Melatonin reduces the expression of alpha-synuclein in the dopamine containing neuronal regions of amphetamine-treated postnatal rats

Alpha-synuclein (α-syn) is a neuronal protein that is involved in various degenerative disorders such as Parkinson's disease. It is found in the presynaptic terminals and perinuclear zones of many brain regions. Amphetamine (AMPH), a psychostimulant drug abused progressively more commonly in recent years, has been known to induce neurotoxicity in the central dopaminergic pathway, which is associated with increased oxidative stress. Recently, AMPH has been shown to significantly increase the level of α-syn in dopaminergic neuroblastoma cell cultures. Melatonin is recognized as an antioxidant for the nervous system. This study tested whether melatonin can attenuate the effect of AMPH on the expression of α-syn in the dopaminergic pathway of the neonatal rat. Four-day old postnatal rats (P4) were injected subcutaneously with either AMPH (increasing dose, 5-10 mg/kg daily) alone or AMPH with melatonin (2 mg/kg) daily at 10:00 AM for 7 consecutive days. As determined using Western blot, the level of α-syn was significantly increased in the substantia nigra, dorsal striatum, nucleus accumbens, and prefrontal cortex of the AMPH-treated group, while melatonin treatment either prior to AMPH or alone decreased the accumulation of the protein to 77%, 96%, 78%, and 77% of the control value, respectively. Furthermore, an immunofluorescent study showed that the α-syn-immunoreactivity increased noticeably in the nuclei of cell bodies and nerve terminals of the AMPH-treated group. Again, melatonin lowered this immunoreactivity. These results indicate that melatonin has a direct or indirect effect in reducing the expression of α-syn in the postnatal rat. The exact mechanism of this mitigation should be further investigated.     

褪黑素对匹罗卡品致癫癎大鼠海马神经发生和突触重建的影响

目的探讨褪黑素(Mel)在匹罗卡品(PILO)致癫癎犬鼠模型中的抗癫癎作用机制。方法将45只Wistar大鼠按癫癎持续状态(SE)后6 h,14、28天分为PILO组(1 5只),PILO+Mel组(15只)和对照组(15只),采用PILO诱导大鼠慢性颞叶癫癎模型,用5溴-2-脱氧尿嘧啶核苷标记增殖细胞,Timms染色评价苔藓纤维发芽(MFS)等技术,动态观察MeI对癫癎大鼠海马神经发生和MFS的影响及其与反复自发性癫癎发作(SRS)发生的关系。结果与对照组比较,PILO组大鼠SE后6 h,14、28天细胞数明显增加,差异有统计学意义(P<0.01);与PILO组比较,PILO+Mel组大鼠在SE后6 h,14、28天,细胞数量明显减少(P<0.05),28天SRS数量明显减少,差异有统计学意义(P<0.05)。与PILO+Mel组比较,PILO组大鼠SE后14天,Timms染色密度开始增强,28天密度明显增强,差异有统计学意义(P<0.05)。结论Mel对SRS的预防作用可能与其对癫癎诱导的神经发生和MFS的抑制作用有关。     

Efficacy of melatonin on offspring liver maturation in pinealectomized pregnant rats subjected to experimental epilepsy

