Melatonin long-lasting beneficial effects on pulmonary vascular reactivity and redox balance in chronic hypoxic ovine neonates

Pulmonary arterial hypertension of the neonate (PAHN) is a pathophysiological condition characterized by maladaptive pulmonary vascular remodeling and abnormal contractile reactivity. This is a multifactorial syndrome with chronic hypoxia and oxidative stress as main etiological drivers, and with limited effectiveness in therapeutic approaches. Melatonin is a neurohormone with antioxidant and vasodilator properties at the pulmonary level. Therefore, this study aims to test whether a postnatal treatment with melatonin during the neonatal period improves in a long-lasting manner the clinical condition of PAHN. Ten newborn lambs gestated and born at 3600 m were used in this study, five received vehicle and five received melatonin in daily doses of 1 mg kg⁻¹ for the first 3 weeks of life. After 1 week of treatment completion, lung tissue and small pulmonary arteries (SPA) were collected for wire myography, molecular biology, and morphostructural analyses. Melatonin decreased pulmonary arterial pressure the first 4 days of treatment. At 1 month old, melatonin decreased the contractile response to the vasoconstrictors K⁺, TX₂, and ET-1. Further, melatonin increased the endothelium-dependent and muscle-dependent vasodilation of SPA. Finally, the treatment decreased pulmonary oxidative stress by inducing antioxidant enzymes and diminishing pro-oxidant sources. In conclusion, melatonin improved vascular reactivity and oxidative stress at the pulmonary level in PAHN lambs gestated and born in chronic hypoxia.     

Melatonin ameliorates low‐grade inflammation and oxidative stress in young Zucker diabetic fatty rats

The aim of this study was to investigate the effects of melatonin on low‐grade inflammation and oxidative stress in young male Zucker diabetic fatty (ZDF) rats, an experimental model of metabolic syndrome and type 2 diabetes mellitus (T2DM). ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups, each composed of 10 rats: naive (N), vehicle treated (V), and melatonin treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Pro‐inflammatory state was evaluated by plasma levels of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and C‐reactive protein (CRP). Also, oxidative stress was assessed by plasma lipid peroxidation (LPO), both basal and after Fe²⁺/H₂O₂ inducement. ZDF rats exhibited higher levels of IL‐6 (112.4 ± 1.5 pg/mL), TNF‐α (11.0 ± 0.1 pg/mL) and CRP (828 ± 16.0 µg/mL) compared with lean rats (IL‐6, 89.9 ± 1.0, P < 0.01; TNF‐α, 9.7 ± 0.4, P < 0.01; CRP, 508 ± 21.5, P < 0.001). Melatonin lowered IL‐6 (10%, P < 0.05), TNF‐α (10%, P < 0.05), and CRP (21%, P < 0.01). Basal and Fe²⁺/H₂O₂‐induced LPO, expressed as malondialdehyde equivalents (µmol/L), were higher in ZDF rats (basal, 3.2 ± 0.1 versus 2.5 ± 0.1 in ZL, P < 0.01; Fe²⁺/H₂O₂‐induced, 8.7 ± 0.2 versus 5.5 ± 0.3 in ZL; P < 0.001). Melatonin improved basal LPO (15%, P < 0.05) in ZDF rats, and Fe²⁺/H₂O₂‐ induced LPO in both ZL (15.2%, P < 0.01) and ZDF rats (39%, P < 0.001). These results demonstrated that oral melatonin administration ameliorates the pro‐inflammatory state and oxidative stress, which underlie the development of insulin resistance and their consequences, metabolic syndrome, diabetes, and cardiovascular disease.     

