Inflammation, which is defined as the complex and essential biological response to tissue injures induced by different harmful stimuli, including pathogens, damaged cells, or irritants, represents a hot topic in biomedical research. Inflammation is essential for homeostasis and is usually finely regulated; however, it sometimes escapes control processes and becomes potentially dangerous for the host organism. Initial stages of inflammation are mediated by the activation of the immune system, and it usually persists for only a short time. However, if the response continues and progresses to chronic inflammation, it predisposes to various chronic pathologies, such as rheumatoid arthritis, septic shock, inflammatory bowel disease, Alzheimer`s or Parkinson’s diseases, cancer.
Usually, acute inflammation is a protective attempt by the organism to remove the injurious stimuli as well as to initiate healing processes for the tissue; this phase is associated with cellular mobilization and migration of neutrophils and macrophages to the inflammatory locus . In recent decades, a large number of mediators, including pro-oxidants, nitric oxide (NO), leukotrienes, adhesion molecules, cytokines, and chemokines, which regulate vascular changes and inflammatory cell recruitment, have been identified . Independent of the initial pro-inflammatory stimulus, cellular responses are related to gene modulation mediated by changes in the activation of transcription factors such as nuclear factor kappa B (NFκB), activator protein 1 (AP-1), or signal transducers and activators of transcription (STATs). Moreover, DNA-binding ability of these transcription factors is frequently modified by the various protein kinases involved in signal transduction, including mitogen-activated protein kinases (MAPK) [3, 4].
Because pathological inflammation has been related to a number of different diseases, and current anti-inflammatory drugs have significant side effects, it is important to identify new pharmacological approaches to reduce chronic inflammation without impairing the physiologic inflammatory response.
Melatonin (N-acetyl-5-methoxytryptamine), an endogenous molecule, is an evolutionary conserved indolamine synthesized from tryptophan that is mainly produced by the pineal gland [5, 6], but also by other nonendocrine organs such as Harderian gland, skin, gut, and immune system among others [7, 8]. In recent decades, a number of anti-inflammatory and antioxidant effects have been noted when melatonin is administer exogenously in vivo or when added to cultured cells [9–14]. Although melatonin may have opposite effects in normal and cancer cells, which makes it important to clearly identify cell types when melatonin’s actions are analyzed [11, 15–17], experimental evidence suggests that melatonin may exert its anti-inflammatory effects by regulating a variety of cellular pathways. Melatonin, because of its antioxidant activity, may combat the onset and progression of inflammation, a process that leads to the production of reactive oxygen species and activation of pro-oxidant enzymes .
Melatonin actions depend in part on receptor-dependent processes as well as independent pathways; the latter relate to its direct radical scavenging functions . Certainly, melatonin exerts many of its actions through specific receptors . Specific membrane and nuclear receptors have been described for melatonin in different tissues from many different species . Melatonin membrane receptors belong to the G protein-coupled receptor superfamily, and two functionally active sites have been cloned and characterized, that is, MT1 (Mel 1a) and MT2 (Mel 1b) . Furthermore, the enzyme quinone reductase type 2 (NQO2) has been identified as the cytosolic melatonin receptor, MT3 . This enzyme belongs to a group of reductases that participate in protection against oxidative stress by preventing electron transfer reactions of quinones. Nuclear-binding sites have also been identified for melatonin; these nuclear receptors belong to the ROR/RZR family (retinoic acid-related orphan receptor/retinoid Z receptor), a group of the steroid hormone receptor superfamily. In contrast to RORβ, which is a brain- and retina-specific receptor, RORα is expressed in many tissues and cells outside the brain .
The present review focuses on recent insights into the modulation of the expression of different inflammatory mediators by melatonin and their effects on cell signaling pathways responsible for melatonin’s anti-inflammatory activity.
Melatonin effects on inflammatory mediators
Nitric oxide (NO) is a key molecule influencing various aspects on the inflammatory cascade, ranging from its own production by immunocompetent cells to the recruitment of leukocytes . Moreover, the formation of peroxynitrite – the product of the diffusion-controlled reaction of NO with superoxide anion radical – leads to increased cytotoxicity . NO is synthesized from l-arginine, with the formation of stoichiometric amounts of l-citrulline, by three nitric oxide synthase (NOS) enzymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Low physiological levels of NO are produced by constitutively expressed eNOS and nNOS, whereas iNOS is responsible for prolonged production of larger amounts of NO . Although the modulation of NOS may contribute to the anti-inflammatory actions of melatonin, the mechanisms involved may differ between tissues (Table 1).
Table 1. Publications related to melatonin’s actions on the modulation of nitric oxide synthases
|iNOS||↓||Brain ischemia|| 29, 35 |
|↓||Brain trauma|| 30 |
|↓||Spinal cord injury|| 31 |
|↓||METH microglia damage|| 33 |
|↓||Neuronal MPTP-induced damage|| 34 |
|↓||Diabetic neuropathy|| 36 |
|↓||LPS-induced inflammation|| 7, 37–39, 44, 57 |
|↓||Liver ischemia|| 47, 48 |
|↓||Kidney ischemia|| 49, 50 |
|↓||IBD|| 51 |
|↓||Aging|| 55, 56 |
|↓||Cerulein-induced pancreatitis|| 58 |
|↓||Lung inflammation by pancreatic fluid|| 59 |
|↓||Physical exercise|| 60–62 |
|nNOS||↓||Brain ischemia|| 29 |
|↓||Neuronal MPTP-induced damage|| 34 |
|eNOS||↑||Brain Ischemia|| 29 |
|↓||Aging|| 54 |
In neuropathologies, both nNOS and iNOS seem to play prominent roles in neurodegeneration, while eNOS may reduce neuronal injury . The neuroprotective effects of melatonin against ischemic brain injury induced in rats by middle cerebral artery occlusion could relate, in part, to its ability to repress upregulation of iNOS and nNOS, and to the downregulation of eNOS; the result of these actions leads to a significant reduction in the infarct volume . Furthermore, melatonin administration after traumatic brain injury – which induces inflammation and oxidative stress – reduces iNOS overexpression in penumbra of the cortical lesion by a mechanism that involves modulation of STAT pathways .
