Melatonin Pretreatment Research Articles

Melatonin alleviates cold-induced oxidative damage by regulation of ascorbate–glutathione and proline metabolism in melon seedlings (Cucumis melo L.)

ABSTRACTCold stress is one of the most detrimental environmental factors affecting plant growth and development. Melatonin (MEL), a natural indoleamine compound, responds to various environmental cues. To explore the role of MEL in the response of melon (Cucumis melo L.) seedlings to cold stress, the effects of exogenous MEL on the ascorbate–glutathione (AsA–GSH) cycle and proline metabolism were investigated. Melon seedlings were sprayed with various concentrations of MEL (0, 50, 100, 200, or 400 μM), then exposed to cold stress, 12/6°C (day/night) for 7 d, followed by recovery at 28/18°C for another 7 d. The results showed that MEL, especially the 200 μM treatment, dramatically alleviated growth inhibition caused by cold stress, manifested by increased plant growth and decreased O2•¯ production rate and malondialdehyde content. Importantly, exogenous application of MEL enhanced the ratios of reduced and oxidized forms of AsA (AsA/DHA) and GSH (GSH/GSSG), and the activities of ascorbate peroxidase (APX), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) involved in the ascorbate–glutathione (AsA–GSH) cycle in melon seedings grown under cold stress. Besides, MEL pretreatment further increased the contents of proline and soluble protein under cold stress. The results reveal that protective effects of MEL against cold stress in melon seedlings are most likely associated with the regulation of the AsA–GSH cycle and proline metabolism as an effective antioxidant system.

Melatonin Pre-treatment Alleviates UVA Radiation Induced Oxidative Stress and Apoptosis in the Skin of a Diurnal Tropical Rodent Funambulus pennanti

UV radiation has been established as a pro-oxidant that mediates tissue injury by triggering the generation of free radicals. However UVA induced oxidative injury has never been investigated before in the skin of a tropical diurnal rodent which spends longer time directly under the sun for foraging. The present study aimed to note the level of oxidative stress induced by 6.36 Jcm-2 UVA radiations on the skin and its possible prevention by melatonin. The oxidative load was assessed by the activities of key antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)) and generation of thiobarbituric acid reactive substances (TBARS). Cutaneous apoptosis if any was checked by the expression of Bcl-2, p53 and Heme oxygenase-I (HO-I). Melatonin membrane receptor expression (MT1) and AANAT activity was also investigated to elucidate the protective effect of melatonin. UVA radiation increased the lipid peroxidation, suppressed the enzymatic antioxidant defense system and increased the HO-I expression. Melatonin restored redox balance and up regulated Bcl-2 and down regulated p53 levels. The restoration of AANAT activity also proved beneficial. In summary melatonin can be a part of topical applications or oral supplements that might help to reduce the UVA radiation mediated cutaneous oxidative damages.

Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells.

Many obstacles compromise the efficacy of bone marrow mesenchymal stem cells (BM-MSCs) by inducing apoptosis in the grafted BM-MSCs. The current study investigates the effect of melatonin on important mediators involved in survival of BM-MSCs in hydrogen peroxide (H2O2) apoptosis model. In brief, BM-MSCs were isolated, treated with melatonin, and then exposed to H2O2. Their viability was assessed by MTT assay and apoptotic fractions were evaluated through Annexin V, Hoechst staining, and ADP/ATP ratio. Oxidative stress biomarkers including ROS, total antioxidant power (TAP), superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH), thiol molecules, and lipid peroxidation (LPO) levels were determined. Secretion of inflammatory cytokines (TNF-α and IL-6) were measured by ELISA assay. The protein expression of caspase-3, Bax, and Bcl-2, was also evaluated by Western blotting. Melatonin pretreatment significantly increased viability and decreased apoptotic fraction of H2O2-exposed BM-MSCs. Melatonin also decreased ROS generation, as well as increasing the activity of SOD and CAT enzymes and GSH content. Secretion of inflammatory cytokines in H2O2-exposed cells was also reduced by melatonin. Expression of caspase-3 and Bax proteins in H2O2-exposed cells was diminished by melatonin pretreatment. The findings suggest that melatonin may be an effective protective agent against H2O2-induced oxidative stress and apoptosis in MSC.

