Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia

The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero-flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial-dependent and -independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham-ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti-inflammatory effect.     

Melatonin protects kidney grafts from ischemia/reperfusion injury through inhibition of NF-kB and apoptosis after experimental kidney transplantation

Abstract:  Free radicals are involved in pathophysiology of ischemia/reperfusion injury (IRI). Melatonin is a potent scavenger of reactive oxygen and nitrogen species. Thus, this study was designed to elucidate its effects in a model of rat kidney transplantation. Twenty Lewis rats were randomly divided into 2 groups (n = 10 animals each). Melatonin (50 mg/kg BW) dissolved in 5 mL milk was given to one group via gavage 2 hr before left donor nephrectomy. Controls were given the same volume of milk only. Kidney grafts were then transplanted into bilaterally nephrectomized syngeneic recipients after 24 hr of cold storage in Histidine–Tryptophan–Ketoglutarate solution. Both graft function and injury were assessed after transplantation through serum levels of blood urea nitrogen (BUN), creatinine, transaminases, and lactate dehydrogenase (LDH). Biopsies were taken to evaluate tubular damage, the enzymatic activity of superoxide dismutase (SOD) and lipid hydroperoxide (LPO), and the expression of NF-kBp65, inducible nitric oxide synthase (iNOS), caspase-3 as indices of oxidative stress, necrosis, and apoptosis, respectively. Melatonin improved survival ( P  < 0.01) while decreasing BUN, creatinine, transaminases, and LDH values up to 39–71% ( P  < 0.05). Melatonin significantly reduced the histological index for tubular damage, induced tissue enzymatic activity of SOD while reducing LPO. At the same time, melatonin down-regulated the expression of NF-kBp65, iNOS, and caspase-3. In conclusion, donor preconditioning with melatonin protected kidney donor grafts from IRI-induced renal dysfunction and tubular injury most likely through its anti-oxidative, anti-apoptotic and NF-kB inhibitory capacity.     

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.     

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.     

A novel electrophysiologic effect of melatonin on ischemia/reperfusion-induced arrhythmias in isolated rat hearts

Abstract:  Reperfusion after a short period of cardiac ischemia triggers ventricular arrhythmias attributable to ionic imbalance and oxidative stress. Melatonin offers some degree of protection, but its effects on the cardiac action potentials are unknown. We evaluated the effects of 5, 10, 20 and 50 μ m melatonin in isolated perfused rat hearts subjected to 10 min of regional ischemia. ECG and membrane potentials were synchronously displayed. After 15 min of reperfusion, total antioxidant capacity (TAC) was determined. Melatonin did not change the ischemic depolarization nor the action potential amplitude depression, but at the end of ischemia the action potential duration (APD) decreased in control and 5 μ m melatonin-treated hearts. By contrast, it returned to preischemic levels in hearts given 20 and 50 μ m melatonin. Melatonin reduced the incidence of reperfusion arrhythmias from 100% in control to 50% in 5 and 10 μ m , to 40% in 20 μ m and 30% in 50 μ m hearts. TAC values were higher at all melatonin concentrations. We conclude that melatonin reduced the incidence of reperfusion arrhythmias because of its antioxidant effects. In addition, at 20 and 50 μ m lengthened APD and promoted an improved protection. This latter effect should be considered when in vivo applications of melatonin are considered.     