NO may aggravate neuronal damage after spinal cord injury (SCI), and the combined therapy of melatonin and exercise reduces mRNA iNOS and glial fibrillary acidic protein (GFAP, a secondary inflammation response marker indicating glial activation) levels and increases hindlimb movement in a SCI rat model . Inflammation, together with other factors such as oxidative/nitrosative stress and mitochondrial dysfunction, seems to play a role in the pathogenesis of Parkinson’s disease (PD). In addition, increases in NO production by activation of microglia induced by chronic or intermittent amphetamine abuse may predispose to Parkinsonism . Melatonin has been shown to inhibit amphetamine-induced iNOS expression in microglial cells, suggesting a potential role as a neuroprotective agent . Studies in a PD disease model that utilized a drug that kills dopaminergic neurons (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or MPTP) indicate that the administration of melatonin or its brain metabolite, N1-acetyl-5-methoxykynuramine (AMK), reduces iNOS, nNOS, and mitochondrial iNOS utilizing an interesting beneficial mechanism . Unexpectedly, the protective effects of melatonin against focal cerebral ischemia, examined in wild-type and MT1/MT2 knockout mice (mt1/2−/−), include decreases in infarct volume and a reduction in brain edema in both groups, but only iNOS activity was impaired in the wild-type mice; this suggests a NO- and membrane receptor-independent beneficial effect in this model . Melatonin also has interesting modulatory effects on neuroinflammation and oxidative stress in diabetic neuropathy and it has been reported in rat model that melatonin administration limits iNOS expression in the sciatic nerve and improves nerve conduction velocity .
In non-neuronal tissues, experimental studies have demonstrated that melatonin inhibits NO production induced by lipopolysaccharide (LPS), a major component of Gram-negative bacteria that induce endotoxic injury. The addition of melatonin, in a micromolar range, prevents LPS-induced iNOS expression in cultured rat endothelial cells and aortic rings through a mechanism not dependent on the activation of G protein-coupled melatonin receptors but an action that is NFκB-dependent . Similar results were noted when this indolamine was administered in vivo (30 mg/kg), where it prevented LPS-induced iNOS expression in rat aorta  and inhibited NFκB transcription factor activation in macrophages . Furthermore, iNOS activation by LPS administration is not only NFκB-dependent but also seems to be stimulated by eNOS expression; eNOS knockout mice are resistant to endotoxic shock . eNOS activation depends on the Ca2+–calmodulin complex, and experiments in endothelial cells stimulated by bradykinin – a molecule involved in a variety of processes including inflammation and endothelial-mediated vasodilation – have demonstrated that the suppression of NO production may be related to intracellular Ca2+ increases. Because eNOS inhibition is not abrogated by the selective MT2 receptor inhibitor (4-phenyl-2-propionamidotetralin or 4P-PDOT), these findings indicate that melatonin effects are not mediated by MT2  and seem to be driven via the stimulation of a G protein-coupled receptor, but not through the opening of receptor-operated ion channels .
In vivo studies show that melatonin exerts beneficial effects against septic shock in humans , and in a mouse model after LPS injection . Melatonin improves survival, induces an important reduction in liver and brain NO production, and modulates the release of pro-/anti-inflammatory cytokines . Moreover, melatonin alleviates the LPS-induced placental cellular response in mice, reducing iNOS and the intensity of placental 3-nitrotyrosine residues when analyzed by immunobloting. These findings are consistent with a protective effect of melatonin against LPS-induced intrauterine fetal death and intrauterine growth restriction [44, 45]. Interestingly, the placenta normally produces melatonin .
Experiments using an in situ liver ischemia/reperfusion model demonstrated that exogenous melatonin is capable of suppressing iNOS expression and NO production with reduced concentrations of TNFα and a restoration of bile production . More recently, it was proposed that the protective effect of melatonin in liver ischemia/reperfusion may be related to the inhibition of toll-like receptor signaling pathway . Also, melatonin protects kidney grafts form ischemia/reperfusion injury and improves survival by a mechanism related to iNOS downregulation . In this model, melatonin was more effective than the iNOS selective inhibitor 1400 W .
Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, and upregulation of different pro-inflammatory markers in the colon. Experimental studies on IBD in rats indicate that melatonin administration inhibits iNOS expression and reduces inflammation. This indicates melatonin may have beneficial effects against IBD .
Aging is frequently associated with an increase in inflammation and oxidative stress, and iNOS expression is elevated in several tissues of aged subjects [52, 53]. Some studies have documented that treatment with a combination of melatonin plus growth hormone partially reduces inflammatory processes in the heart of old mice, decreasing mRNA iNOS expression, and normalizing eNOS expression . Similarly, old male senescence-accelerated (SAMP8) mice show a rise in iNOS expression in the pancreas, a response blocked by melatonin . Moreover, melatonin prevents inflammatory liver damage in old castrated and intact female rats, with significant reductions in iNOS protein expression and cytosolic NO levels . After LPS administration, the inhibitory effects of melatonin against lung and liver iNOS overexpression have been noted in both young and aged animals. Because aged rats have greater organ and metabolic impairment than young animals in response to LPS, the results also suggest an increased efficacy of melatonin in reducing septic shock in aged animals .
