Industrial and academic approaches to the search for alternative melatonin receptor ligands: An historical survey.

The search for melatonin receptor agonists formed the main part of melatonin medicinal chemistry programs for the last three decades. In this short review, we summarize the two main aspects of these programs: the development of all the necessary tools to characterize the newly synthesized ligands at the two melatonin receptors MT1 and MT2, and the medicinal chemist's approaches to find chemically diverse ligands at these receptors. Both strategies are described. It turns out that the main source of tools were industrial laboratories, while the medicinal chemistry was mainly carried out in academia. Such complete accounts are interesting, as they delineate the spirits in which the teams were working demonstrating their strength and innovative character. Most of the programs were focused on nonselective agonists and few of them reached the market. In contrast, discovery of MT1-selective agonists and melatonergic antagonists with proven in vivo activity and MT1 or MT2-selectivity is still in its infancy, despite the considerable interest that subtype selective compounds may bring in the domain, as the physiological respective roles of the two subtypes of melatonin receptors, is still poorly understood. Poly-pharmacology applications and multitarget ligands have also been considered.

Melatonin in the mammalian retina: Synthesis, mechanisms of action and neuroprotection.

Melatonin is an important player in the regulation of many physiological functions within the body and in the retina. Melatonin synthesis in the retina primarily occurs during the night and its levels are low during the day. Retinal melatonin is primarily synthesized by the photoreceptors, but whether the synthesis occurs in the rods and/or cones is still unclear. Melatonin exerts its influence by binding to G protein-coupled receptors named melatonin receptor type 1 (MT1) and type 2 (MT2). MT1 and MT2 receptors activate a wide variety of signaling pathways and both receptors are present in the vertebrate photoreceptors where they may form MT1/MT2 heteromers (MT1/2h). Studies in rodents have shown that melatonin signaling plays an important role in the regulation of retinal dopamine levels, rod/cone coupling as well as the photopic and scotopic electroretinogram. In addition, melatonin may play an important role in protecting photoreceptors from oxidative stress and can protect photoreceptors from apoptosis. Critically, melatonin signaling is involved in the modulation of photoreceptor viability during aging and other studies have implicated melatonin in the pathogenesis of age-related macular degeneration. Hence melatonin may represent a useful tool in the fight to protect photoreceptors-and other retinal cells-against degeneration due to aging or diseases.

Have All of the Phytohormonal Properties of Melatonin Been Verified?

Melatonin is a ubiquitous regulator in plants and performs a variety of physiological roles, including resistance to abiotic stress, regulation of growth and development, and enhancement of plant immunity. Melatonin exhibits the characteristics of a phytohormone with its pleiotropic effects, biosynthesis, conjugation, catabolism, effective concentration, and the shape and location of its dose-response curves. In addition, CAND2/PMTR1, a phytomelatonin receptor candidate belonging to the G protein-coupled receptors (GPCRs), supports the concept of melatonin as a phytohormone. However, the biochemistry of plant melatonin receptors needs to be further characterized. In particular, some of the experimental findings to date cannot be explained by known GPCR signaling mechanisms, so further studies are needed to explore the possibility of novel signaling mechanisms.

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled ß2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

Melatonin injection and red light irradiation affect the antioxidant response and cell damage in disk abalone (Haliotis discus hannai) exposed to high water temperatures.

The effects of red light-emitting diode (LED) light irradiation (630 nm, 0.5 W/m2) and melatonin (10-8 and 10-7 M) on oxidative stress and physiological responses in abalones exposed to high temperatures (28°C) were investigated. Changes in messenger RNA (mRNA) expressions of melatonin receptor (MT-R), heat shock protein 70 (HSP70), and antioxidant enzymes, as well as alterations in H2O2 levels in the hemolymph, were examined. The results revealed that high-temperature-stressed abalones treated with melatonin injections or exposed to red LED light showed a significant increase in MT-R mRNA expression, while HSP70 mRNA expression decreased. Notably, HSP70 mRNA expression levels in the red LED light-irradiated group were similar to those in the group injected with 10-8 M melatonin after 24 h exposure. Abalones treated with melatonin at 20°C or irradiated with red LED light exhibited decreased H2O2 levels and reduced antioxidant enzyme mRNA expression compared with those of the control group. However, the high-temperature environment induced oxidative stress in abalones, leading to increased antioxidant enzyme mRNA expression compared with that under 20°C conditions. Moreover, abalones exposed to high-temperature stress exhibited hepatopancreatic DNA damage, which was attenuated by melatonin treatment or red LED light irradiation. Hence, red LED light reduces oxidative stress, boosts antioxidant enzymes, and alleviates DNA damage in high-temperature-stressed abalones, akin to 10-8 M melatonin treatment. Therefore, considering the practical challenges of continuous melatonin administration to abalones, utilizing red LED light emerges as a practical, effective alternative to protect abalones from oxidative stress compared to 10-8 M melatonin treatment.

