Melatonin is an unusually multitasking molecule that participates in an uncountable number of physiological and pathological actions [ 108 110 ]. Although the modulation of circadian rhythms is considered to be a principal role of melatonin, this indolamine exerts protection in various organs and systems. In the cardiovascular system, it participates in blood pressure (BP) regulation and BP decline during the night and exerts antifibrotic effects in the left ventricle [ 111 112 ]. Melatonin has also been shown to improve glucose and lipid metabolism, modulate body weight and energy metabolism, attenuate neurodegenerative processes and depressive and anxiety behavior and expresses anticancer actions [ 103 113 ]. This indolamine exerts its effects either via specific membrane receptors, cytoplasm or nucleus-bound proteins or via receptor-independent biological actions ( Figure 3 ). Two types of melatonin-specific G-protein-coupled membrane-bound receptors were identified as MT1 and MT2 [ 114 ]. The action via the Gi protein is mediated by a reduction of cyclic adenosine monophosphate, with attenuation of protein kinase activity, and via the Gq protein that is followed by phospholipase and protein kinase C activation. The subsequent signaling may result in an increased level of intracellular calcium and nitric oxide (NO) formation [ 115 ]. Other melatonin-binding sites with obscure biological impacts involve MT3/quinone reductase (QR2) in membranes [ 116 ], nuclear retinoid orphan receptors/Z receptors (RORs/RZRs) [ 117 ] and intracellular proteins, e.g., calmodulin or tubulin [ 118 ].
Melatonin is an ancient molecule associated with the phylogenetic development from unicellular organisms to multiorgan living systems without any structural modification over millions of years [ 107 ]. Primitive photosynthetic bacteria, where melatonin probably evolved as a protection against oxidative stress, were theoretically phagocytized by early eukaryotes and developed into mitochondria and chloroplasts. During evolution, via the distribution of mitochondria, melatonin was subsequently spread to all multicellular organisms with new sites of generation and functional implications [ 103 107 ]. Although the melatonin synthetic pathway is slightly different in plants and animals, the stepwise enzymatic conversion from the amino acid tryptophan to serotonin and melatonin represents the principal metabolic route [ 107 ].
Melatonin (-acetyl-5-methoxytryptamine) is the principal secretory product of the pineal gland which is a major source of this indolamine [ 101 ] ( Figure 2 ). Experimental pinealectomy substantially reduces circulating melatonin levels [ 102 ]; however, it does not lead to complete eradication of melatonin from plasma, indicating that the pineal gland is the main but not the exclusive source of melatonin [ 101 103 ]. Indeed, a number of extrapineal tissues, including the gastrointestinal tract [ 104 ], immune system [ 105 ], kidney, heart and others, were shown to produce melatonin [ 103 ]. Melatonin in the extrapineal tissues acts via intracrine, autocrine and paracrine manners [ 105 ] and is poorly released into circulation; therefore, the circulating indolamine depends mainly on the pineal secretion [ 106 ]. Melatonin was originally discovered as a pineal gland secretory product in vertebrates, but its production was later shown in all animals and also plants, fungi and unicellular organisms [ 103 107 ].
Melatonin has significant antioxidative implications in mitochondria [ 135 137 ]. This indolamine may not only be taken up from circulation by crossing cellular and mitochondrial membranes, but it also seems to be locally synthesized in a pyruvate/serotonin/-acetylserotonin/melatonin pathway [ 137 ]. Melatonin exerts protection at several levels. Besides the direct scavenging action, melatonin stimulates the effect of the major mitochondrial antioxidant enzyme superoxide dismutase 2 via sirtuin 3 activation. This reaction preserves the efficiency of the electron transport chain, thus supporting adenosine triphosphate (ATP) generation and improving cellular energy metabolism. Furthermore, melatonin stimulates the transport of mitochondria from healthy cells to damaged cells through tunneling nanotubules, thus reducing apoptotic cell destruction [ 137 ] ( Figure 3 and Figure 4 ).
Melatonin has been recognized as a modulator of the immune system. A number of inflammatory cells have been shown to produce melatonin, including monocytes, T-lymphocytes and mast cells [ 105 128 ]. Its inflammation-modulatory action seems to depend on the phase of the inflammatory reaction, which acts as a stimulant under basal conditions or as an anti-inflammatory factor in the case of excessive immune response [ 105 129 ]. Although melatonin is not directly viricidal, it can attenuate tissue damage due to exaggerated immune response to a microbial infection and has been suggested as a potential protective means in COVID-19 treatment [ 130 133 ]. Similarly, in non-inflammatory apoptotic cell death, melatonin may exert either an anti- or proapoptotic impact depending on the particular pathological conditions [ 123 134 ].
