Different Concentrations Of Melatonin Research Articles

Screening for Suitable Melatonin Application Scheme to Modulate Drought Stress in Bulgarian Wheat

Drought occupies a dominant share among the environmental factors that directly cause significant economic losses. Wheat is one of the most important crops for humankind, and although it tolerates drought to a certain extent, prolonged water deficit provokes a number of unfavourable alterations in plants, which lead to a significant decrease in productivity. Because of the broadening climate changes, the search for ecological biostimulants as an innovative approach to reduce the harmful effects of drought is gaining more popularity in modern agriculture. Melatonin is a natural product of plant metabolism with growth-regulating properties – it is non-toxic and is safe for the environment. We aimed to investigate the possibilities to modulate the negative effects of drought through application of melatonin using a comparative analysis of the physiological reactions of young wheat seedlings. We studied the effects of melatonin applied before drought on the physiological responses of two Bulgarian wheat cultivars grown as a soil culture. Different concentrations of melatonin ranging from 5 to 500 μM were tested and 75 μM was selected as a promising one. Additionally, we tested the method of application of melatonin – leaf spray or root supplement. Based on the content of selected stress markers we could make a conclusion that leaf spraying of plants before stress had some attenuating effect on the negative consequences of drought, but one dose of root application appeared to be more effective. Abbreviations: MDA – malondialdehyde, Mel – melatonin, ROS – reactive oxygen species

Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies

Dendrobium officinale Kimura & Migo is a perennial herbaceous plant of the genus Dendrobium in the family of Orchidaceae with high medicinal value. Melatonin (MT) is an indole-like tryptamine with functions such as regulating plant growth and development. This experiment investigated the effects of different concentrations of MT on the growth and development of protocorms of D. officinale protocorm-like bodies (PLBs). The results showed that the changes in morphological indicators such as color, cluster size, and surface changes were more significant under 75 µM MT than those of 0 µM (CK), and the appearance of white on the PLB surface was expedited, which was more conducive to the proliferation of PLBs. MT treatment of 100 µM inhibited the differentiation of adventitious buds, and the contents of photosynthetic pigments, polysaccharides, and flavonoids were significantly increased. Moreover, as compared with CK, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PLBs increased significantly, while the content of malondialdehyde (MDA) decreased gradually with 75 µM or less. In conclusion, a concentration of 75 µM melatonin can enhance the rapid propagation rate of D. officinale in vitro, providing insights into the effects of melatonin on the growth of tissue-cultured D. officinale seedlings.

Melatonin Modulates Lipid Metabolism and Reduces Cardiovascular Risk in Apolipoprotein E-Deficient Mice Fed a Western Diet

Melatonin (MLT), a natural compound found in the animal and vegetable kingdom, participates in several physiological processes. MLT exerts antioxidant and anti-inflammatory activities, among others, but information about its action on lipid metabolism is still scarce. For this reason, mice deficient in apolipoprotein E (ApoE−/−) fed a Western diet (WD) were intragastrically treated with different concentrations of MLT (2 and 9 mg/kg) for 12 weeks. The lipid parameters were quantified, and, since links between cardiovascular risk and immune function and oxidative stress have been established, we also analyzed the population of leukocytes and the oxidative stress status. Although there was no change in the weight of the mice, a significant reduction in low-density lipoprotein cholesterol (LDL-C) was observed in mice treated with the higher concentration of MLT tested in this study. Additionally, an improvement in cardiovascular risk indexes was observed. A reduction in the hepatic total cholesterol (TC) and LDL-C levels was also observed in the treated mice. Finally, a decrease in leukocytes and lymphocytes in particular, as well as an increase in the antioxidant status, were shown in MLT-treated mice. In conclusion, MLT is a promising candidate that could be considered as a possible functional ingredient capable of preventing cardiovascular risk.

Impact of exogenous melatonin foliar application on physiology and fruit quality of wine grapes (Vitis vinifera) under salt stress.