Beneficial Roles of Melatonin on Redox Regulation of Photosynthetic Electron Transport and Synthesis of D1 Protein in Tomato Seedlings under Salt Stress.

Melatonin is important in the protection of plants suffering various forms of abiotic stress. The molecular mechanisms underlying the melatonin-mediated protection of their photosynthetic machinery are not completely resolved. This study investigates the effects of exogenous melatonin applications on salt-induced damage to the light reaction components of the photosynthetic machinery of tomato seedlings. The results showed that melatonin pretreatments can help maintain growth and net photosynthetic rate (PN) under salt stress conditions. Pretreatment with melatonin increased the effective quantum yield of photosystem II (ΦPSII), the photochemical quenching coefficient (qP) and the proportion of PSII centers that are “open” (qL) under saline conditions. In this way, damage to the photosynthetic electron transport chain (PET) in photosystem II (PSII) was mitigated. In addition, melatonin pretreatment facilitated the repair of PSII by maintaining the availability of D1 protein that was otherwise reduced by salinity. The ROS levels and the gene expressions of the chloroplast TRXs and PRXs were also investigated. Salt stress resulted in increased levels of reactive oxygen species (ROS), which were mitigated by melatonin. In tomato leaves under salt stress, the expressions of PRXs and TRXf declined but the expressions of TRXm1/4 and TRXm2 increased. Melatonin pretreatment promoted the expression of TRXf and the abundances of TRXf and TRXm gene products but had no effects on the expressions of PRXs. In summary, melatonin improves the photosynthetic activities of tomato seedlings under salt stress. The mechanism could be that: (1) Melatonin controls ROS levels and prevents damaging elevations of ROS caused by salt stress. (2) Melatonin facilitates the recovery of PET and D1 protein synthesis, thus enhancing the tolerance of photosynthetic activities to salinity. (3) Melatonin induces the expression of TRXf and regulates the abundance of TRXf and TRXm gene products, which may facilitate repair of the light reaction parts of the photosynthetic machinery.

Melatonin Attenuates Manganese and Lipopolysaccharide-Induced Inflammatory Activation of BV2 Microglia.

Melatonin, a naturally occurring neurohormone in the pineal gland, has been shown to exert antioxidant and anti-inflammatory effects. This study examined the effects of melatonin on manganese (Mn) and/or lipopolysaccharide (LPS)-induced microglial activation. Melatonin (10μM) inhibited Mn (100μM) and/or LPS (0.5μg/ml)-induced phagocytotic activity of activated BV2 microglia. It also inhibited the lipid peroxidation and intracellular reduced glutathione (GSH) depletion induced by Mn and/or LPS. Melatonin effectively suppressed the upregulation of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) at both mRNA and protein levels in Mn and/or LPS-stimulated BV2 microglia. In addition, melatonin pretreatment attenuated Mn and/or LPS-induced degradation of IκB-α, nuclear translocation of nuclear factor-κB (NF-κB) and its activation, and the expressions of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in BV2 microglial cells. These results suggest that melatonin can effectively modulate phagocytosis and expression of proinflammatory mediators, and can prevent neuroinflammatory disorders accompanied by microglial activation.

Phytanic Acid-Induced Neurotoxicological Manifestations and Apoptosis Ameliorated by Mitochondria-Mediated Actions of Melatonin.