Melatonin ameliorates hepatic fibrosis via the melatonin receptor 2-mediated upregulation of BMAL1 and anti-oxidative enzymes.

Hepatic fibrosis, when left untreated, causes serious health problems that progress to cirrhosis and, in some cases, liver cancer. Activation of hepatic stellate cells may be a key characteristic in the development of hepatic fibrosis. Melatonin, a pineal hormone, exerts anti-fibrotic effects; however, the exact mechanisms remain unclear. In this study, the beneficial effects of melatonin against hepatic fibrosis and the underlying mechanisms were investigated using the human hepatic stellate cell line, LX-2, and in vivo murine animal models. The results showed that melatonin suppressed the expression of transforming growth factor (TGF)-ß1-induced fibrosis markers and production of reactive oxygen species in LX-2 cells. Either 4-phenyl-2-propionamidotetralin, a melatonin receptor 2 selective antagonist, or melatonin receptor 2 small interfering RNA abolished the suppressive effects of melatonin, suggesting the involvement of melatonin receptor 2 in melatonin-induced anti-fibrotic and anti-oxidative actions. Melatonin increased the expression of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), a positive circadian clock gene. BMAL1 knockdown reduced the anti-fibrotic and anti-oxidative effects of melatonin, demonstrating the protective effects of melatonin against TGF-ß1-induced hepatic stellate cell activation by exhibiting melatonin receptor 2-BMAL1-anti-oxidative effects. In high-fat diet-induced and carbon tetrachloride-induced hepatic fibrosis models, oral melatonin administration decreased the expression of hepatic fibrosis markers and increased the expression of messenger RNA and levels of proteins of BMAL1 and melatonin receptor 2. Thus, melatonin exerted protective effects against hepatic fibrosis through melatonin receptor 2 activation, followed by an upregulation of the BMAL1-anti-oxidative enzyme pathways.

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.

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.

Blockade of Melatonin Receptors Abolishes Its Antiarrhythmic Effect and Slows Ventricular Conduction in Rat Hearts.

Melatonin has been reported to cause myocardial electrophysiological changes and prevent ventricular tachycardia or fibrillation (VT/VF) in ischemia and reperfusion. We sought to identify electrophysiological targets responsible for the melatonin antiarrhythmic action and to explore whether melatonin receptor-dependent pathways or its antioxidative properties are essential for these effects. Ischemia was induced in anesthetized rats given a placebo, melatonin, and/or luzindole (MT1/MT2 melatonin receptor blocker), and epicardial mapping with reperfusion VT/VFs assessment was performed. The oxidative stress assessment and Western blotting analysis were performed in the explanted hearts. Transmembrane potentials and ionic currents were recorded in cardiomyocytes with melatonin and/or luzindole application. Melatonin reduced reperfusion VT/VF incidence associated with local activation time in logistic regression analysis. Melatonin prevented ischemia-related conduction slowing and did not change the total connexin43 (Cx43) level or oxidative stress markers, but it increased the content of a phosphorylated Cx43 variant (P-Cx43368). Luzindole abolished the melatonin antiarrhythmic effect, slowed conduction, decreased total Cx43, protein kinase Cε and P-Cx43368 levels, and the IK1 current, and caused resting membrane potential (RMP) depolarization. Neither melatonin nor luzindole modified INa current. Thus, the antiarrhythmic effect of melatonin was mediated by the receptor-dependent enhancement of impulse conduction, which was associated with Cx43 phosphorylation and maintaining the RMP level.

Polarization of Melatonin-Modulated Colostrum Macrophages in the Presence of Breast Tumor Cell Lines.

Human colostrum and milk contain diverse cells and soluble components that have the potential to act against tumors. In breast cancer, macrophages play a significant role in immune infiltration and contribute to the progression and spread of tumors. However, studies suggest that these cells can be reprogrammed to act as an antitumor immune response. This study aimed to evaluate the levels of melatonin and its receptors, MT1 (melatonin receptor 1) and MT2 (melatonin receptor 2), in colostrum and assess the differentiation and polarization of the colostrum macrophages modulated by melatonin in the presence of breast tumor cells. Colostrum samples were collected from 116 mothers and tested for their melatonin and receptor levels. The colostrum cells were treated with or without melatonin and then cultured for 24 h in the presence or absence of breast tumor cells. The results showed that melatonin treatment increased the expression of MT1 and MT2 in the colostrum cells. Furthermore, melatonin treatment increased the percentage of M1 macrophages and decreased the percentage of M2 macrophages. When the colostrum macrophages were cocultured with breast tumor cells, melatonin reduced the percentage of both macrophage phenotypes and the cytokines tumor necrosis factor-alpha (TNF-α) and interleukin 8 (IL-8). These data suggest that melatonin can regulate the inflammatory process via M1 macrophages in the tumor microenvironment and, simultaneously, the progression of M2 macrophages that favor tumorigenesis.