Soil salinisation is an important abiotic stress faced in grape cultivating, leading to weakened plant vigour and reduced fruit quality. Melatonin as a novel hormone has shown positive exogenous application value. Therefore, this study used wine grape (Vitis vinifera ) 'Pinot Noir' as a test material to investigate the changes of foliar spraying with different concentrations of melatonin on the physiology and fruit quality of wine grapes in a field under simulated salt stress (200mmolL-1 NaCl). The results showed that foliar spraying of melatonin significantly increased the intercellular CO2 concentration, maximum photochemical quantum yield of PSII, relative chlorophyll and ascorbic acid content of the leaves, as well as the single spike weight, 100-grain weight, transverse and longitudinal diameters, malic acid, α-amino nitrogen and ammonia content of fruits, and decreased the initial fluorescence value of leaves, ascorbate peroxidase activity, glutathione content, fruit transverse to longitudinal ratio and tartaric acid content of plants under salt stress. Results of the comprehensive evaluation of the affiliation function indicated that 100μmolL-1 melatonin treatment had the best effect on reducing salt stress in grapes. In summary, melatonin application could enhance the salt tolerance of grapes by improving the photosynthetic capacity of grape plants under salt stress and promoting fruit development and quality formation, and these results provide new insights into the involvement of melatonin in the improvement of salt tolerance in crop, as well as some theoretical basis for the development and industrialisation of stress-resistant cultivation techniques for wine grapes.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Winemaking Technologies for the Production of Cabernet Sauvignon and Feteasca Neagra Wines Enriched with Antioxidant Active Principles Due to the Addition of Melatonin.

In recent years, various studies have been carried out to increase the concentration of antioxidant active principles in red wines as a consequence of the effects of winemaking techniques on the polyphenols content. In this study, in order to obtain the most optimal wine in terms of content and efficiency of antioxidant activity, various winemaking technologies (punching-down and pumping-over maceration) were tried with diverse gradations (Feteasca Neagra and Cabernet Sauvignon wines) and the addition of different concentrations of melatonin in must. Suitable HPLC and spectrophotometric methods were used to follow the evolution of the antioxidant compounds from wines during aging (for 12 months). After comparing the acquired results, an increase was observed in the antioxidant compound concentrations, particularly in resveratrol (85%), peonidin-3-glucoside (100%) or cyanidin-3-glucoside (100%), and antioxidant activity (10-40%). The most enriched wine was obtained in the case of Feteasca Neagra by the addition of 0.5 mg of melatonin per 1 kg of must using the punch-down technology and, in the case of Cabernet Sauvignon, by the addition of 0.05 mg of melatonin per 1 kg of must using the pumping-over technique. This study can provide winemakers with an approach to enhance red wines with antioxidant compounds.

Melatonin alleviates ischemic stroke by inhibiting ferroptosis through the CYP1B1/ACSL4 pathway.

This study utilized middle cerebral artery occlusion (MCAO) mouse models and HT-22 cell oxygen and glucose deprivation/reoxygenation (OGD/R) models to investigate the therapeutic effects of melatonin on ischemic brain injury. In the experiments, MCAO mice were treated with 5 and 10 mg/kg doses of melatonin, and H-T22 cells underwent OGD/R treatment and were administered different concentrations of melatonin. The results showed that melatonin significantly reduced ischemic brain area, neural damage, cerebral edema, and neuronal apoptosis in MCAO mice. In the HT-22 cell model, melatonin also improved cell proliferation ability, reduced apoptosis, and ROS production. Further mechanistic studies found that melatonin exerts protective effects by inhibiting ferroptosis, an iron-dependent form of regulated cell death, through regulation of the ACSL4/CYP1B1 pathway. In MCAO mice, melatonin decreased lipid peroxidation, ROS production, and ACSL4 protein expression. Overexpression of CYP1B1 increased ACSL4 ubiquitination and degradation, thereby increasing cell tolerance to ferroptosis, reducing ACSL4 protein levels, and decreasing ROS production. CYP1B1 knockdown obtained opposite results. The CYP1B1 metabolite 20-HETE induces expression of the E3 ubiquitin ligase FBXO10 by activating PKC signaling, which promotes ACSL4 degradation. In the OGD/R cell model, inhibition of CYP1B1 expression reversed the therapeutic effects of melatonin. In summary, this study demonstrates that melatonin protects the brain from ischemic injury by inhibiting ferroptosis through regulation of the ACSL4/CYP1B1 pathway, providing evidence for new therapeutic targets for ischemic brain injury.