Melatonin inhibits type 1 interferon signaling of toll-like receptor 4 via heme oxygenase-1 induction in hepatic ischemia/reperfusion

The cytoprotective mechanisms of melatonin in hepatic ischemia/reperfusion (I/R) injury associated with heme oxygenase-1 (HO-1) induction and type 1 interferon (IFN) signaling pathway downstream of toll-like receptor 4 (TLR4) were investigated. Rats were subjected to 60min of ischemia followed by 5-hr reperfusion. Melatonin (10mg/kg) or vehicle (5% ethanol in saline) was administered intraperitoneally 15min prior to ischemia and immediately before reperfusion. Rats were pretreated with zinc protoporphyrin (ZnPP, 10mg/kg, i.p.), a HO-1 inhibitor, at 16 and 3hr prior to ischemia. Melatonin attenuated the I/R-induced increase in serum alanine aminotransferase activity, and ZnPP reversed this attenuation. Melatonin augmented the levels of HO activity and HO-1 protein and mRNA expression, and this enhancement was reversed by ZnPP. Melatonin enhanced the level of NF-E2-related factor-2 (Nrf2) nuclear translocation, and ZnPP reversed this increase. Overexpression of TLR4 and its adaptor proteins, toll-receptor-associated activator of interferon (TRIF), and myeloid differentiation factor 88 (MyD88), induced by I/R, was attenuated by melatonin; ZnPP reversed the effect of melatonin on TLR4 and TRIF expression. Melatonin suppressed the increased interaction between TLR4/TRIF and TLR4/MyD88, which was reversed by ZnPP. Melatonin attenuated the increased levels of JAK2 and STAT1 activation as well as IFN-β, and ZnPP reversed these inhibitory effects of melatonin. Melatonin inhibited the level of chemokine (C-X-C motif) ligand 10 (CXCL-10), and ZnPP reversed this inhibition. Our findings suggest that melatonin protects the liver against I/R injury by HO-1 overexpression, which suppresses the type 1 IFN signaling pathway downstream of TLR4.     

Erythropoietin promotes hepatic regeneration after extended liver resection in rats

Background and Aim:  It has been proven in various animal studies that recombinant human erythropoietin (rHuEPO) protects renal, cardiac and neuronal, as well as hepatic, tissue from ischemia, and promotes regeneration of adult central nervous system neurons. To date, no data are available as to whether rHuEPO has the ability to stimulate liver regeneration after liver resection.
Methods:  Rats undergoing 70% or 90% hepatectomy received an intraportalvenous administration (i.p.) of rHuEPO prior to resection or a subcutaneous injection (s.c.) for 3 days postoperatively, control animals were treated with surgery and saline injection only. Regeneration capacity of remnant livers was studied over 7 days by histology and immunohistochemistry (Ki-67, proliferating cell nuclear antigen [PCNA]). Polymerase chain reaction was carried out to measure transforming growth factor β (TGF-β), hypoxia induced factor (HIF), signal transducing activator 3 and vascular endothelial growth factor.
Results:  Ten-day survival in rats undergoing 90% hepatectomy significantly increased in i.p.-pretreated animals. After 70% hepatectomy the mitotic index was significantly increased in both rHuEPO-treated groups. These data were confirmed by PCNA and Ki-67 expression, which was significantly increased in the treated groups 24 h and 2 days after liver resection. TGF-β and HIF mRNA both were upregulated in control animals 3 h after surgery.
Conclusion:  rHuEPO effectively increased liver regeneration in rats after 70% liver resection and enhanced survival after 90% hepatectomy. Thus, rHuEPO may increase the regenerative capacity after major hepatectomy.     

Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats

Abstract:  Liver cirrhosis is a critical stage of chronic liver diseases that can produce liver failure, portal hypertension and hepatocarcinoma. Sustained oxidative stress plays a key role in cell damage and fibrosis induced during liver cirrhosis. We evaluated the effect of oxidative stress regulation by melatonin on the development of parenchymal destruction and stellate cell activation in experimental liver cirrhosis. Melatonin was administered to rats with liver cirrhosis induced by thioacetamide (TAA) for 1 or 3 months. Liver injury was assessed by serological analysis, as well as hematoxylin-eosin staining and the in situ apoptosis detection assay in liver sections. Oxidative stress was evaluated by lipoperoxide and reduced glutathione levels, and by the measurement of catalase and superoxide dismutase activities in liver and serum respectively. The activation of stellate cells was evaluated by α -smooth muscle actin expression in liver sections. Our results showed that TAA induced oxidative stress with extensive tissue damage and enhanced α -smooth muscle actin expression in liver. Melatonin prevented the oxidative stress-related changes associated with TAA toxicity. In conclusion, the study showed that melatonin prevents the tissue damage and fibrosis associated with TAA-induced liver cirrhosis in rats.     

