A comparison of melatonin and α-lipoic acid in the induction of antioxidant defences in L6 rat skeletal muscle cells.

Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. The loss of cells during aging in vital tissues and organs is related to several factors including oxidative stress and inflammation. Skeletal muscle degeneration is common in elderly people; in fact, this tissue is particularly vulnerable to oxidative stress since it requires large amounts of oxygen, and thus, oxidative damage is abundant and accumulates with increasing age. Melatonin (N-acetyl-5-methoxytryptamine) is a highly efficient scavenger of reactive oxygen species and it also exhibits beneficial anti-inflammatory and anti-aging effects. This study investigated the susceptibility of rat L6 skeletal muscle cells to an induced oxidative stress following their exposure to hydrogen peroxide (50 μM) and evaluating the potential protective effects of pre-treatment with melatonin (10 nM) compared to the known beneficial effect of alpha-lipoic acid (300 μM). Hydrogen peroxide-induced obvious oxidative stress; it increased the expression of tumour necrosis factor-alpha and in turn promoted nuclear factor kappa-B and overrode the endogenous defence mechanisms. Conversely, pre-treatment of the hydrogen peroxide-exposed cells to melatonin or alpha-lipoic acid increased endogenous antioxidant enzymes, including superoxide dismutase-2 and heme oxygenase-1; moreover, they ameliorated significantly oxidative stress damage and partially reduced alterations in the muscle cells, which are typical of aging. In conclusion, melatonin was equally effective as alpha-lipoic acid; it exhibited marked antioxidant and anti-aging effects at the level of skeletal muscle in vitro even when it was given in a much lower dose than alpha-lipoic acid

Melatonin limits adaptive ER stress and hepatosteatosis in leptin-deficient mice

Non alcoholic fatty liver disease (NAFLD) impacts on about 30% of the population in industrialized countries, associated to the metabolic syndrome may be reversible or dramatically evolve into cirrhosis or hepatocellular cancer (Wree et al., 2011). Leptin-deficient homozygous mice (ob/ob) represent a well-known animal model to study obesity, associated with overweight, liver steatosis and insulinresistance. Recently ER stress has been reported to contribute to hepatic steatosis and cell damage called lipoapoptosis (Flamment et al., 2010). Melatonin, the main pineal indoleamine, has been demonstrated to be useful to limit adipogenesis in many metabolic clinical conditions (de Luxan-Delgado et al., 2014). Therefore major aims of the present study were: 1.To localize ER stress, energy homeostasis and hypoxia markers in the liver of ob/ob mice receiving or not melatonin in drinking water at 100 mg/ kg/day for 8 weeks; 2.To characterize hepatic steatosis and quantify macrosteatosis in different experimental groups. C57BL6 mice treated or not with melatonin were used as controls. Remarkably in ob/ob mice receiving melatonin, macrosteatosis, periportal GRP78 staining decreased while beta catenin became basolateral into hepatocytes. Furthermore melatonin limited nuclear CHOP staining, a recognized index of major sensitivity to apoptosis, but stimulated p62/SQSTM1 signal, involved in reducing lipogenesis. Moreover by TEM analysis, we visualized in ob/ob mice liver mitochondria that displayed more cristae and strict RER adhesion after melatonin intake. In conclusion, our morphological analysis suggests that melatonin might ameliorate NAFLD by anti-oxidative and ER stress modulatory abilities in obese mice

Metabolic syndrome and melatonin: a tool for prevent obesity-associated abnormalities

Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease [1]. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, the major section of the reabsorptive process. To best perform its functions, the kidney requires energy primarily provided by mitochondria. Melatonin, indoleamine and antioxidant, has been identified in mitochondria, and overwhelming evidence has documented its essential role in the prevention of oxidative mitochondrial damage [2]. Herein, we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondria morphology and dynamics as influenced by mitofusin- 2 and the intrinsic apoptotic cascade. Melatonin was dissolved in 1% ethanol and added to the drinking water from postnatal week 5 to 13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-induced morphological alterations were apparent in the proximal tubules; the tubules contained round mitochondria with irregular, short cristae and the lining cells excited and elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria- driven intrinsic apoptotic pathway. The results aid in reducing renal failure. The melatonin-mediated changes probably suggest the use of melatonin to protect against renal morphological damage and dysfunction during metabolic disease