Melatonin protects photoreceptor cells against ferroptosis in dry AMD disorder by inhibiting GSK-3B/Fyn-dependent Nrf2 nuclear translocation

BackgroundFerroptosis is a type of non-apoptotic cell death that relies on iron ions and reactive oxygen species to induce lipid peroxidation. This study aimed to determine whether ferroptosis exists in the pathogenesis of dry age-related macular degeneration (AMD) and to confirm that melatonin (MLT) suppresses the photoreceptor cell ferroptosis signaling pathway. MethodsWe exposed 661W cells to sodium iodate (NaIO3) in vitro and treated them with different concentrations of MLT. In vivo, C57BL/6 mice were given a single caudal vein injection of NaIO3, followed by an intraperitoneal injection of MLT, and eyeballs were taken for subsequent trials. ResultsWe found that NaIO3 could induce photoreceptor cell death and lipid peroxide accumulation, and result in changes in the expression of ferroptosis-related factors and iron maintenance proteins, which were treated by MLT. We further demonstrated that MLT can block Fyn-dependent Nrf2 nuclear translocation by suppressing the GSK-3β signaling pathway. In addition, the therapeutic effect of MLT was significantly inhibited when Nrf2 was silenced. ConclusionsOur findings provide a novel insight that NaIO3 induces photoreceptor cell ferroptosis in dry AMD and suggest that MLT has therapeutic effects by suppressing GSK-3β/Fyn-dependent Nrf2 nuclear translocation.

Effect of melatonin on cryopreservation of Beijing you chicken (gallus gallus) spermatozoa

Beijing You Chicken, a valuable local chicken breed from Beijing, China, was once listed as an endangered breed. From the point of view of conservation, the preservation of this breed is an important task for the local researchers. Semen cryopreservation is a popular method to maintain valuable species. However, during cryopreservation, semen is susceptible to oxidative damage. Melatonin is a potent antioxidant and free radical scavenger, so it has been selected to improve the efficiency of sperm cryopreservation. In this study, the chicken semen was treated with different concentrations of melatonin in the cryopreservation solution. The results showed that melatonin at concentrations of 10−3 M and 10−5 M significantly improved sperm progressive motility and total motility, respectively, compared to the control (P < 0.05). Melatonin at 10−3 M also significantly improved the plasma membrane and acrosome integrity of spermatozoa compared to the control. The mechanisms are that melatonin significantly reduces the level of ROS and preserves sperm mitochondrial membrane potential. Most importantly, the melatonin-treated cryopreserved chicken sperm after artificial insemination significantly increased the hatching rate of chicks compared to the control (p < 0.05). The results show that melatonin has a positive effect on the quality of the cryopreserved spermatozoa. These results provide the theoretical and practical basis for using melatonin to improve Beijing You Chicken conservation, and they may also be applicable to poultry as a whole.

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.

Effects of exogenous melatonin on growth and physiological characteristics of Agropyron mongolicum seedlings under drought stress.

To clarify the alleviation effect of exogenous melatonin (MT) on Agropyron mongolicum under drought stress, we examined the response of A. mongolicum 'Yanchi' seedlings to simulated drought stress with polyethylene glycol 6000 (PEG-6000), by investigating the effects of exogenous addition of different concentrations (0, 1, 10, 50, 100, 150 and 200 mg·L-1) of MT on seedlings growth and physiological characteristics under drought stress. The results showed that drought stress significantly inhibited the growth of A. mongolicum seedlings, and that exogenous addition of different concentrations of MT could alleviate the growth inhibition caused by drought stress, with the strongest mitigation effect observed at MT concentration of 100 mg·L-1. Compared with the drought stress treatment alone, exogenous addition of 100 mg·L-1 MT under drought stress increased plant height, aboveground dry weight, and leaf relative water content by 58.2%, 121.2% and 48.1%. The contents of chlorophyll a, chlorophyll b, carotenoids increased by 48.7%, 80.8% and 38.3%, superoxide dismutase, peroxidase and root activity increased by 12.6%, 33.9% and 39.1%, and the contents of ascorbic acid and glutathione increased by 19.5% and 18.3%, respectively. The contents of proline, soluble sugar and soluble protein were increased by 16.2%, 32.6% and 14.3%, while that of malondialdehyde, hydrogen peroxide and superoxide anion radical were decreased by 45.8%, 65.8% and 30.8%, respectively. In summary, exogenous addition of 100 mg·L-1 MT could improve drought tolerance of A. mongolicum seedlings by promoting growth, enhancing antioxidant capacity, increasing the content of osmoregulation substances, inhibiting the excessive production of reactive oxygen, and reducing membrane peroxide level.