Melatonin improves mitochondrial function in inguinal white adipose tissue of Zücker diabetic fatty rats

Mitochondrial dysfunction in adipose tissue may contribute to obesity‐related metabolic derangements such as type 2 diabetes mellitus (T2DM). Because mitochondria are a target for melatonin action, the goal of this study was to investigate the effects of melatonin on mitochondrial function in white (WAT) and beige inguinal adipose tissue of Zücker diabetic fatty (ZDF) rats, a model of obesity‐related T2DM. In this experimental model, melatonin reduces obesity and improves the metabolic profile. At 6 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control (C‐ZDF and C‐ZL) and treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk (M‐ZDF and M‐ZL). After the treatment period, animals were sacrificed, tissues dissected, and mitochondrial function assessed in isolated organelles. Melatonin increased the respiratory control ratio (RCR) in mitochondria from white fat of both lean (by 26.5%, P < 0.01) and obese (by 34.5%, P < 0.01) rats mainly through a reduction of proton leaking component of respiration (state 4) (28% decrease in ZL, P < 0.01 and 35% in ZDF, P < 0.01). However, melatonin treatment lowered the RCR in beige mitochondria of both lean (by 7%, P < 0.05) and obese (by 13%, P < 0.05) rats by maintaining high rates of uncoupled respiration. Melatonin also lowered mitochondrial oxidative status by reducing nitrite levels and by increasing superoxide dismutase activity. Moreover, melatonin treatment also caused a profound inhibition of Ca‐induced opening of mPTP in isolated mitochondria from both types of fat, white and beige, in both lean and obese rats. These results demonstrate that chronic oral melatonin improves mitochondrial respiration and reduces the oxidative status and susceptibility to apoptosis in white and beige adipocytes. These melatonin effects help to prevent mitochondrial dysfunction and thereby to improve obesity‐related metabolic disorders such as diabetes and dyslipidemia of ZDF rats.     

AZT-Induced Oxidative Cardiovascular Toxicity: Attenuation by Mg-Supplementation

Cardiovascular effects of chronic AZT treatment on SD male rats (185 g) fed either a normal Mg diet (0.1% MgO) or a high Mg diet (0.6% MgO) were examined. AZT treatment (1 mg/ml drinking water) for 3 weeks led to a 5.5-fold (0.88 ± 0.11 nmol/min/10⁶ cells, P < 0.05) elevation in neutrophil basal activity of O₂ ⁻ production versus controls (0.16 ± 0.03 nmol/min, assayed ex vivo as SOD-inhibitable cytochrome c reduction). Concomitantly, plasma 8-isoprostane and PGE₂ levels rose 2.1-fold and 3-fold (both P < 0.05), respectively, compared to control; however, RBC GSH decreased 28% (P < 0.02) with GSSG content increased 3-fold, indicative of systemic oxidative stress. High Mg diet substantially attenuated the AZT-induced neutrophil activation by 70% (0.26 ± 0.05 nmol/min, P < 0.05); reduced plasma 8-isoprostane and PGE₂ to levels comparable to normal; and RBC GSH was restored back to 92% of control. AZT alone caused moderate, but significant vascular inflammatory lesions in the heart (assessed by H&E staining). Immunohistochemical staining revealed significantly higher (about 4-fold) infiltration of CD11b positive cells (WBC surface marker) in the atria and ventricles of AZT-treated rats. However, these inflammatory pathological markers were minimal in samples of rats treated with AZT plus high Mg diet. Moreover, AZT alone significantly (P < 0.02) decreased rat weight gain by 21% at 3 weeks; Mg-supplementation completely prevented (P < 0.05) the weight gain loss due to AZT intake. It is concluded that high dietary Mg may provide beneficial effects against AZT toxicity due to its systemic antioxidative/anti-inflammatory properties.     