BACKGROUND AND AIMS: In clinical practice, maternal epilepsy is a disabling disease for newborn infants, but current data concerning the effect of epileptic phenomena in pregnant mothers on newborns are still limited. This study was undertaken to investigate the effects of pinealectomy (Px) and melatonin treatments on the morphological changes in the liver tissue of newborn rats following experimental epilepsy during pregnancy. METHODS: Female Swiss Albino rats were divided into five groups: intact control group; saline control group; epilepsy group; epilepsy plus Px group; and melatonin-treated epilepsy plus Px group. At one month after Px, an acute grand mal epileptic seizure was induced by penicillin-G during their pregnancy in all animals except the control groups. On the neonatal first day, newborn rats were perfused with intracardiac fixative solution, and then livers were removed and processed for toluidine blue, periodic acid-Schiff (PAS) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reactivity. RESULTS: Normal migration and hepatic maturation were determined in the postnatal rat liver in the control groups, while the morphological structure of the liver in the epilepsy and epilepsy plus Px groups corresponded to the early embryonic period. In the melatonin-treated epilepsy plus Px group, the number of TUNEL positive cells decreased significantly compared to both epilepsy and epilepsy plus Px groups; however, there was no statistically significant difference from the control groups as a result of melatonin activity. CONCLUSIONS: Some histological findings consistent with chronic fetal distress in newborns of mother rats with epilepsy and Px were observed. Melatonin could be a candidate protective drug for the development of liver tissue in pregnant patients with epilepsy.     

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.     

Acutely administered melatonin decreases somatostatin-binding sites and the inhibitory effect of somatostatin on adenylyl cyclase activity in the rat hippocampus

Melatonin is known to increase neuronal activity in the hippocampus, an effect contrary to that of somatostatin (somatotropin release-inhibiting factor, SRIF). Thus, the aim of this study was to investigate whether the somatostatinergic system is implicated in the mechanism of action of melatonin in the rat hippocampus. One group of rats was injected a single dose of melatonin [25 microg/kg subcutaneously (s.c.)] or saline containing ethanol (0.5%, s.c.) and killed 5 hr later. Melatonin significantly decreased the SRIF-like immunoreactivity levels and induced a significant decrease in the density of SRIF receptors as well as in the dissociation constant (Kd). SRIF-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase activity was markedly decreased in hippocampal membranes from melatonin-treated rats. The functional activity of Gi proteins was similar in hippocampal membranes from melatonin-treated and control rats. Western blot analyses revealed that melatonin administration did not alter Gialpha1 or Gialpha2 levels. To determine if the changes observed were related to melatonin-induced activation of central melatonin receptors, a melatonin receptor antagonist, luzindole, was administered prior to melatonin injection. Pretreatment with luzindole (10 mg/kg, s.c.) did not alter the melatonin-induced effects on the above-mentioned parameters and luzindole, alone, had no observable effect. The present results demonstrate that melatonin decreases the activity of the SRIF receptor-effector system in the rat hippocampus, an effect which is apparently not mediated by melatonin receptors. As SRIF exerts an opposite effect to that of melatonin on hippocampal neuronal activity, it is possible that the SRIFergic system could be implicated in the mechanism of action of melatonin in the rat.     

Protective effects of melatonin against myocardial injury induced by isoproterenol in rats

This study was performed to determine whether melatonin could have a protective effect against myocardial injury (MI) induced by isoproterenol (ISO) in rats. Twenty-four rats were divided into three treatment groups: (1) control (n = 8): saline solution. (2) ISO (n = 8): ISO only. (3) melatonin + ISO (n = 8). Melatonin (10 mg/kg/day, i.p.) was administered 30 min before the initiation of ISO (150 mg/kg/day, s.c.). Drugs and saline were given at 14:00 hr for two consecutive days. At the end of the second day, blood samples were taken from the abdominal aorta shortly after the rats were anesthetized for the purpose of measuring cardiac troponins T (cTnT) and I (cTnI); hearts were removed, preserved and examined microscopically. Additionally, based on the histological changes in myocardial tissue, the rats were divided into three groups: no change, mild changes and moderate and/or marked changes. The mean cTnT and cTnI values were significantly increased in ISO group compared with control group [(1.29 +/- 0.22 ng/mL versus 0.46 +/- 0.07 ng/mL, P < 0.0001) and (0.56 +/- 0.11 ng/mL versus 0.21 +/- 0.01 ng/mL, P < 0.001)], respectively, and were significantly reduced in the ISO + melatonin group (0.65 +/- 0.06 ng/mL for cTnT and 0.25 +/- 0.01 ng/mL for cTnI) compared with the ISO only group (P < 0.01), respectively. cTnT and cTnI values were significantly increased in rats with moderate and/or marked cardiac changes compared with hearts where there were mild changes and no change (P < 0.05). ISO + melatonin group showed less histological changes than the ISO group (P < 0.01). In conclusion, this study revealed a protective effect of melatonin against ISO-induced MI in rats, and its potential clinical application in the treatment of MI.     