Exogenous melatonin differentially affects biomass, total carbohydrates, and essential oil production in peppermint upon simultaneous exposure to chitosan-coated Fe3O4 NPs

Melatonin and iron play important roles in the growth processes and production of secondary metabolites in medicinal plants. To evaluate the effects of melatonin and Chitosan-Coated Iron Oxide Nanoparticles (Fe-CTs NPs) on peppermint growth, essential oil content and constituents, total carbohydrates and antioxidant enzyme activities, a factorial experiment was conducted based on randomized complete block design (CRBD) with three replications (n = 3). Treatments consisted of different concentrations of melatonin (0, 100, and 200 mg L−1) and Fe-CTs NPs (0, 7.5, and 15 mg L−1). During the experiment, foliar applications of melatonin and Fe-CTs NPs were performed three times with 20 days’ interval. Treatments with melatonin, and Fe-CTs NPs had significant effects on the studied traits. The results showed that with increasing melatonin and Fe-CTs NPs concentrations, fresh and dry weights, total carbohydrates, and leaf area index were significantly (P < 0.05) increased in treated plants. The highest CAT and APX activities were observed in plants treated with melatonin at 100 mg L−1 and Fe-CTs NPs at 15 mg L−1. Eucalyptol, Menthyl acetate, Thiophene, 2-ethyl-5-(2-methylpropyl), Mint furanone, and Viridiflorol were found as the major essential oil constituents in peppermint plants under experimental treatments. Moreover, analytical results showed that the highest amount of Menthol (38.48 %) was obtained in plants exposed to Fe-CTs NPs at 7.5 mg L−1, however, the highest L-Menthone (30.53 %) was observed in plants treated with melatonin at 200 mg L−1 and Fe-CTs NPs at 15 mg L−1. The findings provide insights into the regulatory mechanisms of melatonin and Fe-CTs NPs on peppermint growth, physiology and biosynthesis of volatile compounds.

Melatonin-induced development of adventitious roots, biosynthesis of secondary cell products and greener synthesis of silver nanoparticles for biological activities using Indigofera heterantha

Indigofera heterantha is a medicinal shrub of the family Fabaceae that naturally produces pharmaceutically important polyphenolics and at the verge of extinction due to ruthless exploitation. The adventitious roots provide bases for the conservation of endangered plants by decreasing pressure on their wild populations and help in the large-scale production of pharmacologically important secondary metabolites. The main objective of the current study was to establish a feasible protocol for adventitious roots and to investigate the effect of melatonin on the biosynthesis of biomass, secondary cell products, and greener synthesis of silver nanoparticles (AgNPs) for various biological activities. Melatonin is an important growth promoter and plays a key role in plant defense that can be used as elicitors for large-scale production of secondary metabolites. Different concentrations of melatonin revealed that 1.5 mg l−1 melatonin was effective for the biosynthesis of maximum biomass during log phases of growth kinetics. The same melatonin concentration enhanced total phenolics content (TPC; 68.28 mg/g-DW), total flavonoids content (TFC; 24.34 mg/g-DW), total phenolics production (TPP; 153.63 g l−1), total flavonoids production (TFP; 54.80 g l−1), antioxidant activity and total secondary metabolites in adventitious roots. The melatonin-treated roots (300 mM) were able to produce maximum biogenic AgNPs, characterized by UV–vis spectroscopy (UV), scanning electron microscope (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) and were used for various biological activities. These NPs displayed maximum antimicrobial activities and further revealed that AgNPs and aqueous root extract displayed higher cytotoxic activities. The protocol developed here provides an alternative strategy for germplasm conservation and melatonin proved to be one of the best elicitors for different activities and has the potential of scaling up by various pharmaceutical industrial applications.

Extended Post-Harvest Effect of Melatonin in Fresh-Cut Broccolini Plants (Bimi®)