Melatonin induces browning of inguinal white adipose tissue in Zucker diabetic fatty rats

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Identification of brown fat (beige/brite) in white adipose tissue (WAT) prompted us to investigate whether melatonin is a brown‐fat inducer. We used Zücker diabetic fatty (ZDF) rats, a model of obesity‐related type 2 diabetes and a strain in which melatonin reduces obesity and improves their metabolic profiles. At 5 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control and those treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk. Melatonin induced browning of inguinal WAT in both ZDF and ZL rats. Hematoxylin–eosin staining showed patches of brown‐like adipocytes in inguinal WAT in ZDF rats and also increased the amounts in ZL animals. Inguinal skin temperature was similar in untreated lean and obese rats. Melatonin increased inguinal temperature by 1.36 ± 0.02°C in ZL and by 0.55 ± 0.04°C in ZDF rats and sensitized the thermogenic effect of acute cold exposure in both groups. Melatonin increased the amounts of thermogenic proteins, uncoupling protein 1 (UCP1) (by ~2‐fold, P < 0.01) and PGC‐1α (by 25%, P < 0.05) in extracts from beige inguinal areas in ZL rats. Melatonin also induced measurable amounts of UCP1 and stimulated by ~2‐fold the levels of PGC‐1α in ZDF animals. Locomotor activity and circulating irisin levels were not affected by melatonin. These results demonstrate that chronic oral melatonin drives WAT into a brown‐fat‐like function in ZDF rats. This may contribute to melatonin′s control of body weight and its metabolic benefits.     

Systemic and transdermal melatonin administration prevents neuropathology in response to perinatal asphyxia in newborn lambs

Perinatal asphyxia remains a principal cause of infant mortality and long‐term neurological morbidity, particularly in low‐resource countries. No neuroprotective interventions are currently available. Melatonin (MLT), a potent antioxidant, anti‐inflammatory and antiapoptotic agent, offers promise as an intravenous (IV) or transdermal therapy to protect the brain. We aimed to determine the effect of melatonin (IV or transdermal patch) on neuropathology in a lamb model of perinatal asphyxia. Asphyxia was induced in newborn lambs via umbilical cord occlusion at birth. Animals were randomly allocated to melatonin commencing 30 minutes after birth (60 mg in 24 hours; IV or transdermal patch). Brain magnetic resonance spectroscopy (MRS) was undertaken at 12 and 72 hours. Animals (control n = 9; control+MLT n = 6; asphyxia n = 16; asphyxia+MLT [IV n = 14; patch n = 4]) were euthanised at 72 hours, and cerebrospinal fluid (CSF) and brains were collected for analysis. Asphyxia resulted in severe acidosis (pH 6.9 ± 0.0; lactate 9 ± 2 mmol/L) and altered determinants of encephalopathy. MRS lactate:N‐acetyl aspartate ratio was 2.5‐fold higher in asphyxia lambs compared with controls at 12 hours and 3‐fold higher at 72 hours (P < .05). Melatonin prevented this rise (3.5‐fold reduced vs asphyxia; P = .02). Asphyxia significantly increased brain white and grey matter apoptotic cell death (activated caspase‐3), lipid peroxidation (4HNE) and neuroinflammation (IBA‐1). These changes were significantly mitigated by both IV and patch melatonin. Systemic or transdermal neonatal melatonin administration significantly reduces the neuropathology and encephalopathy signs associated with perinatal asphyxia. A simple melatonin patch, administered soon after birth, may improve outcome in infants affected by asphyxia, especially in low‐resource settings.     

Daily melatonin protects the endothelial lineage and functional integrity against the aging process,oxidative stress,and toxic environment and restores blood flow in critical limb ischemia area in mice

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide‐induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione‐induced oxidative stress (NOX‐1/NOX‐2), inflammation (TNF‐α/NF‐kB), apoptosis (cleaved caspase‐3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3‐siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C‐kit/CD31+/Sca‐1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow‐derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age‐match mice receiving melatonin (all P < .01). ED‐induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide‐induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging‐, oxidative stress‐, lipopolysaccharide‐, and ischemia‐induced damage probably through upregulating the SIRT signaling pathway.     