Effects of Quercetin on Gene and Protein Expression of NOX and NOS after Myocardial Ischemia and Reperfusion in Rabbit

Previous studies have suggested that reactive oxygen species (ROS), endothelial nitricoxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) are involved in the pathophysiology of myocardial ischemia‐reperfusion injury (MIRI). The NOX family of NADPH oxidases share the capacity to generate superoxide and ROS. Several studies have demonstrated that quercetin possesses a protective effect against MIRI. Our aim is to investigate the effects of quercetin on NOX2, eNOS, and iNOS after MIRI in rabbits. New Zealand rabbits were subjected to 30 min of myocardial ischemia followed by 12 h of reperfusion. They were then randomly assigned to four experimental groups: control, I/R (ischemia/reperfusion), quercetin (Que), I/R + Que. Gene and protein expression of NOX2, eNOS, and iNOS were compared. Both in real‐time PCR and in the Western blotting studies, myocardial ischemia‐reperfusion‐induced NOX2 and iNOS expression were enhanced (P < 0.01) but eNOS mRNA and protein expression in I/R hearts were not significantly different from those in control (P < 0.01). Administration of quercetin reduced NOX2, eNOS, and iNOS mRNA and protein expression both in control and in I/R heart (P < 0.01). Gene and protein expression of NOX2 and iNOS were increased after MIRI. Quercetin not only inhibited myocardial ischemia‐reperfusion‐induced NOX2 and iNOS mRNA and protein expression but also inhibited eNOS mRNA and protein expression.     

Exogenous melatonin positively influences follicular dynamics, oocyte developmental competence and blastocyst output in a goat model

Abstract:  The role of melatonin in modulating mammalian reproduction is of particular interest; however, its effects on ovarian follicles and their oocytes still remain to be characterized. This study determined the influence of melatonin treatment on follicular growth patterns and on in vitro oocyte developmental competence. In a first experiment, the effects of melatonin supplementation on follicular dynamics were evaluated using daily transrectal ultrasonographies for 21 days, in 7 multiparous Sarda goats receiving a subcutaneous implant of 18 mg of melatonin and in 5 control untreated does. Melatonin caused more follicular waves (5.2 ± 0.2 versus 4 ± 0.3; P  < 0.05) as the waves were shortened at around 2 days when compared with the non-melatonin treated control goats ( P  < 0.001). Oocyte developmental competence was evaluated in a second experiment by applying procedures for in vitro embryo production. There were no significant differences in the total number of oocytes obtained from 6 control (n = 192) and 7 melatonin-treated (n = 265) goats given follicle stimulating hormone to induce follicular development. Differences in oocyte developmental competence between the two groups became evident after in vitro fertilization and culture; melatonin increased the rate of cleaved oocytes in comparison with control animals (82.5 versus 63.4%; P  < 0.001), advanced timing of embryo development and enhanced blastocyst output (31.5 versus 16.3%; P  < 0.01). However, blastocyst quality, as evaluated by cryotolerance and gene expression analysis, was not found to be different between the groups. In conclusion, in vivo melatonin treatment is beneficial for increasing ovarian follicle turnover and improving oocyte developmental competence and kinetics of the blastocyst.     

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.     