Melatonin has been shown to be effective as a post-harvest agent in fruits and vegetables. Melatonin has been used in the preservation of Brassicaceae such as broccoli, but not in broccolini. Here, fresh-cut broccolini (baby broccoli or BIMI®) were treated with different concentrations of melatonin (50 and 100 µM) for 15 or 30 min and cold stored for 28 days. Melatonin-treated broccolini had a longer shelf life than control samples, as seen by fresh weight (weight loss rate), hue angle (expresses color quality), and chlorophyll and carotenoid concentrations. Treatments with 50 µM melatonin for 15 min appear to be the most effective, reducing water loss by around 28% compared to the control and increasing the shelf life of fresh-cut broccolini, presenting an optimal conservation time of 7 days, and this could even be extended up to 14 days with acceptable quality. In addition, antioxidant, phenolic, and flavonoid contents were improved in melatonin-treated broccolini plants. Lipid peroxidation damage by cold storage was decreased in melatonin-treated broccolini, with a relevant decrease in malondialdehyde contents, especially 50 µM, on all days of storage. This study demonstrates for the first time the effectiveness of melatonin treatments in extending the shelf life of broccolini during cold storage. The possible commercial use of these treatments will be subject to the processes in the product management and marketing chain. However, more specific research about changes in organoleptic properties is necessary.

Combine with RNA-seq Reveals the Effect of Melatonin in the Synthesis of Melanin in Primary Melanocytes of Silky Fowls Black-Bone Chicken.

(1) Background: It was found that the melanin of black-bone chicken has various effects such as scavenging DPPH free radicals and anti-oxidation, and the synthesis of melanin is affected by various factors including hormones. In addition, several studies have found that melatonin affects the melanoma cell synthesis of melanin, which has not been reported in chicken primary melanocytes; so, relevant studies were conducted. (2) Methods: In this study, chicken primary melanocytes were isolated and characterized, and then melanocytes were treated with different concentrations of melatonin to investigate the effects of melatonin on melanin synthesis in chicken melanocytes in terms of melanin synthesis-related genes, melanin content, and tyrosinase activity, and combined with RNA seq to detect the change in gene expression level of chicken melanocytes after melatonin treatment. (3) Results: We isolated and characterized primary melanocytes, and indirect immunofluorescence assay results showed positive melanocyte marker genes. RT-qPCR results showed that melatonin decreased the expression of melanin synthesis-related genes. In addition, melatonin reduced the melanin content and decreased the tyrosinase activity of melanocytes in the treated group. A total of 1703 differentially expressed genes were screened by RNA-seq, and in addition, in the KEGG results, the signaling pathway associated with melanin synthesis, and the mTOR signaling pathway were enriched. (4) Conclusions: Melatonin could decrease the synthesis of melanin in chicken primary melanocytes.

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats

To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

Melatonin enhances cell death and suppresses the metastatic capacity of ovarian cancer cells by attenuating the signaling of multiple kinases

BackgroundOvarian cancer is a highly aggressive disease that is frequently diagnosed in advanced stages. Melatonin, with its numerous antitumor properties, holds great promise in cancer treatment. Herein, we investigated the effects of melatonin on apoptosis, cell migration, and kinase levels in human ovarian carcinoma SKOV-3 cells and determined whether these effects are mediated by the activation of the MT1 receptor. MethodsSKOV-3 cells were exposed to different concentrations of melatonin based on the presence of MT1 receptor, and we also performed specific silencing of the melatonin receptor gene MTNR1A. ResultsOur findings revealed that melatonin reduced cell viability as shown by the MTT assay, and flow cytometry analysis showed increased rates of apoptosis and necrosis in all melatonin-treated cells. Melatonin significantly decreased the migratory and invasive capacities of the cells. Propidium iodide labeling indicated that melatonin induced cell cycle arrest by reducing DNA content in the S and G2/M phases in SKOV-3 cells. Additionally, the levels of AKT, ERK1/2, JNK, CREB, p70S6K, STAT3/5, and p38 MAP kinase involved in cell survival, proliferation, motility, and stress responses were depressed by melatonin and further reduced after MT1 knockdown. These molecules were found to be associated with lower overall survival in ovarian cancer patients. ConclusionsMelatonin had obvious oncostatic actions on ovarian cancer cells, and MT1 receptor knockdown intensified its antitumor effect. The inhibition of the MT1 receptor resulted in a substantial reduction in the migratory and invasive capacities of the cells, suggesting its potential as a therapeutic target for the treatment of ovarian cancer.