Other models of inflammation have also demonstrated the ability of melatonin to block the overexpression of iNOS. Thus, melatonin significantly reduced iNOS expression in cerulein-induced pancreatitis in the rat by a mechanism related to Nfr2 and NFκB modulation . Oral administration of melatonin also reduced mRNA and protein expression of iNOS in lung inflammation induced by aerosolized pancreatic fluid .
We have recently demonstrated that melatonin prevents cardiac inflammation in a rat model of acute exercise; melatonin caused reduction in iNOS expression and blocked the overexpression of inflammatory cytokines including IL-1, IL-6, C-reactive protein, and TNF-α, possibly through an NFκB-dependent mechanism . Similarly, we have shown that melatonin administration in exercised rats induced a reduction in liver iNOS and cNOS expression , and in muscle iNOS expression .
Finally, it has been shown that melatonin caused a significant reduction in iNOS expression and alleviated inflammatory pathological changes elicited by crude venom from the jelly fish Pelagia noctiluca in rats. Thus, it may be useful in the treatment of local acute inflammation .
Prostanoids and leukotrienes
Prostanoids and leukotrienes are inflammatory mediators synthesized from arachidonic acid, which, in turn, are generated from membrane phospholipid cleavage by phospholipase A2 (PLA2). Prostaglandins (PG) and thromboxan A2 are produced by cyclooxygenases, which exist in two different isoforms (COX-1 and COX-2) and one variant (COX-3) . COX-1 is constitutively expressed in most tissues and produces prostaglandins to regulate physiological processes in response to hormones and other stimuli . Leukotrienes, hydroperoxyeicosatetranoic acids, and hydroxyeicosatetranoic (HETE) acids are generated by lipooxygenases (LOX). While 15-LOX synthetizes the anti-inflammatory 15-HETE, 5- and 12-LOXs are involved in inflammatory processes by producing 5-HETE and leukotrienes, which are potent chemoattractants, and 12-HETE, which aggregates platelet and induces the inflammatory response, respectively .
In vivo and in vitro experiments have shown that melatonin reduces or prevents the inflammatory-derived activation of PLA2, LOX, and COX [66–68]. Mechanisms involved in these effects are presumed to include the engagement of MT1/MT2 or ROR/RZR receptors [66, 67].
In human lymphocytes, melatonin inhibits prostaglandin E2 (PGE2)-stimulated IL-2 production through the MT1 receptor . In rat distal colon, melatonin inhibits PGE2 and sodium nitroprusside-induced ion secretion . Moreover, some authors have demonstrated that melatonin reduces inflammation in a rat model of colitis induced by 2,4,6-trinitro-benzen-sulfonic acid (TNBS) through COX-2 inhibition . In MCF-7 breast cancer cells, melatonin administration limits COX-2 and COX-1 mRNA expression and decreases levels of PGE2, which in turn downregulate aromatase gene expression . Melatonin causes a marked reduction in COX-2 through a NFκB-dependent mechanism in rat cardiac muscle following acute exercise  (Table 2).
Table 2. Published reports related to the effects of melatonin on cyclooxygenases
|COX-1||↓||Breast cancer|| 71 |
|Unchanged||Gastric damage|| 72 |
|COX-2||↓||IBD|| 51, 70 |
|↓||Physical exercise|| 60 |
|↓||LPS-induced macrophages|| 68 |
|↓||Breast cancer|| 71 |
|↑||Gastric damage|| 72–74 |
Surprisingly, a variety of data indicate that melatonin may exert a beneficial action against gastric injury owing to the activation of the COX-PG system . Studies carried out in rats with acetic acid-induced chronic gastric ulcers have shown that the suppression of PGE2 synthesis by pretreatment with indomethacin reduces significantly the healing effects of melatonin. In rats without indomethacin pretreatment and with normal PGE2 mucosal generation, melatonin increases PGE2 biosynthesis and accelerates ulcer healing; this suggests that the effect of the indole could be related to PGE2 stimulation . The gastric effects of melatonin seem to be membrane receptor related because they can be abolished by luzindole, a specific membrane receptor antagonist . However, the beneficial effects of melatonin and its precursor l-tryptophan on gastric mucosal lesions induced by aspirin seem to be independent of PGE2 generation both in humans and in animals . Melatonin is known to reduce both lipid and protein oxidation , showing that it can regulate the healing of different types of wounds .
Additional research has shown that melatonin activates PLA2 and 5-LOX as a consequence of its binding to calmodulin; it also induces the synthesis of arachidonic acid from phospholipids, which then activate HETE production by 5-LOX. PLA2 and 5-LOX activation constitutes a transient effect and extinguishes within 2–3 hr, possibly because of the downregulation of both enzymes. While melatonin could act on early phases by inducing pro-inflammatory mediator production through 5-LOX and PLA2 activation (via indolamine binding to calmodulin), it might induce a later downregulation of these same enzymes (via RZR or MT1/MT2), thereby limiting the inflammatory response .
Cytokines and chemokines
Cytokines are major mediators of local, intercellular communication required for an integrated response to a variety of stimuli during immune and inflammatory processes. Different cytokines are associated with inflammatory diseases, with the clinical outcome partly determined by the balance between pro-inflammatory (i.e., IL-1β, IL-2, IL-6, IL-8, IFN-γ and TNF-α) and anti-inflammatory molecules [i.e., IL-10 and tumor growth factor (TGF-β)] . Melatonin seems to modulate both pro- and anti-inflammatory cytokines in different pathophysiological situations (Tables 3 and 4).