Phytanic acid, a saturated branched chain fatty acid and a major constituent of human diet, is predominantly found in dairy products, meat, and fish. It is a degradation product from the phytol side chain of chlorophyll. Degradation of PA is known to occur mainly in peroxisomes via α-oxidation and in mitochondria via β-oxidation. Due to its β-methyl group present at the 3-position of the carbon atoms, PA cannot be β-oxidized. Although alteration in the metabolism of PA may play an important role in neurodegeneration, the exact mechanism behind it remains to be evaluated. In this study, we have described the potential of PA to induce neurotoxicity as an in vitro model (neuronal cell line, SH-SY5Y cells). Cells were pretreated with melatonin (10μM) for 1h followed by with and without PA (100μM) for 24h. In the present study, our data has confirmed that PA markedly increased both intracellular reactive oxygen species and reactive nitrogen species levels. Our results have shown that PA treatment did not induce cell death by cleavage of caspase-3/PARP-1 mediated by mitochondria through intrinsic pathways; however, PA induced nitric oxide-dependent apoptosis in SH-SY5Y cells. Additionally, melatonin pretreatment reduced the cell death in SH-SY5Y cells. Melatonin also effectively exerted an antiapoptotic and anti-inflammatory action by regulating Bax, Bcl-2, p-NFκB, and iNOS expressions in SH-SY5Y cells. These results suggested that melatonin acted as an antioxidative and antiapoptotic agent by modulating ROS, apoptotic proteins, and inflammatory responses under BCFA-induced neurotoxic conditions. The protective effects of melatonin depend on direct scavenging activity of free radicals and indirect antioxidant effects. Further deciphering of the cellular and molecular mechanism associated with neuroprotection by melatonin is warranted in BCFA-induced neurotoxicity.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

Melatonin reverses H-89 induced spatial memory deficit: Involvement of oxidative stress and mitochondrial function

Oxidative stress and mitochondrial dysfunction play indispensable role in memory and learning impairment. Growing evidences have shed light on anti-oxidative role for melatonin in memory deficit. We have previously reported that inhibition of protein kinase A by H-89 can induce memory impairment. Here, we investigated the effect of melatonin on H-89 induced spatial memory deficit and pursued their interactive consequences on oxidative stress and mitochondrial function in Morris Water Maze model. Rats received melatonin (50 and 100μg/kg/side) and H-89(10μM) intra-hippocampally 30min before each day of training. Animals were trained for 4 consecutive days, each containing one block from four trials. Oxidative stress indices, including thiobarbituric acid (TBARS), reactive oxygen species (ROS), thiol groups, and ferric reducing antioxidant power (FRAP) were assessed using spectrophotometer. Mitochondrial function was evaluated through measuring ROS production, mitochondrial membrane potential (MMP), swelling, outer membrane damage, and cytochrome c release. As expected from our previous report, H-89 remarkably impaired memory by increasing the escape latency and traveled distance. Intriguingly, H-89 significantly augmented TBARS and ROS levels, caused mitochondrial ROS production, swelling, outer membrane damage, and cytochrome c release. Moreover, H-89 lowered thiol, FRAP, and MMP values. Intriguingly, melatonin pre-treatment not only effectively hampered H-89-mediated spatial memory deficit at both doses, but also reversed the H-89 effects on mitochondrial and biochemical indices upon higher dose. Collectively, these findings highlight a protective role for melatonin against H-89-induced memory impairment and indicate that melatonin may play a therapeutic role in the treatment of oxidative- related neurodegenerative disorders.

Melatonin reduces PERK-eIF2α-ATF4-mediated endoplasmic reticulum stress during myocardial ischemia-reperfusion injury: role of RISK and SAFE pathways interaction.