Beneficial effects of melatonin on nicotine‐induced vasculopathy

Abstract: Tobacco smoking is responsible for death of many people each year and increases the risk of developing numerous disorders, particularly cardiovascular disease and cancer. Among the components of cigarette smoke, nicotine is known to excert proatherosclerotic, prothrombotic and proangiogenic effects on vascular endothelial cells. The current study was designed to investigate the mechanisms by which nicotine induces endothelial dysfunction and further to examine whether melatonin protects against nicotine‐induced vasculopathy. Four groups of male rats (controls, melatonin‐treated, nicotine treated [100 μg/mL in drinking water], and nicotine plus melatonin [5 mg/kg/day] treated) were used in this study. After 28 days all the animals were killed by decapitation and the aorta was removed. We evaluated the hydroxyproline content, and the different expression of proteins involved in several types of stress (ERK1/2), in fibrosis (TGF‐β1, NF‐κB) and in recruitment of circulating leukocytes onto the vessel wall, including intercellular adhesion molecule‐1 (ICAM‐1) and vascular cellular adhesion molecule‐1 (VCAM‐1). These metabolic pathways are important in the development of nicotine‐induced atherosclerosis and hypertension. Our results show that nicotine induces marked structural and functional alterations in the aorta. Nicotine receptor binding results in activation and phosphorylation of ERK 1/2. This enzyme, in turn, activates both TGF‐β1 and NF‐κB; they stimulate respectively the synthesis of type I collagen, responsible of fibrosis, and moreover ICAM‐1, VCAM‐1 and reactive oxygen species. Based on these findings, melatonin is able to minimize the negative effects of nicotine by blocking the activation of ERK and the other signalling pathways in which this enzyme is involved.     

塞来昔布对脂多糖诱导的多巴胺能神经元变性的保护作用

目的探讨选择性环氧化酶-2(COX-2)抑制剂塞来昔布对细菌脂多糖(LPS)诱导的多巴胺能神经元变性的保护作用及其机制。方法30只SD大鼠随机分成3组:PBS对照组、生理盐水+LPS(生理盐水治疗组)和塞来昔布+LPS(塞米昔布治疗组),每组10只。黑质内立体定向注射15μg LPS或PBS 14天后,采用免疫组织化学法观察大鼠黑质酪氨酸羟化酶(TH)阳性细胞的减少以及小胶质细胞的形态学变化,免疫印迹法检测黑质COX-2蛋白表达以及放射免疫法测定前列腺素E2(PGE2)和TNF-α水平。结果与PBS对照组比较,生理盐水治疗组TH阳性细胞减少到对侧的16%(P<0.01),黑质COX-2蛋白表达以及PGE2、TNF-α含量明显增加,大部分小胶质细胞呈激活形态;塞来昔布治疗组TH阳性细胞数较生理盐水治疗组明显增多,为对侧的43%(P<0.05),黑质COX-2蛋白表达以及PGE2、TNF-α含量明显减少,激活的小胶质细胞明显减少。结论COX-2涉及炎症介导的多巴胺能神经元变性机制,塞来昔布能抑制小胶质细胞激活,阻止多巴胺能神经元的变性和丢失。     

The neuroprotective and anti‐apoptotic effects of melatonin on hemolytic hyperbilirubinemia‐induced oxidative brain damage