Table 3. A summary of the actions of melatonin on TNF-α and interferon-γ regulation
|TNF-α||↓||Septic shock|| 7 |
|↓||Diabetic neuropathy|| 36 |
|↓||Physical exercise|| 60, 82 |
|↓||Aging|| 54–56 |
|↓||Heat stroke|| 77 |
|↓||Ethanol liver damage|| 78 |
|↓||METH microglia damage|| 79 |
|Interferon-γ||↓||Septic shock|| 7 |
Table 4. Publications related to the effects of melatonin on the control of interleukins
|IL-1||↓||Physical exercise|| 60 |
|↓||Aging|| 54–56 |
|↓||Heatstroke|| 77 |
|↓||Ethanol liver damage|| 78 |
|↓||METH microglia damage|| 79 |
|IL-1ra||↑||Physical exercise|| 82 |
|IL-6||↓||Diabetic neuropathy|| 36 |
|↓||Physical exercise|| 60, 82 |
|↓||Aging|| 54–56 |
|↓||Heatstroke|| 77 |
|↓||Ethanol liver damage|| 78 |
|↓||METH microglia damage|| 79 |
|IL-8||↓||Fibroblasts acrolein induced|| 76 |
|IL-10||↑||Septic shock|| 7 |
|↑||Aging|| 54–56 |
|↑||Heatstroke|| 77 |
|IL-12||↓||Septic shock|| 7 |
Besides the beneficial pleiotropic effects that melatonin has shown in experimental septic shock, this indole also partially counteracts the rise in LPS-induced pro-inflammatory cytokine levels including IL-12, TNF-α, and interferon-γ at the local site of injection. Under the same circumstances, melatonin increases the production of the anti-inflammatory IL-10 both locally and systemically .
We have found that cardiac inflammation associated with acute exercise is associated with a significant rise in mRNA levels of IL-1, IL-6, and TNF-α. Melatonin administration before exercise normalized the mRNA levels for the three cytokines . IL-8 production is enhanced in bronchitis, rhinitis, pulmonary fibrosis, and asthma. Lung and pulmonary fibroblasts secrete IL-8 that plays a critical role in lung inflammation. Recently, it was found that melatonin suppresses acrolein-induced IL-8 overproduction in human pulmonary fibroblasts via extracellular signal-regulated kinases (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt signal inhibition .
Studies using SAMP8 mice indicate that aging augments the production of IL-1β, IL-6, and TNF-α while decreasing the anti-inflammatory cytokine IL-10. Melatonin administration to old rats or old mice reduces the expression of pro-inflammatory cytokines and elevates IL-10 protein levels [54, 55]. Similar changes occur after melatonin administration in a rat model of heat stroke, showing that the indole reduces inflammation and protects against multiorgan injury resulting from severe heat exposure .
Pro-inflammatory cytokines have been implicated in both clinical and experimental alcoholic liver diseases. Acute ethanol administration causes liver tissue damage associated with marked increases in TNF-α, IL-6, and IL-1β hepatic expression in mice. Melatonin dose dependently reduces the expression of pro-inflammatory cytokines, documenting an interesting role in hepatoprotection . It is known that liver inflammation and oxidative stress during aging are more devastating in castrated than in intact rats; liver pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 are overexpressed and anti-inflammatory IL-10 is reduced in female rats during aging and after ovariectomy. Administration of melatonin, both to intact and to ovariectomized animals, normalizes the levels of the cytokines .
Methamphetamine (METH) activates microglia to produce neuroinflammatory molecules including TNF-α, IL-1β, and IL-6; these rises are counteracted by melatonin pretreatment . Also, animal experiments using a model of diabetic neuropathy, which is a complication of diabetes with a high prevalence rate, show that melatonin reduces TNF-α and IL-6 levels in sciatic nerve, confirming its neuroprotective actions . Melatonin inhibits microglial activation and reduces pro-inflammatory cytokine and the frequency of apoptosis in hippocampal neurons of adult rats afflicted by meningitis following inoculation with Klebsiella pneumoniae .
If IL-6 is produced in muscle at low concentrations, it could stimulate the appearance of anti-inflammatory cytokines such as IL-10 and IL-1ra and inhibit TNF-α production, but if muscle IL-6 is produced at higher concentrations, it has a clear pro-inflammatory effect . In humans, it has been shown that oral supplementation of melatonin, before strenuous exercise, was able to reduce plasma pro-inflammatory TNF-α and IL-6, and to increase the anti-inflammatory IL-1ra cytokine .
The vascular endothelial cells, which line the luminal surface of blood vessels, mediate interactions between the blood elements and the cells themselves; thus, they play a key role in a number of important physiological and pathological processes . Endothelial dysfunction is implicated in inflammatory processes, and adhesion of circulating monocytes to vascular endothelial cells constitutes a critical step in both inflammation and atherosclerosis. Endothelial cells characteristically respond to pro-inflammatory stimuli such as IL-1β, TNF-α, or LPS and recruit leukocytes by selectively expressing different adhesion molecules on the surface: these include intercellular adhesion molecules (ICAM-1), vascular cell adhesion molecules (VCAM-1), and endothelial cell selectin (E-selectin) . It is possible that the anti-inflammatory effect of melatonin may be related not only to the inhibition of pro-inflammatory cytokines but also to its ability to reduce the expression of different adhesion molecules .
Physical exertion acts as a trigger of acute myocardial infarction and sudden death in susceptible individuals, and current evidence indicates that prolonged intense exercise can induce transient cardiac dysfunction . Acute exercise is associated with a significant rise in inflammatory mediators, with an elevation of cardiac ICAM-1. These changes are prevented by melatonin administration, suggesting its protective role against cardiac damage caused by acute exercise . Our previous results indicate that melatonin reduces cardiac inflammatory injury induced by acute exercise, also causes normalization in both mRNA and protein levels of ICAM-1 in rats . Elevated levels of soluble cellular adhesion molecules have been reported in patients with acute coronary syndromes, and diurnal variations in endogenous-soluble VCAM-1 production might be related to an attenuated circadian secretion of melatonin .