Recently, we demonstrated that melatonin reduced protein kinase RNA (PKR)-like ER kinase (PERK)-eukaryotic initiation factor 2 alpha (eIF2α)-activating transcription factor-4 (ATF4)-mediated myocardial endoplasmic reticulum (ER) stress and apoptosis during myocardial ischemia-reperfusion (MI/R) injury. However, the underlying mechanisms are still not clear. Myocardial reperfusion injury salvage kinase (RISK) pathway as well as survivor activating factor enhancement (SAFE) pathway are two pivotal intrinsic pro-survival signaling cascades. In this study, we performed in vivo and in vitro experiment to investigate the ameliorative effect of melatonin on ER stress with a focus on RISK and SAFE pathways interaction. Male C57Bl/6 mice received melatonin (300μg/25g/day, 3days before MI/R surgery; 300μg/25g, 25min before the onset of ischemia) pre-treatment with or without the administration of LY294002 (a PI3K/Akt inhibitor), U0126 (an ERK1/2 inhibitor) or AG490 (a STAT3 pathway inhibitor). H9c2 cells were pre-treated with melatonin (100μM, 8h) in the presence or absence of LY294002, U0126 or AG490. Compared with the I/R-injured group, melatonin effectively reduced myocardial apoptosis, oxidative stress and improved cardiac function. In addition, melatonin pre-treatment also increased the phosphorylation of Akt, GSK-3β, ERK1/2 and STAT3 and reduced PERK-eIF2α-ATF4-mediated ER stress. However, these effects were blocked by LY294002, U0126 or AG490. Additionally, either LY294002 or U0126 treatment could inhibit STAT3 phosphorylation, whereas AG490 administration also reduced both Akt and ERK1/2 phosphorylation, indicating an interplay exists between RISK and SAFE pathways in melatonin's cardioprotective effect. In summary, our study demonstrates that RISK and SAFE pathways mediate the cardioprotective effect of melatonin against MI/R injury. Melatonin pre-treatment attenuates PERK-eIF2α-ATF4-mediated ER stress and apoptosis during MI/R injury via RISK and SAFE pathways interaction.

Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition.

Microvascular hyperpermeability that occurs at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema and elevated intracranial pressure following traumatic brain injury (TBI). At a cellular level, tight junction proteins (TJPs) between neighboring endothelial cells maintain the integrity of the BBB via TJ associated proteins particularly, zonula occludens-1 (ZO-1) that binds to the transmembrane TJPs and actin cytoskeleton intracellularly. The pro-inflammatory cytokine, interleukin-1β (IL-1β) as well as the proteolytic enzymes, matrix metalloproteinase-9 (MMP-9) are key mediators of trauma-associated brain edema. Recent studies indicate that melatonin a pineal hormone directly binds to MMP-9 and also might act as its endogenous inhibitor. We hypothesized that melatonin treatment will provide protection against TBI-induced BBB hyperpermeability via MMP-9 inhibition. Rat brain microvascular endothelial cells grown as monolayers were used as an in vitro model of the BBB and a mouse model of TBI using a controlled cortical impactor was used for all in vivo studies. IL-1β (10 ng/mL; 2 hours)-induced endothelial monolayer hyperpermeability was significantly attenuated by melatonin (10 μg/mL; 1 hour), GM6001 (broad spectrum MMP inhibitor; 10 μM; 1 hour), MMP-9 inhibitor-1 (MMP-9 specific inhibitor; 5 nM; 1 hour) or MMP-9 siRNA transfection (48 hours) in vitro. Melatonin and MMP-9 inhibitor-1 pretreatment attenuated IL-1β-induced MMP-9 activity, loss of ZO-1 junctional integrity and f-actin stress fiber formation. IL-1β treatment neither affected ZO-1 protein or mRNA expression or cell viability. Acute melatonin treatment attenuated BBB hyperpermeability in a mouse controlled cortical impact model of TBI in vivo. In conclusion, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP-9 inhibition. In addition, acute melatonin treatment provides protection against BBB hyperpermeability in a mouse model of TBI indicating its potential as a therapeutic agent for brain edema when established in humans.

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition.

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 10(5) each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham-control (SC), IR (60-min left-lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA damage (γ-H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1β/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO(+) /CD68(+) /CD14(+) cells), and DNA damage (γ-H2AX(+) cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin-supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.

Melatonin Improved Anthocyanin Accumulation by Regulating Gene Expressions and Resulted in High Reactive Oxygen Species Scavenging Capacity in Cabbage.