Hepatoprotective actions of melatonin: possible mediation by melatonin receptors

Melatonin,the hormone of darkness and messenger of the photoperiod,is also well known to exhibit strong direct and indirect antioxidant properties. Melatonin has previously been demonstrated to be a powerful organ protective substance in numerous models of injury; these beneficial effects have been attributed to the hormone’s intense radical scavenging capacity. The present report reviews the hepatoprotective potential of the pineal hormone in various models of oxidative stress in vivo,and summarizes the extensive literature showing that melatonin may be a suitable experimental substance to reduce liver damage after sepsis,hemorrhagic shock,ischemia/reperfusion,and in numerous models of toxic liver injury. Melatonin’s influence on hepatic antioxidant enzymes and other potentially relevant pathways,such as nitric oxide signaling,hepatic cytokine and heat shock protein expression,are evaluated. Based on recent literature demonstrating the functional relevance of melatonin receptor activation for hepatic organ protection,this article finally suggests that melatonin receptors could mediate the hepatoprotective actions of melatonin therapy.     

Ischemia/reperfusion-induced arrhythmias in the isolated rat heart: Prevention by melatonin

Abstract: Cardiac arrhythmias during ischemia/reperfusion are believed to be related to free radicals generated in the heart especially during the period of reperfusion. Since melatonin functions as a free radical scavenger and antioxidant, the ability of this molecule to influence cardiac arrhythmias was investigated. The pineal secretory product, melatonin, reduced the incidence and severity of arrhythmias induced by ischemia/reperfusion due to ligation of the anterior descending coronary artery in the isolated rat heart. Melatonin was either infused during both the ischemia and reperfusion periods or only late in the ischemia period and throughout reperfusion. The percentage of hearts that developed cardiac arrhythmias during reperfusion as indicated by the incidence of premature ventricular contraction (PVC) and ventricular fibrillation (VF) were recorded. Melatonin either infused during both the ischemia and reperfusion periods or during essentially the period of reperfusion greatly reduced PVC and VF due to occlusion and reopening the anterior descending coronary artery. Presumably melatonin's beneficial effect in reducing cardiac arrhythmias was due in part to its free radical scavenging activity, which is greatly assisted by the rapidity with which it is taken up into cells. Previous studies have shown that vitamin C is effective in reducing the severity of cardiac arrhythmias induced by ischemia/reperfusion; thus, we also compared the efficacy of melatonin with this well-known antioxidant. Melatonin was more potent than vitamin C in protecting against arrhythmias induced by ischemia/reperfusion. Besides melatonin's function as a broad spectrum free radical scavenger, melatonin may have also reduced cardiac arrhythmias due to its regulation of intracellular calcium levels, i.e., by preventing calcium overloading, or due to its ability to suppress sympathetic nerve function and reduce adrenergic receptor function in the myocardium. Additional studies into the mechanisms of melatonin's action in reducing cardiac arrhythmias due to ischemia/reperfusion or other causes are warranted because of the possible application of this information to humans with heart disease.     

Altered neutrophil apoptosis activity is reversed by melatonin in liver ischemia-reperfusion

Delayed neutrophil apoptosis has been implicated as the mechanism of the systemic inflammatory response. Herein, we examined the effect of melatonin on the neutrophil apoptosis in ischemia and reperfusion of the human liver. We studied seven patients receiving elective hepatectomy for liver tumor and ten patients receiving laparoscopic cholecystectomy for gallstones. Ten milli liters of blood was drawn isolation and incubation of the human neutrophils. Neutrophil apoptosis activity and CD18 expression and respiratory burst activity were assessed flow cytometrically. Another group of neutrophils included those from the patients receiving hepatectomy and isolated and incubated with melatonin. Neutrophil apoptosis is delayed from patients after hepatectomy or laparoscopic cholecystectomy when compared with that of the preoperative state. The decrease in the apoptosis activity is more severe in patients receiving hepatectomy as compared with those receiving laparoscopic cholecystectomy. Neutrophils from patients receiving hepatectomy or laparoscopic cholecystectomy are functionally activated. Melatonin can reverse the delayed process and enhance the apoptosis activity in neutrophils from patients receiving hepatectomy. This study demonstrates that melatonin enhances neutrophil apoptosis in patients receiving hepatectomy involving ischemia and reperfusion of the human liver.     