Melatonin exerts protection in several inflammatory and neurodegenerative disorders. To investigate the neuroprotective effects of melatonin in an experimental hemolysis‐induced hyperbilirubinemia, newborn Sprague–Dawley rats (25–40 g, n = 72) were injected with phenylhydrazine hydrochloride (PHZ; 75 mg/kg) and the injections were repeated at the 24th hour. Rats were treated with saline or melatonin (10 mg/kg) 30 min before the first and second PHZ injections and 24 h after the 2nd PHZ injections. Control rats (n = 24) were injected with saline, but not PHZ. At sixth hours after the last injections of saline or melatonin, all rats were decapitated. Tumor necrosis factor (TNF)‐α, IL–1β, IL‐10 and brain‐derived neurotrophic factor (BDNF) and S100B levels in the plasma were measured. Brain tissue malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase (MPO) activities were measured, and brain tissues were evaluated for apoptosis by TUNEL method. In the saline‐treated PHZ group, hemoglobin, hematocrit levels were reduced, and total/direct bilirubin levels were elevated when compared to control group. Increased plasma TNF‐α, IL‐1β levels, along with decreased BDNF, S100B and IL‐10 values were observed in the saline‐treated PHZ group, while these changes were all reversed in the melatonin‐treated group. Increased MDA levels and MPO activities in the brain tissues of saline‐treated hyperbilirubinemic rats, concomitant with depleted brain GSH stores, were also reversed in the melatonin‐treated hyperbilirubinemic rats. Increased TUNEL(+) cells in the hippocampus of saline‐treated PHZ group were reduced by melatonin treatment. Melatonin exerts neuroprotective and anti‐apoptotic effects on the oxidative neuronal damage of the newborn rats with hemolysis and hyperbilirubinemia.     

Therapeutic effects of melatonin on heatstroke-induced multiple organ dysfunction syndrome in rats

Abstract: Melatonin reportedly exerts beneficial effects to attenuate multiple organ dysfunction syndrome (MODS) in septic shock. Heatstroke resembles septic shock in many aspects. Thus, this study was performed on the anesthetized rats by using heat exposure to induce heatstroke‐associated MODS. We evaluated the effect of melatonin, a versatile molecule synthesized in the pineal gland and in many organs, in heatstroke rats and showed that melatonin (0.2–5.0 mg/kg of body weight, i.v., immediately after the start of heat stress) significantly (i) attenuated hyperthermia, hypotension and hypothalamic ischemia and hypoxia, (ii) reduced plasma index of the toxic oxidizing radicals like nitric oxide metabolites and hydroxyl radicals, (iii) diminished plasma index of hepatic and renal dysfunction like creatinine, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase, (iv) attenuated plasma systemic inflammation response molecules like soluble intercellular and lesion molecule‐1, E‐selectin, tumor necrosis factor‐alpha, interleukin (IL)‐1β, and IL‐6, (v) promoted plasma levels of an anti‐inflammatory cytokine IL‐10, (vi) reduced an index of infiltration of polymorphonuclear neutrophils in the lung like myeloperoxidase activity, and (vii) promoted the survival time to fourfold compared with the heatstroke alone group. Thus, melatonin could be a novel agent for the treatment of heatstroke animals or patients in the early stage.     

Increased melatonin 1a-receptor immunoreactivity in the hippocampus of Alzheimer's disease patients

The pineal secretory product melatonin has, in addition to regulating retinal, circadian and vascular functions, neuroprotective effects. Blood melatonin levels are often decreased in Alzheimer's disease (AD), a progressively disabling neurodegenerative disorder. In this study we provide the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT(1)) in aged human hippocampus and a comparison of AD cases. MT(1) was localized to pyramidal neurons in the hippocampal cornu ammonis (CA)1-4 subfields. There was a distinct increase in staining intensity in all AD cases indicating an up-regulation of the receptor, possibly as a compensatory response to impaired melatonin levels in order to augment melatonin's neuroprotective effects.     