In this work, we found, that exogenous melatonin pretreatment improved anthocyanin accumulation (1- to 2-fold) in cabbage. To verify the relationship with melatonin and anthocyanin, an Arabidopsis mutant, snat, which expresses a defective form of the melatonin biosynthesis enzyme SNAT (Serotonin N-acetyl transferase), was employed. Under cold conditions, the foliage of wild-type Arabidopsis exhibited a deeper red color than the snat mutant. This finding further proved, that exogenous melatonin treatment was able to affect anthocyanin accumulation. To gain a better understanding of how exogenous melatonin upregulates anthocyanin, we measured gene expression in cabbage samples treated with melatonin and untreated controls. We found that the transcript levels of anthocyanin biosynthetic genes were upregulated by melatonin treatment. Moreover, melatonin treatment increased the expression levels of the transcription factors MYB, bHLH, and WD40, which constitute the transcriptional activation complex responsible for coordinative regulation of anthocyanin biosynthetic genes. We found, that free radical generation was downregulated, whereas the osmotic adjustment and antioxidant capacities were upregulated in exogenous melatonin-treated cabbage plants. We concluded, that melatonin increases anthocyanin production and benefits cabbage growth.

Melatonin reverses morphine tolerance by inhibiting microglia activation and HSP27 expression

AimsMelatonin has been reported to attenuate opioid tolerance. In this study, we explored the possible mechanism of melatonin in diminishing morphine tolerance. Main methodsTwo intrathecal (i.t.) catheters were implanted in male Wistar rats for drug delivery. One was linked to a mini-osmotic pump for morphine or saline infusion. On the seventh day, 50μg of melatonin or vehicle was injected through the other catheter instantly after discontinuation of morphine or saline infusion; 3h later, 15μg of morphine or saline was injected. The antinociceptive response was then measured using the tail-flick test every 30min for 120min. Key findingsThe results showed that chronic morphine infusion elicited antinociceptive tolerance and upregulated heat shock protein 27 (HSP27) expression in the dorsal horn of the rat spinal cord. Melatonin pretreatment partially restored morphine's antinociceptive effect in morphine-tolerant rats and reversed morphine-induced HSP27 upregulation. In addition, chronic morphine infusion induced microglial cell activation and was reversed by melatonin treatment. SignificanceThe present study provides evidence that melatonin, acting via inhibiting morphine-induced neuroinflammation, can be useful as a therapeutic adjuvant for patients under long-term opioid treatment for pain relief.

Melatonin attenuates 60 Co γ-ray-induced hematopoietic, immunological and gastrointestinal injuries in C57BL/6 male mice.

Protection of hematopoietic, immunological, and gastrointestinal injuries from deleterious effects of ionizing radiation is prime rational for developing radioprotector. The objective of this study, therefore, was to evaluate the radioprotective potential of melatonin against damaging effects of radiation-induced hematopoietic, immunological, and gastrointestinal injuries in mice. C57BL/6 male mice were intraperitoneally administered with melatonin (50-150mg/kg) 30min prior to whole-body radiation exposure of 5 and 7.5Gy using 60 Co-teletherapy unit. Thirty-day survival against 7.5Gy was monitored. Melatonin (100mg/kg) pretreatment showed 100% survival against 7.5Gy radiation dose. Melatonin pretreatment expanded femoral HPSCs, and inhibited spleenocyte DNA strands breaks and apoptosis in irradiated mice. At this time, it also protected radiation-induced loss of T cell sub-populations in spleen. In addition, melatonin pretreatment enhanced crypts regeneration and increased villi number and length in irradiated mice. Translocation of gut bacteria to spleen, liver and kidney were controlled in irradiated mice pretreated with melatonin. Radiation-induced gastrointestinal DNA strand breaks, lipid peroxidation, and expression of proapoptotic-p53, Bax, and antiapoptotic-Bcl-xL proteins were reversed in melatonin pretreated mice. This increase of Bcl-xL was associated with the decrease of Bax/Bcl-xL ratio. ABTS and DPPH radical assays revealed that melatonin treatment alleviated total antioxidant capacity in hematopoietic and gastrointestinal tissues. Present study demonstrated that melatonin pretreatment was able to prevent hematopoietic, immunological, and gastrointestinal radiation-induced injury, therefore, overcoming lethality in mice. These results suggest potential of melatonin in developing radioprotector for protection of bone marrow, spleen, and gastrointestine in planned radiation exposure scenarios including radiotherapy. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 501-518, 2017.

Effect of exogenous melatonin on physiological and biochemical characteristics of cucumber radicles under p-hydroxybenzoic acid.