Potentially, many biological effects of melatonin are receptor-independent actions ( Figure 3 ). Melatonin is one of the most effective means of limiting the harm of free radical stress both extra- and intracellularly, either by preventing free radical generation or, once produced, by neutralizing them. First, melatonin can directly inactivate free radicals by donating one or more electrons [ 119 120 ]. Furthermore, melatonin enhances the expression and activity of antioxidant enzymes such as catalase, superoxide dismutase or glutathione peroxidase [ 121 ] and enzymes required for glutathione synthesis and recycling, including glutathione synthetase, glutathione reductase and gamma-glutamyl transpeptidase [ 122 ]. Finally, melatonin inhibits prooxidant enzymes, particularly nitric oxide synthase and lipoxygenase [ 123 ]. Importantly, melatonin was shown to exert synergistic antioxidative actions with other common antioxidants, such as vitamin C, vitamin E or glutathione [ 124 ], while melatonin was even more effective in prevention of oxidative DNA damage than vitamin C, alpha lipoic acid and resveratrol [ 125 ]. Additionally, electron donation is the basis for the production of the melatonin derivates, such as 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), that have been defined as melatonin’s antioxidant cascade [ 119 ]. Interestingly, AFMK and AMK exert excellent ·OH radical scavenging activity, but seem to be ineffective in neutralization of ·OOH. AMK seems to be a more potent scavenger of ·OH than melatonin, while melatonin is superior in neutralizing ·OOH compared to AMK [ 126 ]. Of note, 3-hydroxymelatonin (3-OHM) was shown to be a more effective copper chelator than AFMK, AMK or melatonin [ 127 ]. Thus, melatonin and its derivates seem to be complementary players in different conditions [ 126 ] ( Figure 4 ).
The principal receptor-dependent melatonin action is to coordinate the circadian rhythms of various physiological functions in relation to the rotation of Earth on its axis during one day [ 106 138 ]. Specific melatonin receptors express the high density in brain structures, such as the suprachiasmatic nucleus (SCN) [ 139 ], the adenohypophysis [ 140 ] or the PVN [ 141 ], which coordinate circadian rhythm variations in close cooperation with melatonin. Although each organ, system, tissue or even each cell supposedly exerts circadian oscillations, the core regulator and coordination of biological rhythms is located in the hypothalamic SCN, which generates a rhythmic oscillation of 24 h. The coordination of SCN with the periphery is maintained by the nervous system and by humoral factors, whose leader is circulating melatonin of the pineal gland [ 138 ]. This indolamine is supposed to modify and coordinate both SCN-controlled and SCN-independent oscillators, the expression of potentially rhythmic genes and the production of rhythmically secreted hormones, while all these levels work in mutual cooperation [ 106 123 ]. The stimulus for melatonin synthesis and secretion is darkness, and light inhibits its production. Thus, artificial light production at night leads to a shift in melatonin production with potential health disturbances from chronodisruption [ 113 142 ].
3.4. Anxiolytic Effect of Melatonin
144,145,146,147,148,149,150,151,154,155,156,157,158,159,160, Several animal [ 143 152 ] and human [ 153 161 ] studies have shown that melatonin administration exerts an anxiolytic effect. In experimental studies, various paradigms are used to test the anxiety-related behavior of animals. Naturally, rodents prefer dark and closed areas that offer protection and avoid lit and open spaces. Briefly, increased time spent in dark and closed areas is considered to be an anxiety-like behavior. Examples of anxiety behavioral assays include an open field test, an elevated plus maze, a light–dark box or a novelty-suppressed feeding (NSF) [ 162 ].
The surgical removal of the pineal gland, or pinealectomy, compromises the rhythmicity of melatonin release and reduces plasma melatonin concentration [ 101 ]. The melatonin deficit induced by pinealectomy reduced the entry to and the percentage of time spent in the open arm of the EPM in adult male rats [ 163 ] and increased the cumulative burying behavior and burying behavior latency in a burying behavior test [ 146 ], indicating anxiety-like behavior.
Melatonin administration increased the activity in the central area of the OFT in healthy rats [ 143 ]. Melatonin increased the time spent in the open arms of the EPM in healthy rats [ 144 145 ], pinealectomized rats [ 146 ], diazinon-treated rats [ 147 ] and hypertensive rats with an upregulated renin–angiotensin system [ 148 ]. After traumatic brain injury in rats, melatonin increased the time spent in the center of the OFT, the percentage of open arm entries and the percentage of open arm times in the EPM [ 149 ]. In mice, melatonin increased the time spent in the lit box as well as the number of transitions between the two compartments of the LDB [ 150 151 ] and amplified the percentage of time spent in the open arms of the EPM [ 152 ]. All these results support the role of melatonin in attenuating anxiety-related behavior.
156, In humans, melatonin has been tested as a premedication to prevent preoperative and postoperative anxiety, as an adjunct to anesthetic drugs, as an analgesic and for the prevention of postoperative delirium. Clinical studies showed that melatonin given as premedication could reduce preoperative anxiety in patients undergoing abdominal hysterectomy [ 153 ], hand surgery during intravenous regional anesthesia [ 154 ] or other elective surgery [ 155 ] and is equally effective as the standard anxiolytic treatment with benzodiazepines [ 155 157 ]. In children undergoing minor elective surgery, melatonin, equally to midazolam, alleviated preoperative separation anxiety and anxiety associated with the introduction of the anesthesia mask [ 158 159 ].