Oxidative stress in diabetic rats induced by streptozotocin: Protective effects of melatonin

Abstract: We have studied the effect of the administration of two doses of melatonin (melatonin 100 and melatonin 200 μg/kg bw) on diabetes and oxidative stress experimentally induced by the injection of streptozotocin (STZ) in female Wistar rats. STZ was injected as a single dose (60 mg/kg i.p. in buffered citrate solution, pH 4.0) and melatonin (melatonin 100, 100 μg/kg/day i.p.; melatonin 200, 200 μg/kg/day i.p.) beginning 3 days before diabetes induction and continuing until the end of the study (8 weeks). The parameters analysed to evaluate oxidative stress and the diabetic state were a) for oxidative stress, changes of lipoperoxides (i.e., malondialdehyde, MDA) in plasma and erythrocytes and the changes in reduced glutathione (GSH) in erythrocytes and b) for diabetes, changes in glycemia, lipids (triglycerides: TG; total cholesterol: TC; HDL-cholesterol, HDL-c), percentage of glycosylated hemoglobin (Hb%), and plasma fructosamine. The injection of STZ caused significant increases in the levels of glycemia, percentage of glycosylated hemoglobin, fructosamine, cholesterol, triglycerides, and lipoperoxides in plasma and erythrocytes, whereas it decreased the levels of HDL-c and the GSH content in erythrocytes. The melatonin 100 dose reduced significantly all these increases, except the percentage of glycosylated hemoglobin. With regard to the decreases of plasma HDL-c and GSH content in erythrocytes, this melatonin dose returned them to normal levels. The melatonin 200 dose produced similar changes, though the effects were especially noticeable in the decrease of glycemia (55% vs. diabetes), percentage of hemoglobin (P < 0.001 vs diabetes), and fructosamine (31% vs. diabetes). This dose also reversed the decreases of HDL-c and GSH in erythrocytes. Both doses of melatonin caused significant reduction of the percentage of glycosylated hemoglobin in those groups that were non-diabetic. These illustrate the protective effect of melatonin against oxidative stress and the severity of diabetes induced by STZ. In particular, this study confirms two facts: 1) the powerful antioxidant action of this pineal indole and 2) the importance of the severity of oxidative stress to maintain hyperglycemia and protein glycosylation, two pathogenetic cornerstones indicative of diabetic complications. Melatonin reduces remarkably the degree of lipoperoxidation, hyperglycemia, and protein glycosylation, which gives hope to a promising perspective of this product, together with other biological antioxidants, in the treatment of diabetic complications where oxidative stress, either in a high or in a low degree, is present.     

Protective effects of melatonin against caustic esophageal burn injury in rats

Abstract:  Caustic ingestion is one of the most life-threatening events in the pediatric age group, which requires the immediate management and subsequent treatment of its most significant complication, i.e. alterations in esophageal structure. We investigated whether melatonin could reduce the esophageal burn damage induced by sodium hydroxide. It was assumed that melatonin could be effective because of its function as a direct free radical scavenger, its antioxidative actions and its ability to diminish tissue hydroxyproline (HP) levels. Esophageal burns were induced in male rats by the administration of 10% sodium hydroxide. Lipid peroxidation (LPO) products were then measured at the following times: 0, 1, 6, 24, 48 and 72 hr after treatment. Tissue HP concentrations in the injured area were assessed at 14 days after the administration of sodium hydroxide. The groups received either systemic melatonin or normal saline. There were two, non-ischemic, sham control groups treated with or without melatonin. LPO products, malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA), increased immediately after the administration of sodium hydroxide; this indicates the participation of free radicals in the development of damage. Melatonin diminished the oxidative response and the amount of HP in the late phase of the lesion. Melatonin reduced oxidative damage in the early phase of the esophageal burns induced by sodium hydroxide.     