The Effects of Melatonin on Colonic Transit Time in Normal Controls and IBS Patients

Objectives The role of melatonin in regulating gut motility in human subjects is not clear. The aim of this study was to investigate the effects of exogenous melatonin on colonic transit time (CTT) in healthy subjects and in patients with irritable bowel syndrome (IBS). Methods Colonic transit time was measured in 17 healthy controls using the radio-opaque, blue dye, and Bristol stool form score method before and after 30 days of melatonin treatment 3 mg daily. A double blind cross-over study aimed at measuring CTT was also performed in 17 matched IBS patients using the blue dye and Bristol stool form score methods. The patients were randomized and received either melatonin 3 mg or placebo daily for 8 weeks, followed by a 4-week washout, and then placebo or melatonin in the reverse order for a second 8-week period. Results The melatonin treatment of the control subjects caused an increase in CTT (mean ± SD) from 27.4 ± 10.5 to 37.4 ± 23.8 h (P = 0.04). Compared with the CTT of the controls (25.2 ± 7.7), that of the constipation-predominant IBS patients appeared prolonged—65.2 ± 33.3 h (P < 0.01). The CTT did not change significantly in IBS patients after melatonin treatment. Conclusion Melatonin may be a promising candidate for the future research of agents that can modulate bowel motility.     

Melatonin potentiates running wheel‐induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus

This study assessed the role of melatonin in modulating running wheel(RW)‐induced hippocampal neurogenesis in adult C3H/HeN mice. Chronic melatonin (0.02 mg/mL, oral for 12 days) treatment did not affect cell proliferation or cell survival determined by the number of BrdU‐positive cells in dentate gyrus of mice with access to fixed wheel (FW). RW activity significantly increased cell proliferation [RW (n = 8) versus FW (n = 6): dorsal, 199 ± 18 versus 125 ± 12, P < 0.01; ventral, 211 ± 15 versus 123 ± 13, P < 0.01] and newborn cell survival [RW (n = 7) versus FW (n = 8): dorsal, 45 ± 8.5 versus 15 ± 1.8, P < 0.01; ventral, 48 ± 8.1 versus 15 ± 1.4)] in the dorsal and ventral dentate gyrus. Oral melatonin treatment further potentiated RW activity‐induced cell survival in both areas of the dentate gyrus [melatonin (n = 10) versus vehicle (n = 7): dorsal, 63 ± 5.4 versus 45 ± 8.5 P < 0.05; ventral, 75 ± 7.9 versus 48 ± 8.1, P < 0.01] and neurogenesis [melatonin (n = 8) versus vehicle (n = 8): dorsal, 46 ± 3.4, versus 34 ± 4.5, P < 0.05; ventral, 41 ± 3.4 versus 25 ± 2.4, P < 0.01]. We conclude that melatonin potentiates RW‐induced hippocampal neurogenesis by enhancing neuronal survival suggesting that the combination of physical exercise and melatonin may be an effective treatment for diseases affecting the hippocampus neurogenesis.     

Melatonin reduces median nerve injury‐induced mechanical hypersensitivity via inhibition of microglial p38 mitogen‐activated protein kinase activation in rat cuneate nucleus

In this study, we examined the relationships between p38 mitogen‐activated protein kinase (MAPK) activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. We further investigated effects of melatonin administration and pinealectomy on p38 MAPK activation and development of hypersensitivity. Using immunohistochemistry and immunoblotting, low levels of phosphorylated p38 (p‐p38) MAPK were detected in CN of normal rats. As early as 1 day after CCI, p‐p38 MAPK levels in the ipsilateral CN were significantly increased (1.4 ± 0.2‐fold, P < 0.05), which reached a maximum at 7 days (5.1 ± 0.4‐fold, P < 0.001). Double immunofluorescence labeling with cell‐specific markers showed that p‐p38 MAPK immunoreactive cells co‐expressed OX‐42, a microglia activation maker, suggesting the expression of p‐p38 MAPK in microglia. Microinjection of SB203580, a p38 MAPK inhibitor, into the CN 1 day after CCI attenuated injury‐induced behavioral hypersensitivity in a dose‐dependent manner. Furthermore, animals received melatonin treatment at daily doses of 37.5, 75, 150, or 300 mg/kg from 30 min before until 3 days after CCI. Melatonin treatment dose‐dependently attenuated p‐p38 MAPK levels, release of pro‐inflammatory cytokines, and behavioral hypersensitivity following CCI; conversely, pinealectomy that resulted in a reduction in endogenous melatonin levels exacerbated these effects. In conclusion, median nerve injury‐induced microglial p38 MAPK activation in the CN modulated development of behavioral hypersensitivity. Melatonin supplementation eased neuropathic pain via inhibition of p38 MAPK signaling pathway; contrarily, reducing endogenous blood melatonin levels by pinealectomy promoted phosphorylation of p38 MAPK and made rats more vulnerable to nerve injury‐induced neuropathic pain.     