Cytoprotective and anti-inflammatory effects of melatonin in hydrogen peroxide-stimulated CHON-001 human chondrocyte cell line and rabbit model of osteoarthritis via the SIRT1 pathway

Melatonin has potent antioxidant, analgesic, and antinociceptive properties. However, the effects of melatonin against oxidative stress-induced cytotoxicity and inflammatory mediators in human chondrocytes remain poorly understood. This study examined the effects and underlying mechanism of melatonin in hydrogen peroxide (H(2) O(2) )-stimulated human chondrocytes and rabbit osteoarthritis (OA) model. Melatonin markedly inhibited hydrogen peroxide (H(2) O(2) )-stimulated cytotoxicity, iNOS, and COX-2 protein and mRNA expression, as well as the downstream products, NO and PGE(2) . Incubation of cells with melatonin decreased H(2) O(2) -induced Sirtuin 1 (SIRT1) mRNA and protein expression. SIRT1 inhibition by sirtinol or Sirt1 siRNA reversed the effects of melatonin on H(2) O(2) -mediated induction of pro-inflammatory cytokines (NO, PGE(2) , TNF-α, IL-1β, and IL-8) and the expression of iNOS, COX-2, and cartilage destruction molecules. Melatonin blocked H(2) O(2) -induced phosphorylation of PI3K/Akt, p38, ERK, JNK, and MAPK, as well as activation of NF-κB, which was reversed by sirtinol and SIRT1 siRNA. In rabbit with OA, intra-articular injection of melatonin significantly reduced cartilage degradation, which was reversed by sirtinol. Taken together, this study shows that melatonin exerts cytoprotective and anti-inflammatory effects in an oxidative stress-stimulated chondrocyte model and rabbit OA model, and that the SIRT1 pathway is strongly involved in this effect.     

Melatonin accelerates the process of wound repair in full-thickness incisional wounds

Abstract:  The pineal gland hormone melatonin is known to have both anti-inflammatory and immunomodulatory effects. Given this, we propose that melatonin is an ideal candidate to enhance the process of wound healing. The present study assessed the effects of exogenously administered melatonin (1.2 mg/kg intra-dermal), on scar formation using a full-thickness incisional rat model of dermal wound healing. Melatonin treatment significantly improved the quality of scarring, both in terms of maturity and orientation of collagen fibres. An increase in nitric oxide synthase (NOS) activity and therefore nitric oxide production is detrimental during inflammation but is favourable during granulation tissue formation. Melatonin treatment significantly decreased inducible NOS (iNOS) activity during the acute inflammatory phase but significantly increased iNOS activity during the resolving phase. Cyclooxygenase-2, which has been shown to have anti-inflammatory effects, was elevated in the melatonin-treated rats following wounding. In addition, melatonin treatment also accelerated the angiogenic process, increasing the formation of new blood vessels and elevating the level of vascular endothelial growth factor protein expression during granulation tissue formation. Melatonin treatment increased arginase activity (which generates proline, a building block for collagen synthesis) from earlier time points. The protein profiles of hemoxygenase-1 (HO-1) and HO-2 isoforms, vital participants in the repair process, were also up-regulated upon melatonin treatment. This study has therefore demonstrated, for the first time, that melatonin can significantly improve the quality of wound healing and scar formation.     

Melatonin prevents deregulation of the sphingosine kinase/sphingosine 1‐phosphate signaling pathway in a mouse model of diethylnitrosamine‐induced hepatocellular carcinoma