Anti-inflammatory effects of melatonin in heat stroke seem to be related to the serum levels of ICAM-1 and E-selectin; these adhesion molecules could contribute to protection against multiorgan injury . Recently, the beneficial effects of melatonin against nicotine-induced vasculopathy have been described. Studies in rat have shown that nicotine induces marked structural and functional alterations in the aorta with the induction of ICAM-1 and VCAM-1 and their blockade by melatonin .
Ulcerative colitis is also associated with changes in ICAM-1 production. Increases in ICAM-1 protein and mRNA expression have been found in rats with colitis induced by TNBS administration. Melatonin treatment normalizes ICAM-1 expression in a dose-dependent manner by an NFκB-dependent mechanism in these animals . In agreement with this observation, treatment with melatonin was shown to inhibit the immunohistochemical expression of P-selectin in the lower gut in a similar study of experimental colitis with the positive effects being related to a reduction in both oxidative stress and neutrophil infiltration into the colonic mucosa . Similarly, melatonin protects rat liver from intestinal ischemia/reperfusion injury, with a marked reduction in ICAM-1-positive hepatic cells .
In some cases, melatonin alone may have limited actions on the regulation of adhesion molecules. Thus, no anti-inflammatory effect of melatonin was observed in a model of acute lung inflammation (carrageenan-induced pleurisy), but a combination therapy of melatonin and dexamethasone was able to block the overexpression of ICAM-1 and P-selectin in lungs .
In addition to the expression of adhesion molecules, activated endothelial cells release IL-6 that promotes reactive-C protein (CRP) production ; this contributes to the exacerbation of endothelial dysfunction. CRP is an acute phase reactant whose elevation in serum is considered as indicator of chronic inflammation . CRP levels in the blood have a predictive value for cardiovascular disease and it induces adhesion molecule expression . Although the utility of melatonin to treat surgical stress has been proposed, no changes in either CRP or other inflammatory markers have been found in the perioperative period in patients undergoing major vascular surgery . Some studies have reported a relationship between melatonin and light/dark variations in the production of inflammatory systemic markers, including CRP, IL-6, and matrix metalloproteinase-9 (MMP-9) in patients with acute myocardial infarction . Moreover, it has been reported that experimental thermal trauma induces oxidative stress and an intense inflammatory response, with an increase in plasma CRP levels, an effect that is abolished by melatonin administration .
Melatonin and signal transduction pathways
Nuclear factor kappa B pathway
NFκB is a ubiquitous oxidative stress-sensitive transcription factor that plays a critical role in the regulation of a variety of genes important in cellular responses including inflammation, innate immunity, growth, and cell death. NFκB was first described in 1986 as a protein binding to the κ enhancer of lymphocytes . Subsequent studies revealed that NFκB is primarily a cytoplasmic factor that is expressed by virtually all cell types and that it constitutes a major inducible transcription factor whose modulation triggers a cascade of molecular events, some of which may be potential key targets for the treatment of inflammation. NFκB is a dimer, which classically consists of a p50 subunit and a trans-activating subunit p65 (or RelA); other NFkB subunits are p52 RelB or c-Rel. Unlike RelA, RelB, and c-Rel proteins, which are directly synthesized as mature proteins, p50 and p52 are generated by proteolytic processing from their larger precursors NFκB1 (p105) and NFκB2 (p100), respectively [98, 99]. Under basal conditions, NFκB appears in a latent form in the cytoplasm of nonstimulated cells, forming a complex with its inhibitors, the IkappaBs (IκB-α and IκΒ-β) .
When cells are stimulated by various inducers such as ROS, pro-inflammatory cytokines, bacterial products such as LPS or by action of NFκB protein (NFAP), NFκB is activated by means of the phosphorylation and degradation of the IκBs. The phosphorylation of IκB involves a specific IκB kinase (IKK) complex consisting of a core of three subunits, two catalytic subunits (IKK-α and IKK-β), and the regulatory subunit NFκB essential modifier (NEMO or IKKγ). IKK-α and IKK-β contain functional kinase domains that are capable of phosphorylating IκB at specific N-terminal serine residues to initiate its ubiquitination. The degradation of IκB-α results in rapid changes in the induction of NFκB, whereas the degradation of IκB-β is associated with a prolonged activation of NFκB [101, 102]. After its liberation, NFκB translocates to the nucleus and binds to specific elements within the promoters of responsive genes (κB-sites) to activate their transcription .
Various studies have shown that melatonin modulates the NFκB signaling pathway during inflammation and that modulation can occur during early (regulation of oxidant levels, IKK activation) as well as late (binding of NFκB to DNA) stages of the response [104, 105]. Modulation of NFκB modifies the expression of genes involved in the inflammatory process including iNOS, COX-2, and proinflammatory cytokines .
As mentioned earlier, among the wide range of genes involved in inflammation that contain functional κB-sites is iNOS. Once activated, NFκB stimulates the expression of iNOS, with an increase in NO formation . The basis for the protective effects of antioxidants such as melatonin is consistent with interference at the NFκB transcriptional pathway. For example, experimental models of ischemia reperfusion have shown that antioxidants such as glycine simultaneously block oxidative stress, the activation of NFκB and NO production [108, 109]. Moreover, research on models of inflammation in isolated hepatocytes also confirms that the inhibition of NFκB activation by immunosuppressive agents is related to the reduction in iNOS . Studies documenting the ability of melatonin to reduce NFκB activation and iNOS expression have been carried out using J774 and RAW 264.7 murine macrophages . Experiments using old SAMP8 mice have determined that melatonin reduces the expression of NFκB, induces mRNA downregulation of iNOS, and improves liver function . Melatonin administration lowers exercise-related inflammation and oxidative stress in both skeletal and cardiac muscle, by preventing NFκB activation and normalizing IkBα and IKKα protein expression [60, 62]. Other studies have also shown that reduction in NFκB activation by melatonin protects against liver thioacetamide damage or ischemia/reperfusion after kidney transplantation [49, 111].