A PERIOD3 variable number tandem repeat polymorphism modulates melatonin treatment response in delayed sleep-wake phase disorder

We examined whether a polymorphism of the PERIOD3 gene (PER3; rs57875989) modulated the sleep-promoting effects of melatonin in Delayed Sleep-Wake Phase Disorder (DSWPD). One hundred and four individuals (53 males; 29.4 ±10.0 years) with DSWPD and a delayed dim light melatonin onset (DLMO) collected buccal swabs for genotyping (PER3^4/4 n = 43; PER3 5 allele [heterozygous and homozygous] n = 60). Participants were randomised to placebo or 0.5 mg melatonin taken 1 hour before desired bedtime (or ~1.45 hours before DLMO), with sleep attempted at desired bedtime (4 weeks; 5-7 nights/week). We assessed sleep (diary and actigraphy), Pittsburgh Sleep Quality Index (PSQI), Insomnia Severity Index (ISI), Patient-Reported Outcomes Measurement Information System (PROMIS: Sleep Disturbance, Sleep-Related Impairment), Sheehan Disability Scale (SDS) and Patient- and Clinician-Global Improvement (PGI-C, CGI-C). Melatonin treatment response on actigraphic sleep onset time did not differ between genotypes. For PER3^4/4 carriers, self-reported sleep onset time was advanced by a larger amount and sleep onset latency (SOL) was shorter in melatonin-treated patients compared to those receiving placebo (P = .008), while actigraphic sleep efficiency in the first third of the sleep episode (SE T1) did not differ. For PER3 5 carriers, actigraphic SOL and SE T1 showed a larger improvement with melatonin (P < .001). Melatonin improved ISI (P = .005), PROMIS sleep disturbance (P < .001) and sleep-related impairment (P = .017), SDS (P = .019), PGI-C (P = .028) and CGI-C (P = .016) in PER3^4/4 individuals only. Melatonin did not advance circadian phase. Overall, PER3^4/4 DSWPD patients have a greater response to melatonin treatment. PER3 genotyping may therefore improve DSWPD patient outcomes.     

Melatonin rescues cardiovascular dysfunction during hypoxic development in the chick embryo

There is a search for rescue therapy against fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia, particularly following clinical diagnosis of fetal growth restriction (FGR). Melatonin protects the placenta in adverse pregnancy; however, whether melatonin protects the fetal heart and vasculature in hypoxic pregnancy independent of effects on the placenta is unknown. Whether melatonin can rescue fetal cardiovascular dysfunction when treatment commences following FGR diagnosis is also unknown. We isolated the effects of melatonin on the developing cardiovascular system of the chick embryo during hypoxic incubation. We tested the hypothesis that melatonin directly protects the fetal cardiovascular system in adverse development and that it can rescue dysfunction following FGR diagnosis. Chick embryos were incubated under normoxia or hypoxia (14% O₂) from day 1 ± melatonin treatment (1 mg/kg/day) from day 13 of incubation (term ~21 days). Melatonin in hypoxic chick embryos rescued cardiac systolic dysfunction, impaired cardiac contractility and relaxability, increased cardiac sympathetic dominance, and endothelial dysfunction in peripheral circulations. The mechanisms involved included reduced oxidative stress, enhanced antioxidant capacity and restored vascular endothelial growth factor expression, and NO bioavailability. Melatonin treatment of the chick embryo starting at day 13 of incubation, equivalent to ca. 25 wk of gestation in human pregnancy, rescues early origins of cardiovascular dysfunction during hypoxic development. Melatonin may be a suitable antioxidant candidate for translation to human therapy to protect the fetal cardiovascular system in adverse pregnancy.     

