Rapid modulation of the silent information regulator 1 by melatonin after hypoxia‐ischemia in the neonatal rat brain

Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia‐ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin‐treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI‐induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin‐induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated.     

Melatonin regulates the autophagic flux via activation of alpha‐7 nicotinic acetylcholine receptors

Our previous study suggested that melatonin‐mediated neuroprotective effects are related with the activation of autophagy. However, the mechanism of melatonin‐mediated autophagic activation in prion‐mediated mitochondrial damage is not reported. Alpha‐7 nicotinic acetylcholine receptors (α7nAchR) is a member of nicotinic acetylcholine receptors, and α7nAchR activation regulates via melatonin. Thus, we hypothesized that melatonin‐mediated neuroprotective effect related with to autophagy pathway as a result of α7nAchR regulation. Inactivation of α7nAchR inhibited melatonin‐mediated autophagic activation and protective effect against prion‐mediated mitochondrial neurotoxicity. Also, knockdown of ATG5 blocked the melatonin‐mediated neuroprotection and did not influence to the activation of α7nAchR caused by melatonin. This report is the first study demonstrating that melatonin‐mediated autophagic activation regulates via modulation of α7nAchR signals, and upregulation of α7nAchR signals induced by melatonin plays a pivotal role in neuroprotection of prion‐mediated mitochondrial neurotoxicity. Our results suggested that regulator of α7 nAChR signals including melatonin may have used for neuroprotective strategies for the neurodegenerative disorders including prion diseases.     

Melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway

Abstract: Although melatonin treatment following trauma‐hemorrhage or ischemic reperfusion prevents organs from dysfunction and injury, the precise mechanism remains unknown. This study tested whether melatonin prevents liver injury following trauma‐hemorrhage involved the protein kinase B (Akt)‐dependent heme oxygenase (HO)‐1 pathway. After a 5‐cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), or melatonin plus phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin (1 mg/kg). At 2 hr after trauma‐hemorrhage, the liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and aspartate aminotransferase levels were significantly increased compared with sham‐operated control. Trauma‐hemorrhage resulted in a significant decrease in the Akt activation in comparison with the shams (relative density, 0.526 ± 0.031 versus 1.012 ± 0.066). Administration of melatonin following trauma‐hemorrhage normalized liver Akt phosphorylation (0.993 ± 0.061), further increased mammalian target of rapamycin (mTOR) activation (5.263 ± 0.338 versus 2.556 ± 0.225) and HO‐1 expression (5.285 ± 0.325 versus 2.546 ± 0.262), and reduced cleaved caspase‐3 levels (2.155 ± 0.297 versus 5.166 ± 0.309). Coadministration of wortmannin abolished the melatonin‐mediated attenuation of the shock‐induced liver injury markers. Our results collectively suggest that melatonin prevents hemorrhagic shock‐induced liver injury in rats through an Akt‐dependent HO‐1 pathway.     

Melatonin receptor‐mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1

Melatonin confers cardioprotective effect against myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress. Activation of silent information regulator 1 (SIRT1) signaling also reduces MI/R injury. We hypothesize that melatonin may protect against MI/R injury by activating SIRT1 signaling. This study investigated the protective effect of melatonin treatment on MI/R heart and elucidated its potential mechanisms. Rats were exposed to melatonin treatment in the presence or the absence of the melatonin receptor antagonist luzindole or SIRT1 inhibitor EX527 and then subjected to MI/R operation. Melatonin conferred a cardioprotective effect by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase release, upregulating SIRT1, Bcl‐2 expression and downregulating Bax, caspase‐3 and cleaved caspase‐3 expression. Melatonin treatment also resulted in reduced myocardium superoxide generation, gp91^phox expression, malondialdehyde level, and increased myocardium superoxide dismutase (SOD) level, which indicate that the MI/R‐induced oxidative stress was significantly attenuated. However, these protective effects were blocked by EX527 or luzindole, indicating that SIRT1 signaling and melatonin receptor may be specifically involved in these effects. In summary, our results demonstrate that melatonin treatment attenuates MI/R injury by reducing oxidative stress damage via activation of SIRT1 signaling in a receptor‐dependent manner.     

Neuroprotective effect of melatonin against hypoxia‐induced retinal ganglion cell death in neonatal rats

The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) concentrations, expression of vascular endothelial growth factor receptors, Flt‐1 and Flk‐1, release of cytochrome c from mitochondria, and caspase‐3 expression were examined in the retinas of 1‐day‐old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt‐1 and Flk‐1 and the tissue concentration of LPO, TNF‐α, and IL‐1β were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF‐α and IL‐1β expression was specifically located on microglial cells, whereas Flt‐1 and Flk‐1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase‐3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti‐inflammatory, and anti‐apoptotic effects. Melatonin suppressed Flt‐1 and Flk‐1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity.     