Neuroprotective effect of melatonin in experimental optic neuritis in rats

Optic neuritis (ON) is an inflammatory, demyelinating, and neurodegenerative condition of the optic nerve, which might induce permanent vision loss. Currently, there are no effective therapies for this disorder. We have developed an experimental model of primary ON in rats through a single microinjection of 4.5 μg of bacterial lipopolysaccharide (LPS) into the optic nerve. Since melatonin acts as a pleiotropic therapeutic agent in various neurodegenerative diseases, we analyzed the effect of melatonin on LPS‐induced ON. For this purpose, LPS or vehicle were injected into the optic nerve from adult male Wistar rats. One group of animals received a subcutaneous pellet of 20 mg melatonin at 24 hr before vehicle or LPS injection, and another group was submitted to a sham procedure. Melatonin completely prevented the decrease in visual evoked potentials (VEPs), and pupil light reflex (PLR), and preserved anterograde transport of cholera toxin β‐subunit from the retina to the superior colliculus. Moreover, melatonin prevented microglial reactivity (ED1‐immunoreactivity, P < 0.01), astrocytosis (glial fibrillary acid protein‐immunostaining, P < 0.05), demyelination (luxol fast blue staining, P < 0.01), and axon (toluidine blue staining, P < 0.01) and retinal ganglion cell (Brn3a‐immunoreactivity, P < 0.01) loss, induced by LPS. Melatonin completely prevented the increase in nitric oxide synthase 2, cyclooxygenase‐2 levels (Western blot) and TNFα levels, and partly prevented lipid peroxidation induced by experimental ON. When the pellet of melatonin was implanted at 4 days postinjection of LPS, it completely reversed the decrease in VEPs and PLR. These data suggest that melatonin could be a promising candidate for ON treatment.     

Effects of melatonin on macrophages/microglia in postnatal rat brain

The present study examined the response of macrophages/microglia to multiple injections of melatonin in the pineal gland and different regions of the brain. The macrophages/microglia showed a significant increase in cell numbers and upregulation of complement type 3 receptors (CR3), major histocompatibility complex class I (MHC I) and class II (MHC II) antigens, and antigens of monocyte/macrophage lineage, as detected by the antibodies OX-42, OX-18, OX-6, and ED1, respectively. The upregulation of the above antigens was observed in 1-d-old rats given daily injections of melatonin and killed at 7-11 d of age; no noticeable change was observed at earlier time intervals. The macrophages/microglia expressing the above antigens appeared round and showed a vacuolated cytoplasm compared with ramified cells in the control rats. Upregulation of CD4 antigens as detected with the antibody W3/25 was also observed in macrophages/microglia in the corpus callosum and epiplexus cells in the lateral ventricles, but not in the pineal gland and the cerebral cortex in the same age group. In rats killed between 2 and 5 d, and at 14 d of age after melatonin treatment, the immunoreactivities of macrophages/microglia with the above mentioned antibodies were comparable to cells in the control rats. Immunoreactive cells were not detected in any of the age groups in melatonin-treated or control rats with the antibodies W3/13 and OX-33, which are markers for T and B lymphocytes. It is concluded that CR3 receptors, MHC antigens, and CD4 antigens on macrophages/microglia are upregulated following melatonin administration. On the other hand, once the melatonin treatment is discontinued the expression of the various antigens/receptors returns to normal levels, suggesting that increased immune potentiality and its maintenance in these cells require the continuous action of the drug.     

The protective effect of melatonin on smoke‐induced vascular injury in rats and humans: a randomized controlled trial