Melatonin alleviates weanling stress in mice: Involvement of intestinal microbiota

Melatonin influences intestinal microbiota and the pathogenesis of various diseases. This study was conducted to explore whether melatonin alleviates weanling stress through intestinal microbiota in a weanling mouse model. Melatonin supplementation in weanling mice (provided in the drinking water at a dosage of 0.2 mg/mL for 2 weeks) significantly improved body weight gain (1.4 ± 0.03 g/day in melatonin group vs 1.2 ± 0.06 g/day in control group) and intestinal morphology (ie, villus length, crypt depth, and villus to crypt ratio), but had little effect on the proliferation or apoptosis of intestinal cells, the numbers of Paneth cells and goblet cells, as well as the expression of makers related to enterocytes (sucrase) and endocrine cells (chromogranin A and peptide YY) in the ileum. Melatonin supplementation had little effect on serum levels of amino acids or stress‐related parameters (eg, SOD, TNF‐α, and angiotensin I). 16S rRNA sequencing suggested that melatonin supplementation increased the richness indices of intestinal microbiota (observed species, Chao 1, and ACE) and shaped the composition of intestinal microbiota (eg, increase in the abundance of Lactobacillus [19 ± 3% in melatonin group vs 6 ± 2% in control group]), which was demonstrated using an ex vivo proliferation assay and colonic loop proliferation assay. Melatonin supplementation also significantly influenced the metabolism of intestinal microbiota, such as amino acid metabolism and drug metabolism. More importantly, in antibiotic‐treated weanling mice and germ‐free weanling mice, melatonin failed to affect body weight gain or intestinal morphology. Melatonin significantly reduced (by about 60%) the bacterial load in enterotoxigenic Escherichia coli (ETEC)‐infected weanling mice, but had little effect on ETEC load in antibiotic‐pretreated animals. In conclusion, melatonin affects body weight gain, intestinal morphology, and intestinal ETEC infection through intestinal microbiota in weanling mice. The findings highlight the importance of intestinal microbiota in mediating the various physiological functions of melatonin in the host.     

Melatonin and Exposure to Constant Light/Darkness Affects Ovarian Follicular Kinetics and Estrous Cycle in Indian Desert GerbilMeriones hurrianae(Jerdon)

Melatonin mediates photoperiodic influence on reproduction and constant light and darkness affect pineal biosynthesis of melatonin. The present study was undertaken to assess the effects of melatonin and drastic photoperiodic changes on reproduction in a tropical desert species with a fossorial lifestyle. Ovarian follicular kinetics and estrous cycle were studied in the Indian desert gerbilMeriones hurrianae,after treatment with melatonin and exposure to constant light (LL) and darkness (DD) regimes. Melatonin treatment increased (P < 0.001) ovarian weights without changing the uterine weights. While exposure to LL decreased (P < 0.001) both ovarian and uterine weights, exposure to DD had no effect on these weights. Follicular kinetics of growing and regressing follicles revealed that ovaries of melatonin-treated and DD-exposed animals had significantly more growing follicles. Melatonin treatment increased all types of growing follicles, especially antral and Graafian follicles. Exposure to DD increased all types of growing follicles, with the medium sized antral and Graafian follicles being significant (P < 0.01). In contrast to stimulation of follicular growth by melatonin and DD, LL caused regression of all stages of follicular growth and also reduced the number of small preantral follicles. Melatonin treatment increased (P < 0.001) the length of estrous cycle (5.08 to 7.29 days). Gerbils treated with melatonin, exposed to LL and DD, had a longer (P < 0.001) metestrus. Animals held in LL, had the least number (P < 0.001) of estrous smears (1 in 30 days). The results suggest that melatonin is involved in growth of ovarian follicles in the Indian desert gerbil.     