The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia/reperfusion injury

Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R‐induced reduction of RORα, we subjected RORα‐deficient staggerer mice and wild‐type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R‐induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R‐induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin‐exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα‐deficient mice, but negligibly affected by cardiac‐specific silencing of RORγ. Finally, to determine cell type‐specific effects of RORα, we generated mice with cardiomyocyte‐specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin‐exerted cardioprotection; melatonin‐RORα axis signaling thus appears important in protection against ischemic heart injury.     

Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin‐conferred cardioprotection against myocardial ischemia/reperfusion injury

Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin‐conferred cardioprotection remains unclear. In this study, we employed both loss‐of‐function and gain‐of‐function approaches to reveal the answer. Mice (wild‐type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild‐type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R‐initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2‐silenced heart, but not the MT1 subtype. Furthermore, AAV9‐mediated cardiomyocyte‐specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.     

Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1

In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10^?11, 10^?9, 10^?7, 10^?5, 10^?3 m ) were incubated with fertilized embryos. Melatonin in the range of 10^?11 to 10^?5 m significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10^?9 and 10^?7 m . Using quantitative real‐time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin‐induced embryo development, while 4‐P‐PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up‐regulated the expression of antioxidative (Gpx4, SOD1, bcl‐2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down‐regulating expression of pro‐apoptotic genes (P53, BAX, and Caspase‐3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.     

Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway

Abstract: This study investigated the immunomodulating effect of melatonin on toll‐like receptor (TLR)‐stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high‐mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF‐κB) and phosphorylated c‐Jun, a component of activator protein 1. The increased level of toll‐receptor‐associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN‐β was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase‐1 (HO‐1) was augmented. Our results suggest that melatonin ameliorates I/R‐induced liver damage by modulation of TLR‐mediated inflammatory responses.     

Proteomic identification of proteins differentially expressed by melatonin in hepatic ischemia‐reperfusion injury

Abstract: Hepatic ischemia‐reperfusion (I‐R) injury induces hepatic dysfunction or failure. Melatonin is a potent free radical scavenger and a strong antioxidant. Although many studies have demonstrated the protective effect of melatonin in hepatic injury, the molecular mechanisms of this protection are unclear. We identified specific proteins that are differentially expressed by melatonin treatment in hepatic I‐R injury. Adult mice were subjected to 1 hr of ischemia and 3 hr of reperfusion. Animals were treated with vehicle or melatonin (10 mg/kg, i.p.) 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I‐R group than in sham‐operated group, and these increases were reduced by melatonin treatment. Proteins that were differentially expressed following melatonin treatment during hepatic I‐R injury were detected using two‐dimensional gel electrophoresis. Hepatic I‐R injury induced down‐regulation of glyoxalase I, glutaredoxin‐3, spermidine synthase, proteasome subunit beta type‐4, and dynamin like protein‐1 (DLP‐1). However, melatonin prevented the reductions in these proteins induced by I‐R injury. Among the identified proteins, we focused on DLP‐1, which is essential for the maintenance of mitochondrial and endoplasmic reticulum morphology. Western blot analysis confirmed that melatonin prevents the hepatic I‐R injury‐induced decrease in DLP‐1. These results suggest that melatonin protects hepatic cells against hepatic I‐R injury and that its protective effects involve the regulation of specific proteins.     

The mechanism for the neuroprotective effect of melatonin against methamphetamine‐induced autophagy

Abstract: Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson’s disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug‐induced neurotoxicity. Therefore, we hypothesized that METH‐generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK–N–SH dopaminergic cell line as our model system, we found that METH‐induced autophagy by inhibiting dissociation of Bcl‐2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti‐apoptotic protein Bcl‐2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl‐2 was activated by c‐Jun N‐terminal kinase 1 (JNK 1), which is upstream of Bcl‐2 phosphorylation, to induce Bcl‐2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl‐2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl‐2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.     