'Jinyan No. 4'cucumber was used as experimental material. The growth of cucumber radicles treated with different concentrations of p-hydroxybenzoic acid (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 mmol·L-1) was investigated firstly, and 10 mmol·L-1 p-hydroxybenzoic acid was chosen for the further experiment. To investigate if melatonin alleviate p-hydroxybenzoic acid stress on cucumber radical elongation, 0, 1, 10, 25, 50, 75 and 100 μmol·L-1 melatonin were used to pretreat cucumber seeds for 24 h before 10 mmol·L-1 p-hydroxybenzoic acid treatment. The results suggested that exogenous melatonin could alleviate the radicle growth inhibition induced by p-hydroxybenzoic acid, and 75 μmol·L-1 melatonin showed the best effect. The growth of cucumber radical was significantly inhibited with an increase in p-hydroxybenzoic acid concentration, which was accompanied with lower activity of amylase. The melatonin pretreatment could significantly increase the activities of amylase and antioxidant enzymes in cucumber seeds resulting in lower accumulation of O2-·, H2O2 and MDA. Exogenous melatonin effectively reduced the PHBA stress on cucumber germination, which might be due to its function in decreasing oxidative stress and promoting starch catabolism.

Melatonin prevents abnormal mitochondrial dynamics resulting from the neurotoxicity of cadmium by blocking calcium-dependent translocation of Drp1 to the mitochondria.

Cadmium (Cd) is a persistent environmental toxin and occupational pollutant that is considered to be a potential risk factor in the development of neurodegenerative diseases. Abnormal mitochondrial dynamics are increasingly implicated in mitochondrial damage in various neurological pathologies. The aim of this study was to investigate whether the disturbance of mitochondrial dynamics contributed to Cd-induced neurotoxicity and whether melatonin has any neuroprotective properties. After cortical neurons were exposed to 10 μM cadmium chloride (CdCl2 ) for various periods (0, 3, 6, 12, and 24 hr), the morphology of their mitochondria significantly changed from the normal tubular networks into punctuated structures within 3 hr. Following this pronounced mitochondrial fragmentation, Cd treatment led to signs of mitochondrial dysfunction, including excess reactive oxygen species (ROS) production, decreased ATP content, and mitochondrial membrane potential (▵Ψm) loss. However, 1 mM melatonin pretreatment efficiently attenuated the Cd-induced mitochondrial fragmentation, which improved the turnover of mitochondrial function. In the brain tissues of rats that were intraperitoneally given 1 mg/kg CdCl2 for 7 days, melatonin also ameliorated excessive mitochondrial fragmentation and mitochondrial damage in vivo. Melatonin's protective effects were attributed to its roles in preventing cytosolic calcium ([Ca(2+) ]i ) overload, which blocked the recruitment of Drp1 from the cytoplasm to the mitochondria. Taken together, our results are the first to demonstrate that abnormal mitochondrial dynamics is involved in cadmium-induced neurotoxicity. Melatonin has significant pharmacological potential in protecting against the neurotoxicity of Cd by blocking the disbalance of mitochondrial fusion and fission.

Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.

Adipogenic differentiation is characterized by an increase in two major transcription factors: peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT/enhancer binding protein alpha (C/EBPα). These two signals are influenced by C/EBPβ and C/EBPδ and cross-regulate each other's expression during the initial stages of adipogenesis. Melatonin has been known to act as not only a direct scavenger of free radicals but also an inhibitor of glycogen synthase kinase 3β (GSK-3β). Here, we report that melatonin inhibits the adipogenic differentiation of human mesenchymal stem cells (hMSCs) which is due to the regulations of C/EBPβ in the early stage of adipogenic differentiation. Melatonin reduced the lipid accumulation, adiponectin, and lipoprotein lipase (LPL) during the adipogenic differentiation of hMSCs. Since C/EBPβ has been associated with the activation of PPARγ and the consensus site of ERK/GSK-3β, PPARγ and β-catenin were detected by immunofluorescence staining after pretreatment of melatonin. Melatonin blocked the activation of PPARγ which induced the degradation of β-catenin. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate (cAMP) and reactive oxygen species (ROS). The cAMP triggered the activity of C/EBPβ which is a critical inducer of PPARγ and C/EBPα activation in the early stage of adipogenic differentiation, and this is further affected by ROS production. The adipogenic marker proteins such as PPARγ, C/EBPα, C/EBPβ, and pERK were also decreased by melatonin. In summary, melatonin inhibited the cAMP synthesis through ROS reduction and the phosphorylation of the ERK/GSK-3β site which is known to be responsible for C/EBPβ activation for adipogenic differentiation in hMSCs.