LPS-induced NO production in rat endothelial cells is blocked by melatonin through reduction in iNOS expression by an NFκB-dependent mechanism . Similar results were obtained when the indolamine was administered in vivo (30 mg/kg); it prevented LPS-induced iNOS expression in rat aorta  and inhibited NFκB transcription factor activation in macrophages .
Equivalent changes in NFκB activation and COX expression were also reported following melatonin use in different models. In vitro studies confirmed that melatonin can suppress both iNOS and COX-2 expression by the inhibition of p52 NFκB-binding ability in LPS-stimulated macrophages . Experiments using C6 glioma cells exposed to LPS/interferon-γ showed that melatonin blocks NFκB activation, with a minor IκB-α degradation, and reduces iNOS and COX-2 expression . Interestingly, NFκB activation but not COX-2 overexpression is abolished by melatonin administration in RAW264.7 macrophage-like cells when previously stimulated with the fimbriae of the oral anaerobe Porphyromonas gingivalis .
It has been reported that melatonin blocks pro-inflammatory cytokine production in rats by reducing NFκB and AP-1 translocation into the nucleus . In line with this, reduction in mRNA and protein levels of TNF-α and IL-1β, via NFκB inhibition, has been reported in old SAMP8 melatonin-treated mice where the indoleamine exerts a protective role on the age-related degeneration of the liver, pancreas, and after injury to the heart [55, 106, 115]. Acute ethanol administration to mice not only causes liver tissue damage and pro-inflammatory cytokine overexpression, but also induces NFκB nuclear translocation and increased IκB degradation, effects that are significantly inhibited by melatonin . Melatonin modulation of inflammation in experimental diabetic neuropathy is also related to the inhibition of the NFκB cascade, induction of a marked reduction in TNF-α and IL-6 levels, and downregulation of iNOS and COX-2 . In Alzheimer’s disease, melatonin and its metabolites regulate both proinflammatory signals and oxidative stress mediators, such as COX2 and iNOS, by restricting NFκB full integration . Blockade by melatonin of cardiac iNOS, COX-2, TNF-α, IL-1, and IL-6 overexpression following acute exercise is also related to its ability to inactivate the NFκB pathway . Moreover, it has been reported that melatonin reduces Prevotella intermedia LPS-induced production of NO and IL-6 through the inhibition of nuclear translocation and DNA-binding activity of NFκB p50 subunit .
NFκB activation also appears to be a necessary step in the transcriptional induction of adhesion molecules, as confirmed by the fact that blockers of NFκB inhibit VCAM-1 expression in endothelial cells stimulated by the TNF-α . Melatonin diminishes not only ICAM-1 but also pro-inflammatory cytokine expression in a rat model of IBD, via NFκB inhibition .
CREB-binding protein (CBP) and its homologue p300 are large nuclear molecules that have been implicated in the coordination of a variety of transcriptional pathways with chromatin remodeling. They possess histone acetylase activity, which is necessary to open the chromatin structure through an acetylation-induced conformational change in histone protein. Via this action, CBP/p300 acts as an activator of NFκB and CRE-binding proteins [119, 120]. Activation of the noncanonical NFκB signaling pathway involved in the proteolytic processing of NFκB p100 to p52 is tightly regulated, and overproduction of p52 leads to lymphocyte hyperplasia and transformation. In vitro studies have demonstrated that melatonin suppresses p52 NFκB binding by the inhibition of CBP/p300-dependent acetylation, impairing its transactivation in LPS-stimulated macrophages .
The nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 catalyzes the formation of poly(ADP-ribose) polymers from its substrate NAD+, leading to a depletion of this dinucleotide and a reduction in the rate of glycolysis. Because NAD+ functions as a cofactor in the tricarboxylic acid cycle and in glycolysis, NAD+ depletion could lead to a rapid fall in intracellular ATP and, ultimately, cell injury . PARP-1 was initially known for its facilitating role in the repair of DNA damage induced by oxidative/nitrosative stress , but has subsequently been shown to be involved in the pathophysiology of acute and chronic inflammatory diseases. Currently, it is recognized that PARP-1 also participates in the regulation of NFκB-mediated production of pro-inflammatory cytokines, and inhibition of PARP-1 with synthetic inhibitors reportedly reduces the DNA-binding activity of NFκB and the transcription of NFκB-mediated genes . It has been shown, using zymosan and carrageenan-induced experimental models of inflammation, that melatonin reduces PARP-1 activation and attenuates the reduction in NAD+. The overall effect of melatonin is a significant protection of cellular viability . Moreover, some investigations have demonstrated an important role of peroxynitrite and subsequent PARP-1 activation in arthritis, which is blocked by melatonin administration . Furthermore, melatonin’s anti-inflammatory effects in experimental colitis are also associated with a marked reduction in PARP-1 immunoreactivity . Experimental studies carried out by subplantar injected superoxide into the hindpaw, which evokes a potent thermal hyperalgesia, found that PARP-1 was activated at the time of maximal hyperalgesia, and this was blocked by melatonin. This suggests that the antihyperalgesic effect of melatonin is derived partly by the inhibition of superoxide-driven PARP-1 activation .