The sphingosine kinase (SphK)/sphingosine 1‐phosphate (S1P) pathway is involved in multiple biological processes, including carcinogenesis. Melatonin shows beneficial effects in cell and animal models of hepatocellular carcinoma, but it is unknown if they are associated with the modulation of the SphK/S1P system, along with different downstream signaling pathways modified in cancer. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight i.p) once a week for 8 weeks. Melatonin was given at 5 or 10 mg/kg/day i.p. beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Melatonin alleviated the distortion of normal hepatic architecture, lowered the incidence of preneoplastic/neoplastic lesions, and inhibited the expression of proliferative/cell cycle regulatory proteins (Ki67, PCNA, cyclin D1, cyclin E, CDK4, and CDK6). S1P levels and expression of SphK1, SphK2, and S1P receptors (S1PR1/S1PR3) were significantly elevated in DEN‐treated mice. However, there was a decreased expression of S1P lyase. These effects were significantly abrogated in a time‐ and dose‐dependent manner by melatonin, which also increased S1PR2 expression. Following DEN treatment, mice exhibited increased phosphorylation of PI3K, AKT, mTOR, STAT3, ERK, and p38, and a higher expression of NF‐κB p50 and p65 subunits. Melatonin administration significantly inhibited those changes. Data obtained suggest a contribution of the SphK/S1P system and related signaling pathways to the protective effects of melatonin in hepatocarcinogenesis.     

Age-dependent lipopolysaccharide-induced iNOS expression and multiorgan failure in rats: effects of melatonin treatment

Senescence amplifies the sensitivity to endotoxemia, which correlates with increased nitric oxide (NO) levels and mortality. Melatonin displays antioxidant and anti-inflammatory effects, but its levels decrease with age. Lipopolysaccharide (LPS) (10 mg/kg) was injected to 3- and 18-month-old rats 6 h before they were killed, and melatonin (60 mg/kg) was injected before and/or after LPS. Inducible nitric oxide synthase (iNOS) expression and activity, nitrite content, lipoperoxidation (LPO) levels, and serum markers of liver, renal, and metabolic dysfunction, were measured in liver and lung of these animals. An age-dependent increase in iNOS activity, NO content, and LPO levels was observed, and these changes were augmented further by LPS. Melatonin decreased the expression and activity of iNOS, reducing NO and LPO levels to basal values in both septic LPS-treated groups. Liver, kidney, and metabolic dysfunctions were also significantly higher in aged that in young rats and further increased by LPS. Melatonin treatment counteracted these alterations in young and aged septic rats. Melatonin reduced LPS-dependent iNOS expression and multiorgan failure in a similar extent in young and aged rats. Because aged rats showed higher organ and metabolic impairment than young animals in response to LPS, the results also suggest an increased efficacy of the anti-septic properties of melatonin in the aged animals.     

Melatonin plus porcine bone on discrete calcium deposit implant surface stimulates osteointegration in dental implants

Abstract:  The aim of this study was to evaluate the effect of the topical application of melatonin mixed with collagenized porcine bone to accelerate the osteointegration on the rough discrete calcium deposit (DCD) surface implants in Beagle dogs 3 months after their insertion. In preparation for subsequent insertion of dental implants, lower premolars and molars were extracted from 12 Beagle dogs. Each mandible received three parallel wall implants with discrete calcium deposit (DCD) surface of 4 mm in diameter and 10 mm in length. The implants were randomly assigned to the distal sites on each side of the mandible in three groups: group I implants alone, group II implants with melatonin and group III implants with melatonin and porcine bone. Prior to implanting, 5 mg lyophylized powdered melatonin was applied to one bone hole at each side of the mandible. None was applied at the control sites. Ten histological sections per implant were obtained for histomorphometric studies. After a 4-wk treatment period, melatonin significantly increased the perimeter of bone that was in direct contact with the treated implants ( P <  0.0001), bone density ( P <  0.0001), new bone formation ( P <  0.0001) in comparison with control implants. Topical application of melatonin on DCD surface may act as a biomimetic agent in the placement of endo-osseous dental implants and enhance the osteointegration. Melatonin combined with porcine bone on DCD implants reveals more bone to implant contact at 12 wk (84.5 ± 1.5%) compared with melatonin treated (75.1 ± 1.4%) and nonmelatonin treated surface implants (64 ± 1.4%).