Smoking is one of the most harmful lifestyles in the world. Very few studies have investigated the effects of melatonin in smoke‐induced vascular injury. This study was designed to investigate whether melatonin could protect rats and humans from smoke‐induced vascular injury. 32 male rats and a double‐blind randomized controlled trial (RCT) containing 63 participants formed the subjects of this study. In rats, 10 mg/kg of melatonin was intraperitoneally injected. Blood samples and abdominal artery were harvested two weeks later. Melatonin decreased the expression of platelet endothelial cell adhesion molecule‐1 (CD31), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and endothelin‐1 (ET‐1) compared with the smoke exposed group (P < 0.05), whereas endothelial nitric oxide synthase (eNOS), nuclear erythroid 2‐related factor 2 (Nrf2), NAD(P)H quinone oxidoreductase 1 (NQO‐1), catalytic glutamate cysteine ligase (GCLC) and heme oxygenase‐1 (HO‐1) recovered markedly (P < 0.05). In humans, 3 mg/day of melatonin was taken orally by the participants. Blood samples were drawn at baseline and after two weeks of treatment. Compared with the oral placebo group, melatonin decreased the concentration of fibrinogen (Fbg) (P = 0.04) and free fatty acids (FFA) (P = 0.04) in smokers, along with the decreased expression of ICAM‐1, VCAM‐1 and ET‐1 (P = 0.004, P = 0.001, P < 0.0001, respectively). In contrast, Nrf2 and HO‐1 expression were markedly increased (P = 0.0001, P = 0.0049, respectively) after smokers took melatonin orally. In summary, our present data suggest that melatonin could ameliorate smoke‐induced vascular injury.     

Fetal/placental regulation of maternal melatonin in rats

Abstract:  We investigated how maternal melatonin is regulated in pregnant rats. To examine the involvement of the conceptus (fetus and placenta) in serum melatonin concentrations, the number of conceptuses was experimentally reduced to one on day 7 of pregnancy (1-conceptus group). Maternal circulating nighttime melatonin levels increased toward day 21 of pregnancy and rapidly decreased to the non-pregnancy levels after parturition, whereas the maternal serum nighttime melatonin levels of the 1-conceptus group on day 21 of pregnancy were significantly lower than normal pregnancy bearing dams more than 10 conceptuses. When the fetuses were removed by fetectomy (all fetuses but not the placentae) on day 12 of pregnancy, serum melatonin concentrations were not decreased. To examine the source of circulating maternal melatonin, mRNA expression of N -acetyltransferase (NAT), which is a late limiting enzyme for melatonin synthesis, was examined in the placenta and fetal pineal. NAT was not expressed in the placenta and was negligible in the pineal gland of the fetus compared with the mother's pineal gland. To examine the effect of placental hormones on maternal melatonin production, a conditioned medium, which was made by incubating placenta of day 20 of pregnancy with medium, was injected into the 1-conceptus dams from day 17 to day 20 of pregnancy. Injection of conditioned medium significantly increased serum melatonin concentrations compared with the control values whereas charcoal treatment abolished the stimulatory effect of conditioned medium. In conclusion, maternal circulating melatonin is from the maternal pineal gland and is increased by placental hormones during pregnancy.     

Olanzapine‐induced early cardiovascular effects are mediated by the biological clock and prevented by melatonin

Second generation antipsychotics (SGA) are associated with adverse cardiometabolic side effects contributing to premature mortality in patients. While mechanisms mediating these cardiometabolic side effects remain poorly understood, three independent studies recently demonstrated that melatonin was protective against cardiometabolic risk in SGA‐treated patients. As one of the main target areas of circulating melatonin in the brain is the suprachiasmatic nucleus (SCN), we hypothesized that the SCN is involved in SGA‐induced early cardiovascular effects in Wistar rats. We evaluated the acute effects of olanzapine and melatonin in the biological clock, paraventricular nucleus and autonomic nervous system using immunohistochemistry, invasive cardiovascular measurements, and Western blot. Olanzapine induced c‐Fos immunoreactivity in the SCN followed by the paraventricular nucleus and dorsal motor nucleus of the vagus indicating a potent induction of parasympathetic tone. The involvement of a SCN‐parasympathetic neuronal pathway after olanzapine administration was further documented using cholera toxin‐B retrograde tracing and vasoactive intestinal peptide immunohistochemistry. Olanzapine‐induced decrease in blood pressure and heart rate confirmed this. Melatonin abolished olanzapine‐induced SCN c‐Fos immunoreactivity, including the parasympathetic pathway and cardiovascular effects while brain areas associated with olanzapine beneficial effects including the striatum, ventral tegmental area, and nucleus accumbens remained activated. In the SCN, olanzapine phosphorylated the GSK‐3β, a regulator of clock activity, which melatonin prevented. Bilateral lesions of the SCN prevented the effects of olanzapine on parasympathetic activity. Collectively, results demonstrate the SCN as a key region mediating the early effects of olanzapine on cardiovascular function and show melatonin has opposing and potentially protective effects warranting additional investigation.     