Other transcriptional pathways
A number of other critical factors may participate in melatonin’s anti-inflammatory effects. These include inhibition of transcription factors and related proteins such as hypoxia-inducible factor (HIF), nuclear factor erythroid 2-related factor 2 (Nrf2), CREB, activating transcription factor 1 (ATF-1), CCAAT/enhancer binding protein (C/EBP), STATs, peroxisome proliferator-activated receptors (PPARs), AP-1, and actions on the phosphoinositol phosphate (PI3K)/Akt signaling pathway.
The best characterized response to hypoxia is the induction of HIF-1, which governs the oxygen-dependent induction of erythropoyetin, vascular endothelial cell growth factor (VEGF), and glycolytic enzymes. HIF-1 consists of two subunits (α- and β-subunit). Whereas HIF-1β is constitutively expressed, HIF-1α is strictly regulated by the cellular oxygen tension. Under normoxic conditions, HIF-1α protein is post-translationally hydroxylated on specific proline residues to enable binding to von-Hippel–Lindau protein, which targets HIF-1α for ubiquitinylation and proteasomal degradation . Important roles for HIF-1 in inflammation also include the activation of the immune response and stimulation of angiogenesis and have been proposed, and different groups have provided evidence that HIFs play an integrative role in conditions of hypoxia and inflammation by increasing the expression of iNOS or pro-inflammatory cytokines [126, 127]. Melatonin reportedly downregulates HIF-1α protein expression, under both normoxic and hypoxic conditions in DU145, PC-3, and LNCaP prostate cancer cells, without changes in HIF-1α mRNA levels. These effects may be related to a downregulation on HIF-1α protein translation through a p70S6K inhibitory mechanism .
More recently, it was found that melatonin destabilizes hypoxia-induced HIF-1α protein levels owing to its antioxidant activity in HCT116 human colon cancer cells. Moreover, in the same study under hypoxia, melatonin suppressed HIF-1α transcriptional activity, leading to reduction in different angiogenic processes such as VEGF expression or in vitro tube formation, invasion, and migration of human umbilical vein endothelial cells (HUVEC) by hypoxia-stimulated conditioned media of HCT116 cells . These findings suggest that the indolamine could be useful in tumor suppression via the inhibition of HIF-1α-mediated angiogenesis.
In Alzheimer’s disease, HIF-1 may upregulate BACE-1 protein, a transmembrane aspartic protease that is the major β-secretase facilitating the Aβ cytotoxic peptide generation. Melatonin administration reportedly prevents the Aβ peptide production by reducing both the BACE-1 protein and its mRNA . Finally, it has been proposed that melatonin may protect foetuses from LPS-induced placental cellular stress by a pleiotropic action involving a significant attenuation in both HIF-1α and VEGF expressions .
Another transcription factor involved in combating oxidative stress and inflammation is Nrf2. Nrf2 is a basic leucine zipper transcription factor that regulates the expression of a number of detoxifying, antioxidant genes and modulates some inflammatory processes [130, 131]. The Nrf2 pathway increases heme oxygenase-1 (HO-1) expression, which strengthens antioxidant defense. It has been suggested that melatonin modulates neuroinflammation by limiting the NFκB activation cascade and oxidative stress through elevated Nrf2 expression, which might be responsible, at least in part, for its neuroprotective effect in diabetic neuropathy . Moreover, data obtained in a model of acute liver injury using dimethylnitrosamine indicate that melatonin administration increases Nrf2 expression, normalizes antioxidant activities, reduces NFκB activation, and leads to the inhibition of iNOS and pro-inflammatory cytokine expression . In a rabbit model of hepatic failure of viral origin, it has been recently demonstrated that melatonin administration activates Nrf2 signaling, which causes the normalization of antioxidant enzymes expression, and supports the potential hepatoprotective role of this indole . Other studies, using cyclophosphamide, have documented that melatonin treatment reduces bladder damage and apoptosis, increases Nrf2, HO-1, and quinone oxidoreductase-1 expressions, and inhibits the NFκB pathway . Furthermore, melatonin significantly reduced iNOS expression in cerulein-induced pancreatitis in rat by a mechanism related to Nfr2 and NFκB modulation .
The CREB family of transcription factors consists of cAMP-responsive activators in mammalian systems. These include CREB and ATF-1, which are induced by a variety of growth factors and inflammatory signals, and subsequently mediate the transcription of genes containing a cAMP-responsive element (CRE) transcription factor. Several inflammation-related genes possess this CRE, including IL-2, IL-6, IL-10, and TNF-α. All CREB family members have a basic region leucine zipper dimerization domain located at the carboxy-terminal end, and they bind to DNA target sequences, such as the CRE, by dimerization through a leucine zipper . The activation of CRE-binding proteins is modulated owing to phosphorylation by several kinases and is mediated by coactivators such as CBP and p300 . Melatonin decreases phosphorylation of CREB and ATF-1 transcription factors not only in wild-type animals but also in mt1/2−/− after focal cerebral ischemia, indicating the presence of a MT1/MT2 receptor-independent mechanism on CREB and ATF-1 activation .
The C/EBP family belongs to the large group of basic leucine zipper (bZip) transcription factors. All members of the C/EBP family have a C-terminal leucine zipper domain for dimerization and a basic domain for DNA binding, respectively . Several C/EBP family members are involved in regulating various aspects of inflammation and immunity in the liver and in cells of the myelomonocytic lineage . Moreover, C/EBPβ has emerged as a key transcriptional transactivator for COX-2 and iNOS expression in murine and human cells induced by proinflammatory mediators [138, 139]. Some studies have indicated that melatonin inhibits the C/EBPβ transcriptional activity working as a negative regulator of adipogenesis in mice . Recently, it was reported that in LPS-stimulated CRL1999 human vascular smooth muscle cells melatonin inhibited phosphorylation of C/EBPβ, reduced its DNA binding, and caused an important reduction in both COX-2 and iNOS expression, which attenuates inflammatory damage .