Effect of mercury in virgin, pregnant and lactating rats

Mercury is a toxic metal widely used in industrial activities. In Brazil, cases of mercury contamination occur mainly in the Amazon region, where mercury is used in mining to amalgamate the gold; people is so exposed to mercury occupationally and through the consumption of contaminated fish and water [1]. Mercury has a non uniform distribution after absorption, being accumulated mainly in the kidneys causing renal injury [2]. Numerous metabolic changes occur physiologically in pregnancy and lactation periods and induce a different response to exogenous substances, respect virgin animals. At this aim, we evaluated the pathogenetic mechanism, at kidney level, involved in the different responses of virgin, pregnant and lactating rats exposed to a single dose of mercury. Interestingly, the mercury-induced nephrotoxicity differs among pregnant or lac- tating respect virgin rats. In particular, virgin rat showed kidney histopathological alterations including interstitial fibrosis and tubular damages and an altered modulation of heat shock proteins, damages that correlates with the overall loss of renal function. The distinctive responses between pregnant and lactating respect virgin rats observed in our study may be associated with some of several physiological changes that occur during pregnancy or lactation periods

Protective effects of melatonin against nicotine-induced oxidative damage of kidney

Several studies demonstrated that melatonin treatment prevents tissue damage in various models of oxidative stress (1). Experiments have shown that chronic nicotine administration caused oxidant damage in various organs by increasing lipid peroxidation products and decreasing the activity of endogenous antioxidants (2). The aim of this study was to investigate the effects of melatonin treatment on nicotine-induced oxidative changes in rat kidney and to explore the possible mechanisms of action. Three groups of rats were used as controls (the first without treatment, the second with melatonin alone and the third with nicotine alone). The last group of rats was orally treated with nicotine and melatonin for 28 days. Morphological changes in kidney were evaluated by histological procedures and immunohistochemical analysis using inflammation (NFkB and IL-6) and oxidative stress (SOD, CAT and iNOS) markers. Experiments performed demonstrated that nicotine administration increases inflammation and oxidative stress. Melatonin has a protective effect against nicotine kidney toxicity through an inhibition of inflammation and consequent oxidative damage. These data suggest that melatonin supplementation effectively counteracts the deleterious effect of chronic nicotine administration on kidney and attenuates oxidative damage possibly by its anti-inflammatory and antioxidant effects.This work was supported by grants from University of Brescia (EX 60%)

Pineal gland and neuropathic pain

The pineal gland is a small neuroendocrine organ involved primarily in the circadian rhythm by the secretion of melatonin [1]. In addition, the pain modulatory properties of melatonin are generally recognized but its involvement in neuropathic pain regulation is not fully understood. In fact, it is known that the activation of the endogenous melatonin system in the spinal cord can reduce the generation, development and maintenance of central sensitization [2]. Moreover, melatonin showed an analgesic effect, in fact several works in animals [2] and in humans [3] underline its ability to inhibit hyperalgesia. In particular, intracerebroventricular and intraperitoneal melatonin, with its higher doses, produces a blockade of thermal hyperalgesia in mice with partial tight ligation of the sciatic nerve. The aim of our work is to characterize the morphological changes in peripheral structures, such as plantar skin and dorsal root ganglia (DRG) of rats in a neuropathic pain model (chronic constriction injury) after a single melatonin treatment monitoring the behaviour and the changes in NO-system using immunohistochemical techniques. The behavioural results show an increase of withdrawal latency during plantar test already after 30 min from melatonin administration. The immunohistochemical results suggest that melatonin plays a crucial role in keratinocytes-mediated neuropathic pain transmission through the modulation of nitroxidergic system, which could have also a protective role at this site. In addition, at DRG level the NO-system is maintained at low level. These results suggest that melatonin administration or modulation of pineal gland activity may have clinical utility in neuropathic pain therapy in the future