Synergistic neuroprotective effect of combined low concentrations of galantamine and melatonin against oxidative stress in SH‐SY5Y neuroblastoma cells

Abstract: Melatonin is a potent free radical scavenger, antioxidant and neuroprotective drug. On the other hand, galantamine is a cholinergic drug with antioxidant and neuroprotective properties linked to inhibition of acetylcholinesterase and allosteric modulation of nicotinic receptors. This investigation evaluated a possible synergistic neuroprotective effect of subeffective concentrations of combined galantamine and melatonin. Human neuroblastoma SH‐SY5Y cells were subjected to a mitochondrial oxidative stress, by blockade of mitochondrial complexes I and V with rotenone and oligomycin‐A (R/O); cells were treated for 24 hr with R/O. This caused 40% of the cell to die as measured by lactate dehydrogenase (LDH) release. Cell incubation with increasing concentrations of galantamine (10–300 nm ) or melatonin (0.3–10 nm ) for 24 hr, followed by a 24‐hr period with R/O, caused a concentration‐dependent protection; maximum protection was achieved with 300 nm galantamine (56% protection) and 10 nm melatonin (50% protection). Combination of subeffective concentrations of melatonin (0.3 nm ) and galantamine (30 nm ) caused a synergistic and significant protection that was similar to the maximum protection afforded by effective concentrations of melatonin or galantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors were blocked respectively by mecamylamine and luzindole. The neuroprotective effect was prevented by chelerythrine, LY294002, and Sn (IV) protoporphyrin IX dichloride (SnPP), indicating the participation of the PKC/PI3K/Akt activation and induction of the antioxidant enzyme heme oxygenase‐1. The synthesis of novel multitarget compounds having in a single molecule the combined neuroprotective properties of galantamine and melatonin could be a new strategy for potential therapeutic agents in neurodegenerative diseases.     

Gliptin‐mediated neuroprotection against stroke requires chronic pretreatment and is independent of glucagon‐like peptide‐1 receptor

Gliptins are anti‐type 2 diabetes (T2D) drugs that regulate glycaemia by preventing endogenous glucagon‐like peptide‐1 (GLP‐1) degradation. Chronically administered gliptins before experimental stroke can also induce neuroprotection, and this effect is potentially relevant for reducing brain damage in patients with T2D and high risk of stroke. It is not known, however, whether acute gliptin treatment after stroke (mimicking a post‐hospitalization treatment) is neuroprotective or whether gliptin‐mediated neuroprotection occurs via GLP‐1‐receptor (GLP‐1R) activation. To answer these two questions, wild‐type and glp‐1r^?/? mice were subjected to transient middle cerebral artery occlusion (MCAO). Linagliptin was administered acutely (50 mg/kg intravenously), at MCAO time or chronically (10 mg/kg orally) for 4 weeks before and 3 weeks after MCAO. Neuroprotection was assessed by stroke volume measurement and quantification of NeuN‐positive surviving neurons. Plasma/brain GLP‐1 levels and dipeptidyl peptidase‐4 activity were also measured. The results show that the linagliptin‐mediated neuroprotection against stroke requires chronic pretreatment and does not occur via GLP‐1R. The findings provide essential new knowledge with regard to the potential clinical use of gliptins against stroke, as well as a strong impetus to identify gliptin‐mediated neuroprotective mechanisms.     

Protection of melatonin in experimental models of newborn hypoxic‐ischemic brain injury through MT1 receptor

The function of melatonin as a protective agent against newborn hypoxic‐ischemic (H‐I) brain injury is not yet well studied, and the mechanisms by which melatonin causes neuroprotection in neurological diseases are still evolving. This study was designed to investigate whether expression of MT1 receptors is reduced in newborn H‐I brain injury and whether the protective action of melatonin is by alterations of the MT1 receptors. We demonstrated that there was significant reduction in MT1 receptors in ischemic brain of mouse pups in vivo following H‐I brain injury and that melatonin offers neuroprotection through upregulation of MT1 receptors. The role of MT1 receptors was further supported by observation of increased mortality in MT1 knockout mice following H‐I brain injury and the reversal of the inhibitory role of melatonin on mitochondrial cell death pathways by the melatonin receptor antagonist, luzindole. These data demonstrate that melatonin mediates its neuroprotective effect in mouse models of newborn H‐I brain injury, at least in part, by the restoration of MT1 receptors, the inhibition of mitochondrial cell death pathways and the suppression of astrocytic and microglial activation.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to post‐traumatic cardiac dysfunction in rats and the protective effect of melatonin