Agomelatine inhibits platelet aggregation through melatonin receptor-dependent and independent mechanisms.

AIMS: Melatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion. MAIN METHODS: Human platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B2 (TxB2), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays. KEY FINDINGS: Our data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B2 (TxB2) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole. SIGNIFICANCE: The present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion.

Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus-oocyte complexes.

BACKGROUND: Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non-human primate cumulus-oocyte complexes (COCs). METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate-limiting enzyme in melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs. RESULTS: RT-PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)- and metaphase II (MII)-stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV- and MII-stage COCs. CONCLUSIONS: Our results provide the first evidence for the existence of the rate-limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non-human primate COCs.

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.

Melatonin and Health: Insights of Melatonin Action, Biological Functions, and Associated Disorders.

Melatonin is ubiquitous molecule with wide distribution in nature and is produced by many living organisms. In human beings, pineal gland is the major site for melatonin production and to lesser extent by retina, lymphocytes, bone marrow, gastrointestinal tract, and thymus. Melatonin as a neurohormone is released into circulation wherein it penetrates all tissues of the body. Melatonin synthesis and secretion is supressed by light and enhanced by dark. Melatonin mostly exerts its effect through different pathways with melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2) being the predominant type of receptor that are mainly expressed by many mammalian organs. Melatonin helps to regulate sleep patterns and circadian rhythms. In addition, melatonin acts as an antioxidant and scavenges excessive free radicals generated in the body by anti-excitatory and anti-inflammatory properties. A multiple array of other functions are displayed by melatonin that include oncostatic, hypnotic, immune regulation, reproduction, puberty timing, mood disorders, and transplantation. Deficiencies in the production or synthesis of melatonin have been found to be associated with onset of many disorders like breast cancer and neurodegenerative disorders. Melatonin could be used as potential analgesic drug in diseases associated with pain and it has quite promising role there. In the past century, a growing interest has been developed regarding the wide use of melatonin in treating various diseases like inflammatory, gastrointestinal, cancer, mood disorders, and others. Several melatonin agonists have been synthesized and are widely used in disease treatment. In this review, an effort has been made to describe the biochemistry of melatonin along with its therapeutic potential in various diseases of humans.

Multiplatform-Integrated Identification of Melatonin Targets for a Triad of Psychosocial-Sleep/Circadian-Cardiometabolic Disorders.

Several psychosocial, sleep/circadian, and cardiometabolic disorders have intricately interconnected pathologies involving melatonin disruption. Therefore, we hypothesize that melatonin could be a therapeutic target for treating potential comorbid diseases associated with this triad of psychosocial-sleep/circadian-cardiometabolic disorders. We investigated melatonin's target prediction and tractability for this triad of disorders. The melatonin's target prediction for the proposed psychosocial-sleep/circadian-cardiometabolic disorder triad was investigated using databases from Europe PMC, ChEMBL, Open Targets Genetics, Phenodigm, and PheWAS. The association scores for melatonin receptors MT1 and MT2 with this disorder triad were explored for evidence of target-disease predictions. The potential of melatonin as a tractable target in managing the disorder triad was investigated using supervised machine learning to identify melatonin activities in cardiovascular, neuronal, and metabolic assays at the cell, tissue, and organism levels in a curated ChEMBL database. Target-disease visualization was done by graphs created using "igraph" library-based scripts and displayed using the Gephi ForceAtlas algorithm. The combined Europe PMC (data type: text mining), ChEMBL (data type: drugs), Open Targets Genetics Portal (data type: genetic associations), PhenoDigm (data type: animal models), and PheWAS (data type: genetic associations) databases yielded types and varying levels of evidence for melatonin-disease triad correlations. Of the investigated databases, 235 association scores of melatonin receptors with the targeted diseases were greater than 0.2; to classify the evidence per disease class: 37% listed psychosocial disorders, 9% sleep/circadian disorders, and 54% cardiometabolic disorders. Using supervised machine learning, 546 cardiovascular, neuronal, or metabolic experimental assays with predicted or measured melatonin activity scores were identified in the ChEMBL curated database. Of 248 registered trials, 144 phase I to IV trials for melatonin or agonists have been completed, of which 33.3% were for psychosocial disorders, 59.7% were for sleep/circadian disorders, and 6.9% were for cardiometabolic disorders. Melatonin's druggability was evidenced by evaluating target prediction and tractability for the triad of psychosocial-sleep/circadian-cardiometabolic disorders. While melatonin research and development in sleep/circadian and psychosocial disorders is more advanced, as evidenced by melatonin association scores, substantial evidence on melatonin discovery in cardiovascular and metabolic disorders supports continued R&D in cardiometabolic disorders, as evidenced by melatonin activity scores. A multiplatform analysis provided an integrative assessment of the target-disease investigations that may justify further translational research.