Melatonin atheroprotective effects in vivo

Chronic inflammatory fibro-proliferative changes leading to atherosclerotic plaques are considered hallmark of cardiovascular diseases [1]. Atherosclerosis pathogenesis is a complex entity, which has not been fully understood; however, many studies have demonstrated the role of oxidative stress and inflammation in its development. Melatonin effects on inflammation and oxidative stress process have been demonstrated in the last ten-year literature [2]. However, its role(s) and mechanism(s) of action as a therapeutic tool against atherosclerosis remain largely unexplored. Our aims were to assess the role of melatonin in the onset and developing of atherosclerotic plaques through radiologic and morphometrical tools in 20 apolipoprotein-E knockout (ApoE) mice fed with Western diet (42% calories from fat). 10/20 mice were treated with melatonin (10 mg/kg per os). 18F-FDG PET-CT is a widely used tool to assess inflammatory changes, even before macroscopic changes have taken place. Glucose metabolism is known to be higher in areas of inflammation due to an increased expression of GLUT transporters on the cell membranes both in animals and humans. Using this feature PET/CT is able to determinate metabolic cellular changes and therefore it can be used as biomarker of atherosclerosis. All mice were scanned both before starting melatonin treatment and at the end of the study. After the last scan mice were sacrificed and vascular remodeling, oxidative stress and inflammation at aortic arch level were evaluated. CT-corrected PET datasets were used for computation of SUVmax. Atherosclerotic vascular remodeling, oxidative stress and inflammation levels were significantly more conspicuous in the control cohort, compared to the treated mice (p≤0.05). 18F-FDG PET/CT did not show significant difference in SUVmax. In summary, also in vivo, melatonin may have a protective effect in the atherosclerotic pathogenesis. Flamma S.p.A.-Italy (www.flammagroup.com) provided with melatonin. Financial supports: Fondazione Cariplo e Regione Lombardia “New opportunities and ways towards ERC” (Project 2014-2256) and University of California - Radiology and Biomedical Imaging Nuclear Medicine Section

Melatonin oral supplementation against fibromyalgia-related skeletal muscle alterations

Fibromyalgia is a chronic idiopathic pain syndrome characterized by widespread musculoskeletal pain and a deep range of other symptoms including disordered sleep, paresthesia, depression and anxiety (1). To date, its aetiopathogenesis and pathophysiology are still not understood, but the musculoskeletal, neuroendocrine and central nervous systems appear to play major roles in the development and progression of fibromyalgia (2). Important factors involved in the pathogenic process of fibromyalgia are oxidative stress and inflammation suggesting that antioxidative supplementation might be important in the management and modulation of fibromyalgia. Recent evidences suggest that melatonin may be suitable for this purpose. Melatonin is a small, highly conserved pineal indoleamine and due to its important and well known antioxidant and antinflammatory properties, together with also its analgesic effects, our research group studied the beneficial effects of the melatonin oral supplementation against the pathogenetic process of fibromyalgia. In detail, Sprague Dawley rats were randomly treated with reserpine, to reproduce the pathogenic process of fibromyalgia (3), and/or with melatonin (MelapureTM by Flamma S.p.A.). At the end of the treatments, the animals treated with reserpine showed moderate alteration at hind limb skeletal muscle level with difficult in moving, together with a significant expression of several oxidative stress and inflammatory markers at the gastrocnemius muscle level. Interestingly, melatonin, dose and time dependently, reduced the difficulties in walking and the musculoskeletal oxidative stress and inflammatory processes. In summary, this pilot study suggested that melatonin could be an in vivo effective tool against muscoloskeletal morphofunctional damages and dysfunctions in the management of fibromyalgia-related complications.Sincere thanks to Flamma S.p.A.- Italy (www.flammagroup.com) for courteously providing the melatonin and for the precious economic support to this study

Metabolic syndrome and beneficial vascular effect of melatonin

The metabolic syndrome is a cluster of metabolic abnormalities including abdominal obesity, hypertension, altered levels of triglycerides and of cholesterol and high fasting glucose levels (Sohet and Delzenne, 2012). In this study, we hypothesized that melatonin, due to its ability to neutralize a number of toxic reactants, including reactive oxygen species and free radicals (Reiter et al. 2008; Agil et al. 2011), can minimize obesity-related alterations in aorta morphology and vasocostriction in an animal model of obesity (ob/ob mice). The animals were divided in four groups: (i) control lean mice, (ii) control lean mice treated with melatonin, (iii) ob/ob mice and (iv) ob/ob mice treated with melatonin. The melatonin (kindly provided by Chronolife S.r.l.) was added to the drinking water from postnatal week 5 to 13. Compared with the obese mice, melatonin intake was associated with a significant decrease in body weight and water consumption. Histological analysis showed that the aortic wall of ob/ob mice had a high tunica media/lumen ratio and that the elastic fibers in the medial layer appeared disrupted and degraded. Moreover, the aorta of ob/ob mice displayed both a higher degree of collagen accumulation in the tunica media and an elevated expression of endothelin-1, marker of vasoconstriction, respect to the normal aorta. Whereas, the aorta of ob/ob mice treated with melatonin had a lower tunica media/lumen ratio, collagen accumulation and endothelin-1 expression in comparison with untreated ob/ob mice. In conclusion, our results showed that melatonin had no apparent effects on the aorta of lean mice, but its administration in ob/ob mice can lead to a reduction in body weight and can ameliorate aorta histopathological dysfunctions; so melatonin could be an effective tool in the management of obesity-related vascular complications