STAT proteins participate in the regulation of cellular responses to cytokines and growth factors. With the aid of Janus kinases (JAKs), the pathway transduces the signal carried by these extracellular polypeptides to the cell nucleus, where activated STAT proteins modify gene expression . Inflammatory cytokines can bind to receptors and activate the STAT pathway . In addition, STAT may be negatively regulated by the suppressors of cytokine signaling (SOCS) family proteins . Melatonin protects neurons from brain-contusion-induced oxidative insults and reduces upregulation of SOCS3 and IL-6 mRNA expression, and STAT1 inactivation; these actions could contribute to the neuroprotective effects of melatonin . Studies in LPS-stimulated BV2 microglial cells have shown that melatonin induces anti-inflammatory effects through STAT activation and downregulation of chemokine expression by the inhibition of NFκB . Melatonin inhibition of P. intermedia LPS-mediated production of NO and IL-6 is related not only to an NFκB-related mechanisms but also suppression of STAT1 signaling .
PPARs belong to the nuclear hormone receptor superfamily of ligand-activated transcription factors. Three isoforms (α, β/δ, and γ), encoded by different genes, have been identified. Moreover, it has highlighted the role of PPARs in regulating inflammatory responses . Because of this, PPAR-γ functions as a central regulator of differentiation, apoptosis, and inflammatory responses, etc. . PPAR-related melatonin anti-inflammatory effects remain unclear, but it has been noted that this indolamine is able to suppress PPAR-γ expression when human mesenchymal stem cell (hMSC) differentiation was analyzed .
AP-1 is a heterodimeric protein, composed of members of the basic region leucine zipper protein superfamily, specifically, the Jun, Fos, and activating transcription factor proteins, which regulate gene expression in response to a variety of stimuli including cytokines, growth factors, stress, and bacterial and viral infections . Melatonin blocks pro-inflammatory cytokine production in rats by reducing not only NFκB but also AP-1 translocation into the nucleus .
PI3K/Akt is another important signaling pathway related to both acute and chronic inflammatory disorders . PI3K inhibition seems to play a role in the modulation of HUVEC by melatonin . Studies using C6 glioma cells have demonstrated a melatonin antioxidant effect associated with inhibition of PI3K/Akt and NFκB . Finally, melatonin was able to inhibit PI3K phosphorylation in LPS-stimulated BV2 microglial cells, documenting its important anti-inflammatory effect .
Mitogen-activated protein kinases
Effects of melatonin on the binding capacity of transcription factors such as NFκB or AP-1 may be regulated through the inhibition of protein kinases involved in signal transduction, such as MAPKs. MAPKs have been implicated in many physiologic processes including cell proliferation, differentiation, and death, and their activation seems to be a key component in signal transduction associated with cell migration. MAPKs are a family of serine/threonine kinases with three major types in mammalian cells: the extracellular signal-regulated protein kinases (ERK), the p38 MAPKs, and the c-Jun NH2-terminal kinases (JNK) . MAPKs are activated by specific MAPK kinases (MAPKK), such as MEK1/2 for ERK, MKK3/6 for p38, or MKK4/7 for JNK . Among the MAPK family members, the ERK pathway is frequently activated by mitogens and growth factors, while inflammation is a main trigger for JNK and p38 . In the last few years, melatonin has been shown to modulate MAPKs by acting on several steps of the activation cascade, and consequently on downstream effectors.
Antiproliferative melatonin effects on HUVEC and C6 glioma cells seem to be associated with inactivation of not only PI3K/Akt and NFκB but also ERK1/2 and protein kinase C (PKC) pathways [150, 151]. More recently, it has been demonstrated that melatonin attenuates apoptotic liver damage in a fulminant hepatic failure model in rabbits by a mechanism implicating a reduction in phosphorylated JNK and tumor necrosis receptor-1 (TNFR-1) expression . However, increases in JNK1/2/3 and p38 expression, mainly through a MT1 receptor pathway, have been reported in HepG2 hepatocarcinoma cells, which may be related to the ability of melatonin to induce apoptosis and cell cycle arrest in these and other cancer cells [17, 155]. These apparently contradictory effects may be related to MT1/MT2 as a mechanism determining the receptor-mediated biological actions of melatonin. As an example, ERK activity is increased by melatonin in mouse neuroblastoma cells that express only MT1 receptors , whereas in HUVEC expressing both MT1 and MT2 receptors, ERK is inhibited by melatonin . Furthermore, it has been reported that melatonin decreased p38 in both mt1/2−/− and wild-type mice after focal cerebral ischemia, but only reduced JNK1/2 in wild type .
This brief review summarizes the results of investigations showing that melatonin constitutes an interesting pharmacological agent in the treatment of inflammatory pathophysiological conditions. A number of different pharmacological targets of melatonin have been described including iNOS, COX-2, and cytokine and adhesion molecule production. Melatonin’s anti-inflammatory effects are driven via a number of different transcriptional pathways including not only NFκB but also HIF, Nrf2, cAMP, CREB, ATF-1, C/EBP, STAT, PPARs, or AP-1. Moreover, melatonin also modulates pathways dependent of different kinases such as PI3K/Akt or MAPKs and it induces anti-inflammatory effects both under in vitro and in vitro conditions. Definition of the role of melatonin in the pathophysiological mechanisms of inflammation is a large and growing research field, but further studies are necessary to elucidate its complex regulatory mechanisms in different cellular types and tissues.