Methylation of melatonin receptors in patients with unipolar and bipolar depression.

Disturbances of melatonin secretion alter the circadian rhythm and sleep-wake cycle, which is observed among patients with depression. Melatonin acts via melatonin receptors MT1 and MT2, which are present in many tissues, including peripheral blood mononuclear cells (PBMC). We assume that disturbances of the melatonin pathway in the brain may be reflected by molecular changes in peripheral organs. The study objective was to evaluate the methylation profile of CpG island in the promoter region of melatonin receptor genes MTNR1A and MTNR1B in PBMC of patients with depression and compare it with healthy volunteers. The study group comprised 85 patients with unipolar (UP) and bipolar disorders (BP) and 83 controls. The methylation pattern of CpG island in the promoter region was analyzed using the quantitative methylation-specific real-time PCR (qMSP-PCR) method. We found that the methylation profile of the patients with depression varied in comparison to the control group. The methylation level of MTNR1A was significantly lower among depressed patients compared to controls. Additionally, melatonin concentration was negatively correlated with MTNR1B methylation level among the UP patients. The study may suggest that the methylation profile of melatonin receptors in PBMC may be used as a complementary molecular marker in depression diagnosis.

Effect of a hyaluronic acid-based mesotherapeutic injectable on the gene expression of CLOCK and Klotho proteins, and environmentally induced oxidative stress in human skin cells.

OBJECTIVE: Normal circadian rhythms are essential to the repair mechanisms of oxidative stress implicated in skin aging. Given reports that hyaluronic acid (HA) homeostasis exhibits a different profile in chronological skin aging, as compared to environmental or extrinsic aging, an improved understanding of the way HA interacts with its surroundings, and the impact of HA injectables in replacing lost HA and encouraging rejuvenation, is of key benefit to skin aging treatments. The objectives of these current studies were twofold. Firstly, to demonstrate the in vitro effects of two lightweight hyaluronic-based injectables on the expression of CLOCK protein in human skin fibroblasts, and their effects on Klotho protein expression as a marker for circadian rhythms in a combined human keratinocyte and Merkel cell model. Secondly, to ascertain whether these findings could be correlated with in vitro effects on various environmental oxidative stress aging markers (blue light, UVA/UVB, Urban Dust, and IR exposures). METHODS: Oxidative stress studies were aimed to highlight possible protective effects through different challenge conditions in two models, ex vivo human skin explants and in vitro monolayer cultures of normal human dermal fibroblasts (NHDF). The protective effects of the test products were evaluated against an increase of cyclobutene pyrimidine dimers (CPDs) abundance within epidermal section of ex vivo skin explants after UVA/UVB radiation; effects of blue light on gene expression from NHDFs fibroblasts; effects of pollutants (Urban dust, UbD) on gene expression in NHDFs fibroblasts; and an increase of reactive oxygen species (ROS) production by NHDFs fibroblasts after infrared-A radiation. Gene expression was assayed and analyzed utilizing microfluidic TaqMan qPCR arrays. CLOCK expression was measured in young and senescing NHDFs by immunostaining, and Klotho and melatonin expression by immunostaining in Merkel cell-enriched normal adult human epidermal cell cultures. RESULTS: In an aging culture of mixed keratinocyte and Merkel skin cells, activation of Klotho expression was induced by the application of both HA test products. Moreover, the HA products increase Klotho protein expression in both Merkel cells and keratinocytes. The observed positive effect of the tested products on melatonin receptors 1A and 1B expression in aging Merkel cell culture and keratinocytes is also interesting. HA-Y (developed for patients 25+ years old) stimulated melatonin receptors type 1B expression in aging cell cultures more strongly than HA-S (developed for patients 35-65 years old). In age (stressed) cells, a lower expression of Klotho protein and melatonin receptors 1A and 1B is apparent. The addition of HA-Y and HA-S stimulates their expression thus providing a "protective" effect. The blue light irradiation at 40 J/cm2 performed in NHDF fibroblast cultures led to a modification of the expression of several genes, all involved in mechanisms known to be modulated in case of solar radiation stress. CONCLUSIONS: Although these are preliminary findings, they are the first we know of that demonstrate HA facial injectables having a benefit and possibilities beyond the "physical filling" of the skin. As regards the beneficial effects against blue light-induced oxidative stress, and a return to cellular homeostasis, there is a need to conduct further and more precise investigations into HA-S. Furthermore, the benefit of these HA injectables (Novacutan®) in the modulation of oxidative stressed circadian rhythms widens their potential benefit.