Melatonin actions on ovaprim (synthetic GnRH and domperidone)-induced oocyte maturation in carp.

The major objective of the present study was to demonstrate the actions of exogenous melatonin on ovaprim (synthetic GnRH and domperidone)-induced final oocyte maturation focusing on the oxidative status of pre-ovulatory follicles in the carp Catla catla. Accordingly, gravid carp during the early spawning phase of the reproductive cycle were injected with melatonin and/or ovaprim at different time intervals or luzindole (a pharmacological blocker of melatonin receptors) before their administration. We studied their effects on the latency period, the rate of germinal vesicle breakdown (GVBD; a visual marker of final oocyte maturation) in oocytes, and the levels of maturation-promoting factor (MPF), as well as oxidative stress, different antioxidants, melatonin and MT1 melatonin receptor protein in the extracts of pre-ovulatory follicles. Notably, melatonin treatment 2 h before the injection of ovaprim resulted in the shortest latency period as well as the highest rate of GVBD and MPF formation. Exogenous melatonin, irrespective of the injection schedule, caused a significant reduction in intra-follicular oxidative stress and an increase in the levels of both enzymatic and non-enzymatic antioxidants, melatonin and its receptor protein. Concentrations of ovarian melatonin in each fish exhibited a significant negative correlation with the level of oxidative stress, but a positive correlation with the rate of GVBD and the activity/level of different antioxidants. However, no significant effects of melatonin and/or ovaprim were detected in luzindole-pretreated carp. Collectively, the present study provides the first evidence that melatonin pretreatment in carp ameliorates ovaprim actions on the process of final oocyte maturation by the formation of MPF and alleviates oxidative stress in pre-ovulatory follicles by stimulating different antioxidants.

Identify Melatonin as a Novel Therapeutic Reagent in the Treatment of 1-Bromopropane(1-BP) Intoxication.

1-Bromopropane (1-BP) has been used as an alternative for fluoride compounds and 1-BP intoxication may involve lung, liver, and central neural system (CNS). Our previous studies showed that 1-BP impaired memory ability by compromising antioxidant cellular defenses. Melatonin is a powerful endogenousantioxidant, and the objective of this study was to explore the therapeutic role of melatonin in the treatment of 1-BP intoxication. Rats were intragastrically treated with 1-BP with or without melatonin, and then sacrificed on 27th day after 1-BP administration. The Morris water maze (MWM) test was used to evaluate the spatial learning and memory ability of the experimental animals, and NeuN staining was performed to assess neuron loss in hippocampus. We found that rats treated with 1-BP spent more time and swam longer distance before landing on the hidden platform with a comparable swimming speed, which was markedly mitigated by the pretreatment with melatonin in a concentration-dependent manner. In addition, 1-BP-induced notable decrease in neuron population in hippocampus by promoting apoptosis, and melatonin pretreatment attenuated those changes in brain. The GSH/GSSG ratio was proportionately decreased and heme oxygenase 1 was increased in the rats exposed to 1-BP (Figure 6), and administration of melatonin restored them. Meanwhile, MDA, the level of lipid peroxidation product, was significantly increased upon exposed to 1-BP, which was significantly attenuated by melatonin pretreatment, indicating that administration of 1-BP could interfere with redox homeostasis of brain in rat, and such 1-BP-induced biomedical changes were reversed by treatment with melatonin.We conclude that treatment with melatonin attenuates 1-BP-induced CNS toxicity through its ROS scavenging effect.

Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.