Mechanical trauma (MT ) causes myocardial injury and cardiac dysfunction. However, the underlying mechanism remains largely unclear. This study investigated the role of mitochondrial dynamics in post‐traumatic cardiac dysfunction and the protective effects of melatonin. Adult male Sprague Dawley rats were subjected to 5‐minute rotations (200 revolutions at a rate of 40 rpm) to induce MT model. Melatonin was administrated intraperitoneally 5 minute after MT . Mitochondrial morphology, myocardial injury, and cardiac function were determined in vivo. There was smaller size of mitochondria and increased number of mitochondria per μm² in the hearts after MT when the secondary myocardial injury was induced. Melatonin treatment at the dose of 30 mg/kg reduced serine 616 phosphorylation of Drp1 and inhibited mitochondrial Drp1 translocation and mitochondrial fission in the hearts of rats subjected to MT , which contributed to the reduction of myocardial injury and the improvement of cardiac function. In vitro, H9c2 cells cultured in 20% traumatic plasma (TP ) for 12 hour showed enhanced mitochondrial fission, mitochondrial membrane potential (?Ψm) loss, mitochondrial cytochrome c release, and decreased mitochondrial complex I‐IV activities. Pretreatment with melatonin (100 μmol/L) efficiently inhibited TP ‐induced mitochondrial fission, ?Ψm loss, cytochrome c release, and improved mitochondrial function. Melatonin's protective effects were attributed to its role in suppressing plasma TNF ‐α overproduction, which was responsible for Drp1‐mediated mitochondrial fission. Taken together, our results demonstrate for the first time that abnormal mitochondrial dynamics is involved in post‐traumatic cardiac dysfunction. Melatonin has significant pharmacological potential in protecting against MT ‐induced cardiac dysfunction by preventing excessive mitochondrial fission.     

Long‐term enteral administration of melatonin reduces plasma insulin and increases expression of pineal insulin receptors in both Wistar and type 2‐diabetic Goto‐Kakizaki rats

Abstract: This paper represents an essential aspect of recent investigations into the functional and clinical implications of insulin–melatonin interrelationships. The aim of the study was to analyze whether melatonin reduces insulin secretion in an animal in a manner comparable to the pattern observed in previous in vitro experiments; to this end, we used two models: Wistar and type 2‐diabetic Goto‐Kakizaki (GK) rats. Thirty‐two Wistar and 32 GK rats were divided into two subgroups of 16 rats each; each subgroup was treated either with or without melatonin. The daily administration of melatonin, starting in 8‐ wk‐old rats, was adjusted to 2.5 mg/kg body weight. Melatonin was given daily during the dark period for 12 hr. After 9 wk of treatment, the rats were sacrificed in the middle of the dark period. Melatonin administration strongly enhanced the plasma melatonin level and diminished the expression of pancreatic melatonin receptor‐mRNA, whereas the expression of pineal AA‐NAT and HIOMT was unchanged. Furthermore, the experiments showed in agreement with recent in vitro results of pancreatic islets that plasma insulin levels were diminished after melatonin treatment. However, the pineal insulin receptor expression was increased after melatonin administration. The pancreatic expression of glucagon, GLUT2, and glucokinase was decreased in GK rats, whereas the glucose levels, as well as the parameters of glucose sensing, GLUT2‐mRNA, and glucokinase‐mRNA, were unchanged after melatonin administration in both Wistar and GK rats. In summary, the results show that melatonin administration decreases plasma insulin levels in vivo and, furthermore, that an insulin–melatonin antagonism exists.     

Protective effect of melatonin on oxaliplatin‐induced apoptosis through sustained Mcl‐1 expression and anti‐oxidant action in renal carcinoma Caki cells

Abstract: Melatonin is an indolamine initially found to be produced in the pineal gland but now known to be synthesized in a variety of other tissues as well. The mechanisms whereby melatonin regulates the apoptotic program remain only partially understood. Anti‐/pro‐apoptotic effects of exogenous melatonin on various stimuli‐mediated apoptosis were investigated in this report. We investigated the combined effect of melatonin and death receptor–mediated ligands (TNF‐α, TRAIL, and anti‐Fas antibody) or endoplasmic reticulum (ER) stress‐inducing agents (thapsigargin, brefeldin A, and tunicamycin) on apoptosis of cancer cells. Death receptor– or ER stress–induced apoptosis was not significantly influenced by melatonin treatment. However, pretreatment with melatonin significantly inhibited DNA damage–induced apoptosis and glutathione (GSH) depletion, suggesting the reactive oxygen species mediate oxaliplatin/etoposide‐induced apoptosis. Interestingly, we also found the involvement of myeloid cell leukemia‐1 (Mcl‐1) downregulation in oxaliplatin‐induced apoptosis; thus, pretreatment with melatonin inhibited Mcl‐1 downregulation, and ectopic expression of Mcl‐1 attenuated oxaliplatin‐induced apoptosis. Taken together, the results demonstrate that melatonin attenuates oxaliplatin‐induced apoptosis in cancer cells by inhibition of GSH depletion and Mcl‐1 downregulation.