Systemic combined melatonin–mitochondria treatment improves acute respiratory distress syndrome in the rat

Despite high in‐hospital mortality associated with acute respiratory distress syndrome (ARDS), there is no effective therapeutic strategy. We tested the hypothesis that combined melatonin–mitochondria treatment ameliorates 100% oxygen‐induced ARDS in rats. Adult male Sprague‐Dawley rats (n = 40) were equally categorized into normal controls, ARDS, ARDS‐melatonin, ARDS with intravenous liver‐derived mitochondria (1500 μg per rat 6 hr after ARDS induction), and ARDS receiving combined melatonin–mitochondria. The results showed that 22 hr after ARDS induction, oxygen saturation (saO₂) was lowest in the ARDS group and highest in normal controls, significantly lower in ARDS‐melatonin and ARDS‐mitochondria than in combined melatonin–mitochondria group, and significantly lower in ARDS‐mitochondria than in ARDS‐melatonin group. Conversely, right ventricular systolic blood pressure and lung weight showed an opposite pattern compared with saO₂ among all groups (all P < 0.001). Histological integrity of alveolar sacs showed a pattern identical to saO₂, whereas lung crowding score exhibited an opposite pattern (all P < 0.001). Albumin level and inflammatory cells (MPO+, CD40+, CD11b/c+) from bronchoalveolar lavage fluid showed a pattern opposite to saO₂ (all P < 0.001). Protein expression of indices of inflammation (MMP‐9, TNF‐α, NF‐κB), oxidative stress (oxidized protein, NO‐1, NOX‐2, NOX‐4), apoptosis (mitochondrial Bax, cleaved caspase‐3, and PARP), fibrosis (Smad3, TGF‐β), mitochondrial damage (cytochrome C), and DNA damage (γ‐H2AX+) exhibited an opposite pattern compared to saO₂ in all groups, whereas protein (HO‐1, NQO‐1, GR, GPx) and cellular (HO‐1+) expressions of antioxidants exhibited a progressively increased pattern from normal controls to ARDS combined melatonin–mitochondria group (all P < 0.001). In conclusion, combined melatonin–mitochondrial was superior to either treatment alone in attenuating ARDS in this rat model.     

Dietary antioxidant supplementation reduces lipid peroxidation but impairs vascular function in small mesenteric arteries of the streptozotocin-diabetic rat

Summary Impaired endothelium-dependent relaxation could underlie many of the vascular complications associated with insulin-dependent diabetes mellitus, and may be mediated by increased oxidative stress. The effect of antioxidants on vascular endothelial function and oxidative stress of streptozotocin-diabetic rats was assessed by dietary supplementation with vitamins E and C. Diabetic (i. v. streptozotocin, 45 mg/kg) male Sprague-Dawley rats were fed one of six supplemented diets containing 75.9, 250, or 500 mg vitamin E/kg chow, 250 mg vitamin C/kg H₂0, 250 mg vitamin E/kg chow plus 250 mg vitamin C/kg H₂O, or chow deficient in vitamin E, and then compared to standard-fed control rats. After 4 weeks, small mesenteric arteries were dissected and mounted on a small vessel myograph, concentration response curves were then constructed to noradrenaline, acetylcholine and sodium nitroprusside. Acetylcholine-mediated relaxation was impaired in arteries from diabetic rats (pEC₅₀ 6.701 ± SEM 0.120, n = 8) compared to controls (7.386 ± 0.078, n = 6; p < 0.05). The 500 mg/kg vitamin E diet further impaired maximum relaxation to acetylcholine (58.2 ± 10.5 vitamin E, n = 7 vs 84.4 ± 5.3 % standard, p < 0.05), and the combined vitamin E plus C diet impaired maximum relaxation to sodium nitroprusside (48.5 ± 4.1 in vitamin E + C, n = 8 vs 75.6 ± 3.9 % standard; p < 0.01). However, plasma 8-epi-prostaglandin (PG)F₂α (measured as an estimate of oxidative stress) was dose-dependently decreased in rats on vitamin E supplemented diets. Dietary antioxidant supplementation did not reverse impaired endothelial function in this model of uncontrolled diabetes despite a concomitant decrease in oxidative stress. [Diabetologia (1998) 41: 148–156] Received: 8 July 1997 and in revised form: 29 September 1997     

