascular stiffness indices and the results of blood test was carried out in 85 healthy donors aged 19-64 years, carriers of polymorphic variants of type 1 and type 2 melatonin receptor genes. The associations of polymorphic markers of type 1 MTNR1A (rs34532313) and type 2 MTNR1B (rs10830963) melatonin receptor genes with parameters of vascular stiffness and blood parameters in healthy patients were studied. Genotyping was performed using allele-specific PCR. In all patients, 24-h BP monitoring with assessment of arterial stiffness was performed. Allele C homozygotes of MTNR1A differed significantly from carriers of the major T allele by elevated triglyceride, LDL, and fibrinogen levels. The major allele C of the rs10830963 polymorphic variant of the MTNR1B gene is associated with elevated LDL and triglycerides, as well as with individual differences in the elastic properties of the vascular wall in the examined subjects.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

Novel Therapeutics for Treating Sleep Disorders: New Perspectives on Maydis stigma.

Sleep is a restorative period that plays a crucial role in the physiological functioning of the body, including that of the immune system, memory processing, and cognition. Sleep disturbances can be caused by various physical, mental, and social problems. Recently, there has been growing interest in sleep. Maydis stigma (MS, corn silk) is a female maize flower that is traditionally used as a medicinal plant to treat many diseases, including hypertension, edema, and diabetes. It is also used as a functional food in tea and other supplements. ß-Sitosterol (BS) is a phytosterol and a natural micronutrient in higher plants, and it has a similar structure to cholesterol. It is a major component of MS and has anti-inflammatory, antidepressive, and sedative effects. However, the potential effects of MS on sleep regulation remain unclear. Here, we investigated the effects of MS on sleep in mice. The effects of MS on sleep induction were determined using pentobarbital-induced sleep and caffeine-induced sleep disruption mouse models. MS extracts decreased sleep latency and increased sleep duration in both the pentobarbital-induced sleep induction and caffeine-induced sleep disruption models compared to the positive control, valerian root extract. The butanol fraction of MS extracts decreased sleep latency time and increased sleep duration. In addition, ß-sitosterol enhances sleep latency and sleep duration. Both MS extract and ß-sitosterol increased alpha activity in the EEG analysis. We measured the mRNA expression of melatonin receptors 1 and 2 (MT1/2) using qRT-PCR. The mRNA expression of melatonin receptors 1 and 2 was increased by MS extract and ß-sitosterol treatment in rat primary cultured neurons and the brain. In addition, MS extract increased the expression of clock genes including per1/2, cry1/2, and Bmal1 in the brain. MS extract and ß-sitosterol increased the phosphorylation of ERK1/2 and αCaMKII. Our results demonstrate for the first time that MS has a sleep-promoting effect via melatonin receptor expression, which may provide new scientific evidence for its use as a potential therapeutic agent for the treatment and prevention of sleep disturbance.

Synthesis, In Silico, and Biological Evaluation of a Borinic Tryptophan-Derivative That Induces Melatonin-like Amelioration of Cognitive Deficit in Male Rat.

Preclinical and clinical evidence supports melatonin and its analogues as potential treatment for diseases involving cognitive deficit such as Alzheimer's disease. In this work, we evaluated by in silico studies a set of boron-containing melatonin analogues on MT1 and MT2 receptors. Then, we synthesized a compound (borolatonin) identified as potent agonist. After chemical characterization, its evaluation in a rat model with cognitive deficit showed that it induced ameliorative effects such as those induced by equimolar administration of melatonin in behavioral tests and in neuronal immunohistochemistry assays. Our results suggest the observed effects are by means of action on the melatonin system. Further studies are required to clarify the mechanism(s) of action, as the beneficial effects on disturbed memory by gonadectomy in male rats are attractive.

Melatonin ameliorates aging-related impaired angiogenesis in gastric endothelial cells via local actions on mitochondria and VEGF-survivin signaling.

Tissue injury healing is impaired in aging, and this impairment is caused in part by reduced angiogenesis. Melatonin, a neuroendocrine hormone that regulates sleep and circadian rhythm, is also produced in the gastrointestinal tract. The expression of melatonin receptors MT1 and MT2 in gastric endothelial cells and their roles in aging-related impairment of gastric angiogenesis have not been examined. We hypothesized that MT1 and MT2 expression is reduced in gastric endothelial cells of aging rats and that melatonin treatment can upregulate their expression and improve angiogenesis. We examined the expression of MT1 and MT2 in gastric endothelial cells (GECs) isolated from young and aging rats. We also examined the effects of melatonin treatment on angiogenesis, GEC mitochondrial function, expression of vascular endothelial growth factor (VEGF), its signaling receptor (VEGFR-2), and the inhibitor of apoptosis protein, survivin. Young and aging GECs expressed MT1 (in the cytoplasm and mitochondria) and MT2 (in nucleus and mitochondria). In aging GECs, MT1 and MT2 levels, in vitro angiogenesis, and mitochondrial membrane potential were significantly reduced (by 1.5-fold, 1.9-fold, 3.1-fold, and 1.63-fold, respectively) compared with young GECs. Melatonin treatment of aging GECs significantly increased MT1 and MT2 expression compared with the controls, induced nuclear translocation of MT1, and significantly ameliorated the aging-related impairment of angiogenesis and mitochondrial function. Aging GECs have significantly reduced MT1 and MT2 expression, angiogenesis, and mitochondrial membrane potential compared with young GECs. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function.NEW & NOTEWORTHY This study showed reduced expression of melatonin receptors MT1 and MT2, angiogenesis, and mitochondrial function in gastric endothelial cells (GECs) isolated from aging rats. Treatment of aging GECs with melatonin increases expression of VEGF receptor and survivin and ameliorates aging-related impaired angiogenesis and mitochondrial function. These studies provide new insight into the mechanisms of the aging-related impairment of angiogenesis and delayed tissue injury healing and provide a rationale for melatonin treatment to reverse these abnormalities.

Melatonin multifaceted pharmacological actions on melatonin receptors converging to abrogate COVID-19.

Data indicate that controlling inflammatory responses to COVID-19 may be as important as antiviral therapies or could be an important adjunctive approach. Melatonin possesses anti-inflammation, antioxidation, and immune-enhancing features directly and/or indirectly through its own receptor signaling and is therefore well suited to reduce the severity of COVID-19. Studies have proposed that melatonin regulates COVID-19-associated proteins directly through regulation of molecules such as calmodulin (CALM) 1 and CALM 2, calreticulin (CalR), or myeloperoxidase (MPO) and/or indirectly through actions on GPCR (eg, MTNR1A, MTNR1B) and nuclear (eg, RORα, RORß) melatonin receptor signaling. However, the exact mechanism(s) and doses by which melatonin reduces the severity of COVID-19 is still open for debate, warranting the need for further testing of melatonin in placebo-controlled randomized clinical trials for COVID-19.