Melatonin blunted the angiogenic activity in 3D colon cancer tumoroids by the reduction of endocan

BackgroundComplexity and heterogeneity of the tumor niche are closely associated with the failure of therapeutic protocols. Unfortunately, most data have been obtained from conventional 2D culture systems which are not completely comparable to in vivo microenvironments. Reconstructed 3D cultures composed of multiple cells are valid cell-based tumor models to recapitulate in vivo-like interaction between the cancer cells and stromal cells and the oncostatic properties of therapeutics. Here, we aimed to assess the tumoricidal properties of melatonin on close-to-real colon cancer tumoroids in in vitro conditions.MethodsUsing the hanging drop method, colon cancer tumoroids composed of three cell lines, including adenocarcinoma HT-29 cells, fibroblasts (HFFF2), and endothelial cells (HUVECs) at a ratio of 2: 1: 1, respectively were developed using 2.5% methylcellulose. Tumoroids were exposed to different concentrations of melatonin, from 0.005 to 0.8 mM and 4 to 10 mM, for 48 h. The survival rate was measured by MTT and LDH leakage assays. Protein levels of endocan and VEGF were assessed using western blotting. Using histological examination (H & E) staining, the integrity of cells within the tumoroid parenchyma was monitored.ResultsDespite the reduction of viability rate in lower doses, the structure of tumoroids remained unchanged. In contrast, treatment of tumoroids with higher doses of melatonin, 4 and 10 mM, led to disaggregation of cells and reduction of tumoroid diameter compared to the non-treated control tumoroids (p < 0.05). By increasing melatonin concentration from 4 to 10 mM, the number of necrotic cells increased. Data showed the significant suppression of endocan in melatonin-treated tumoroids related to the non-treated controls (p < 0.05). According to our data, melatonin in higher doses did not alter protein levels of VEGF (p > 0.05).ConclusionsMelatonin can exert its tumoricidal properties on colon cancer tumoroids via the reduction of tumor cell viability and inhibition of the specific pro-angiogenesis factor.

Physiological and Biochemical Mechanisms of Exogenous Melatonin Regulation of Saline–Alkali Tolerance in Oats

Saline–alkali stress is one of the major factors limiting oat seed germination. The regulatory role of melatonin (MT) as a naturally occurring active substance is well known, but the mechanism of MT-mediated intrinsic physiological regulation of oat seed germination under saline–alkali stress is unclear. Therefore, this study investigated (1) the variability of different MT seed soaking concentrations and times on the germination of oat seeds under saline–alkali stress, and (2) the possible physiological regulatory mechanisms of MT on the germination of oat seeds under saline–alkali stress. The results showed that seed vigor was significantly reduced under saline–alkali stress, and seed germination of oats was significantly inhibited; different concentrations of MT seed soaking treatments improved the germination rate, germination potential, germination index, vigor index, root length, germ length, fresh weight, and dry weight; and, overall, treatment improved seed germination and exhibited the phenomenon of “low promotion and high inhibition”. The 100 μmol·L−1 MT seed soaking treatment effectively enhanced the activities of seed antioxidant enzymes (SOD, POD, CAT, and APX) and nonenzymatic antioxidants (GSH and AsA), affected the AsA-GSH cycle, and effectively increased the contents of osmoregulatory substances (proline, protein, and soluble sugar) by reducing levels of H2O2, OH−, and MDA, thus enhancing the tolerance of oats to saline–alkali and promoting seed germination. In conclusion, MT has a positive effect on the saline–alkali tolerance of oat seeds.

Formulation and Characterization of Electrospun Nanofibers for Melatonin Ocular Delivery.

The poor ocular bioavailability of melatonin (MEL) limits the therapeutic action the molecule could exert in the treatment of ocular diseases. To date, no study has explored the use of nanofiber-based inserts to prolong ocular surface contact time and improve MEL delivery. Here, the electrospinning technique was proposed to prepare poly (vinyl alcohol) (PVA) and poly (lactic acid) (PLA) nanofiber inserts. Both nanofibers were produced with different concentrations of MEL and with or without the addition of Tween® 80. Nanofibers morphology was evaluated by scanning electron microscopy. Thermal and spectroscopic analyses were performed to characterize the state of MEL in the scaffolds. MEL release profiles were observed under simulated physiological conditions (pH 7.4, 37 °C). The swelling behavior was evaluated by a gravimetric method. The results confirmed that submicron-sized nanofibrous structures were obtained with MEL in the amorphous state. Different MEL release rates were achieved depending on the nature of the polymer. Fast (20 min) and complete release was observed for the PVA-based samples, unlike the PLA polymer, which provided slow and controlled MEL release. The addition of Tween® 80 affected the swelling properties of the fibrous structures. Overall, the results suggest that membranes could be an attractive vehicle as a potential alternative to liquid formulations for ocular administration of MEL.