Melatonin improves bone mineral density at the femoral neck in postmenopausal women with osteopenia: a randomized controlled trial

Melatonin is known for its regulation of circadian rhythm. Recently, studies have shown that melatonin may have a positive effect on the skeleton. By increasing age, the melatonin levels decrease, which may lead to a further imbalanced bone remodeling. We aimed to investigate whether treatment with melatonin could improve bone mass and integrity in humans. In a double‐blind RCT, we randomized 81 postmenopausal osteopenic women to 1‐yr nightly treatment with melatonin 1 mg (N = 20), 3 mg (N = 20), or placebo (N = 41). At baseline and after 1‐yr treatment, we measured bone mineral density (BMD) by dual X‐ray absorptiometry, quantitative computed tomography (QCT), and high‐resolution peripheral QCT (HR‐pQCT) and determined calciotropic hormones and bone markers. Mean age of the study subjects was 63 (range 56–73) yr. Compared to placebo, femoral neck BMD increased by 1.4% in response to melatonin (P < 0.05) in a dose‐dependent manner (P < 0.01), as BMD increased by 0.5% in the 1 mg/day group (P = 0.55) and by 2.3% (P < 0.01) in the 3 mg/day group. In the melatonin group, trabecular thickness in tibia increased by 2.2% (P = 0.04), and volumetric bone mineral density (vBMD) in the spine, by 3.6% (P = 0.04) in the 3 mg/day. Treatment did not significantly affect BMD at other sites or levels of bone turnover markers; however, 24‐hr urinary calcium was decreased in response to melatonin by 12.2% (P = 0.02). In conclusion, 1‐yr treatment with melatonin increased BMD at femoral neck in a dose‐dependent manner, while high‐dose melatonin increased vBMD in the spine. Further studies are needed to assess the mechanisms of action and whether the positive effect of nighttime melatonin will protect against fractures.     

Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats

Abstract: The study objective was to investigate the effects of melatonin on obesity and obesity‐associated systolic hypertension and dyslipidemia in young male Zucker diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome. ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups (n = 10): naive (N), vehicle‐treated (V), and melatonin‐treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Melatonin reduced mean weight gain (51 ± 2/100 g BW) versus N‐ZDF group (58 ± 3, P < 0.05) without food intake differences. M‐ZDF rats showed an apparent reduction in systolic hypertension that proved not to be statistically significant, and a significant improvement in dyslipidemia, with a reduction in hypertriglyceridemia from 580 ± 40 to 420.6 ± 40.9 mg/dL (P < 0.01). Melatonin raised high‐density‐lipoprotein (HDL) cholesterol in ZDF (from 81.6 ± 4.9 to 103.1 ± 4.5 mg/dL, P < 0.01) and ZL rats (from 62.8 ± 4.8 to 73.5 ± 4.8 mg/dL, P < 0.05) and significantly reduced low‐density‐lipoprotein (LDL) cholesterol in ZDF rats from 5.20 ± 0.4 to 4.14 ± 0.3 mg/dL (P < 0.05) but had no effect on total cholesterol levels. To our knowledge, this is the first evidence of a positive effect of melatonin on overweight and lipid pattern of obese Zucker diabetic rats, supporting the proposition that melatonin administration may ameliorate overweight and lipid metabolism in humans. Because these benefits occurred in youth, before advanced metabolic and vascular complications, melatonin might help to prevent cardiovascular disease associated with obesity and dyslipidemia.