Modulation of Melatonin Receptors Regulates Reproductive Physiology: The Impact of Agomelatine on the Estrus Cycle, Gestation, Offspring, and Uterine Contractions in Rats.

Agomelatine is a pharmaceutical compound that functions as an agonist for melatonin receptors, with a particular affinity for the MT1 and MT2 receptor subtypes. Its mode of action is integral to the regulation of diverse physiological processes, encompassing the orchestration of circadian rhythms, sleep-wake cycles, and mood modulation. In the present study, we delve into the intricate interplay between agomelatine and the modulation of estrus cycles, gestation periods, offspring numbers, and uterine contractions, shedding light on their collective impact on reproductive physiology. Both in vivo and in vitro experiments were performed. Wistar Albino rats, divided into four groups: two non-pregnant groups (D1 and D2) and two pregnant groups (G1 and G2). The D1 and G1 groups served as control groups, while the D2 and G2 groups received chronic agomelatine administration (10 mg/kg). Uterine contractions were assessed in vitro using myometrial strips. Luzindole, a melatonin receptor antagonist, was employed to investigate the pathway mediating agomelatine's effects on uterine contractions. In in vivo studies, chronic agomelatine administration extended the diestrus phase (p<0.05) in non-pregnant rats, prolonged the gestational period (p<0.01), and increased the fetal count (p<0.01) in pregnant rats. Additionally, agomelatine reduced plasma oxytocin and prostoglandin-E levels (p<0.01) during pregnancy. In vitro experiments showed that agomelatine dose-dependently inhibited spontaneous and oxytocin-induced myometrial contractions. Luzindole (2 µM) reverse the agomelatine-induced inhibition of myometrial contractions. These findings suggest that agomelatine holds the potential to modulate diverse reproductive parameters during the gestational period, influencing estrus cycling, gestational progression, offspring development, and the orchestration of uterine contractions.

Melatonin in Endometriosis: Mechanistic Understanding and Clinical Insight.

Endometriosis is defined as the development of endometrial glands and stroma outside the uterine cavity. Pathophysiology of this disease includes abnormal hormone profiles, cell survival, migration, invasion, angiogenesis, oxidative stress, immunology, and inflammation. Melatonin is a neuroendocrine hormone that is synthesized and released primarily at night from the mammalian pineal gland. Increasing evidence has revealed that melatonin can be synthesized and secreted from multiple extra-pineal tissues where it regulates immune response, inflammation, and angiogenesis locally. Melatonin receptors are expressed in the uterus, and the therapeutic effects of melatonin on endometriosis and other reproductive disorders have been reported. In this review, key information related to the metabolism of melatonin and its biological effects is summarized. Furthermore, the latest in vitro and in vivo findings are highlighted to evaluate the pleiotropic functions of melatonin, as well as to summarize its physiological and pathological effects and treatment potential in endometriosis. Moreover, the pharmacological and therapeutic benefits derived from the administration of exogenous melatonin on reproductive system-related disease are discussed to support the potential of melatonin supplements toward the development of endometriosis. More clinical trials are needed to confirm its therapeutic effects and safety.

GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue.

The [35S]GTPγS assay is a method that measures the level of G protein activation by determining the binding of [35S]GTPγS, a non-hydrolyzable and radioactively labeled GTP analog, to Gα subunit of heterotrimeric G protein upon activation of G protein-coupled receptors (GPCR). The power of this assay lies in the fact that it measures an early event of GPCR signaling in cells expressing recombinant receptors and cells and tissues expressing endogenous receptors. The present protocol describes a sensitive method for studying G protein activation by melatonin receptors MT1 and MT2, in membranes prepared from mouse brain. Immunoprecipitation of [35S]GTPγS-labeled G proteins with Gα subunit specific antibodies (Gi, Gq, etc.) allows to determine the activation of specific G proteins. The assay can be easily applied to other tissues.