Pulmonary function changes in rats with taurocholate‐induced pancreatitis are attenuated by pretreatment with melatonin

Melatonin is a free radical scavenger and broad‐spectrum antioxidant with immunomodulatory effects. We studied the effects of melatonin on changes in lung function, oxidative/nitrosative stress, and inflammatory cell sequestration in an acute pancreatitis (AP)‐associated lung inflammation model. Acute pancreatitis was induced by injection of 5% sodium taurocholate into the pancreatic duct of rats. Animals were randomized into control, AP, and a melatonin pretreatment (10 mg/kg)/AP group. Functional residual capacity (FRC), lung compliance (Cchord), expiratory flow rate at 50% (FEF50), airway resistance index (RI), and peak expiratory flow rate (PEF) were evaluated. White blood cell count (WBC) and hydrogen peroxide, lung lavage fluid WBC, methylguanidine, protein, lactic dehydrogenase (LDH), nitric oxide (NO), and leukotriene B4 (LTB4) levels were determined. Lung wet‐to‐dry weight ratio, peroxynitrite, and inducible nitric oxide synthase (NOS) mRNA and protein were measured. AP induction resulted in reductions in FRC, Cchord, FEF50, and PEF, and increase in RI and lung wet‐to‐dry weight ratio. Blood and lung lavage fluid WBC, lavage fluid LDH, protein, and blood hydrogen peroxide also increased. Levels of hydroxyl radicals, nitric oxide, and LTB4 in lung lavage fluid, inducible NOS mRNA, protein expression, and peroxynitrite in lung tissue also were significantly elevated. Pretreatment with melatonin attenuated obstructive and restrictive ventilatory insufficiency induced by AP. Blood and lavage WBC, lavage LDH and protein, lung edema, oxidative/nitrosative stress, and lipoxygenase pathway derivatives were also significantly attenuated by melatonin. We conclude that melatonin decreases AP‐induced obstructive and restrictive lung function changes via its antioxidant and anti‐inflammatory properties.     

Effect of continuous melatonin infusions on steady-state plasma melatonin levels in rats under near physiological conditions

Abstract: It was the aim of this study to measure the actual amount of melatonin required for elevating the circulating hormone from low daytime levels to the 10-fold higher nocturnal steady-state concentrations in rats. For this purpose, escalating doses of melatonin were continuously infused into the right jugular vein and blood samples were repeatedly drawn from the left jugular vein for a period of 2 hr in freely moving catheterized rats. In order to achieve an about 10-fold elevation of the plasma melatonin concentration, 500 ng melatonin/hr had to be infused, i.e., about 300 times the normal nocturnal melatonin content of the pineal. Infusions of up to 61 ng melatonin/hr (equivalent to the melatonin content of 40 pineals at darkness) failed to cause a significant rise of the low daytime steady-state concentrations in the blood. If the dose of 500 ng melatonin/h was infused at night, a less-pronounced rise of the blood levels was observed, as compared to that caused by the infusion of the same dose during daytime. No differences were found in the rate of metabolism between daytime and nighttime. The results of this study indicate 1) that the low basal concentrations of melatonin in the blood are not affected by an increased melatonin supply up to a certain critical threshold, 2) that the rat pineal gland would have to release all its melatonin content almost every 10 sec in order to sustain the elevated steady-state level of melatonin in the circulation during the dark period, and 3) that significant day/night differences exist in the disposition of circulating melatonin if administered in near physiological amounts and under near physiological conditions.