The postoperative anxiety following laminectomy [ 160 ] and colorectal surgeries [ 161 ] was reduced after melatonin premedication. Melatonin diminished the postoperative emergence delirium in children undergoing elective infraumbilical surgery [ 164 ] and after sevoflurane anesthesia in children undergoing elective ambulatory procedures [ 165 ]. Moreover, some clinical studies observed melatonin’s superiority to midazolam in reducing the incidence of postoperative excitement [ 158 ], emergence delirium [ 165 ] and sleep disturbances [ 158 ]. Melatonin also reduced postoperative pain in patients with colorectal surgery [ 158 161 ] and abdominal hysterectomy [ 153 ], while decreasing morphine consumption [ 153 ] as well as in children undergoing elective infraumbilical surgery [ 164 ]. Moreover, melatonin is associated with faster recovery, lower incidence of excitement and a lower incidence of sleep disturbance postoperatively [ 158 ].
In summary, melatonin reduces both preoperative and postoperative anxiety, while working as effectively as benzodiazepines [ 157 ]. While benzodiazepines impair psychomotor and cognitive function after anesthesia, melatonin does not [ 155 ]. The randomized, placebo-controlled trials identified only a few mild adverse effects after melatonin treatment, such as daytime sleepiness and other sleep-related adverse effects, headache, dizziness and hypothermia [ 166 ]. Only a few serious or clinically significant adverse events were reported, such as agitation, fatigue, mood swings, nightmares, skin irritation and palpitations. Almost all adverse effects resolved spontaneously within a few days or immediately after melatonin discontinuation, while the rate of adverse effects was not markedly different from that for placebo. Importantly, no life-threatening adverse effects of melatonin or those of major clinical significance were identified [ 166 ]. Of note, melatonin was effective in counteracting antipsychotic drug-induced metabolic side effects. Melatonin reduced BP, weight gain or cholesterol levels induced by antipsychotic treatment [ 167 ]. In conclusion, melatonin seems to be an attractive alternative to benzodiazepines in relieving the anxiety associated with surgical procedures in children and adults while being considered a safe and well-tolerated drug.
The mechanisms of melatonin’s observed anxiolytic effect are not entirely understood. Melatonin’s anxiolytic effects might be associated with direct or indirect mechanisms. Direct ones are related to melatonin receptors in the brain and indirect ones to melatonin’s ability to modulate various neurohumoral systems, including the SNS, the RAAS, glucocorticoids and neurotransmitters, thus potentially interfering with the stress reaction and circadian rhythms, and modifying oxidative and nitrosative stress and inflammation.
MT1 and MT2 receptors have been shown to play a role in anxiety-like responses. These melatonin receptors were identified in brain areas involved in fear processing, such as the amygdala, hippocampus and prefrontal cortex [ 168 ]. The melatonin MT1 receptor knockout mice took a longer time to eat in the novel environment in the NSF test [ 169 170 ] and buried more marbles in the marble-burying test [ 170 ]. Female mice with a genetic deletion of the MT2 receptor increased thigmotaxis and reduced time spent in the center of the cage [ 171 ]. MT2 receptor knockout mice displayed a longer latency to feed in the NSF test [ 87 170 ] and shorter latency to enter the dark compartment of the LDB [ 87 ]. Transgenic mice with genetic deletion of the MT1/MT2 receptors showed increased latency to feed in the NSF test [ 170 ]. In contrast, MT1/MT2 receptor stimulation by S23478 reduced the duration of immobility and ultrasonic vocalization in defeated rats and increased wall-climbing and rearing in anticipating a social defeat test [ 172 ]. The selective stimulation of MT2 receptors by UCM765 increased the time spent in the open arms of the EPM and decreased the latency to eat in the NSF test in rats [ 145 ]. These data indicate the potential anxiolytic-like behavior mediated by MT1 and MT2 receptors.
The following sections discuss the possible indirect pathways of melatonin’s anxiolytic effect.
3.4.1. Melatonin–Sympathetic Nervous System Interactions Production and effects of melatonin are interrelated with the sympathetic nervous system on various levels. The retinohypothalamic tract leads from melanopsin-containing retinal neurons to the SCN, generating rhythmic oscillations. The neurons of the PVN in the hypothalamus pass directly or after interpolation in the rostral ventrolateral medulla (RVLM) to the medullar intermediolateral cell column, which innervates sympathetic ganglia generating a sympathetic tone enhancing the peripheral vascular tone and cardiac contractility with a blood pressure increase [ 173 ]. Simultaneously, the sympathetic impulses from the intermediolateral column project to the superior cervical ganglia by a preganglionic sympathetic axon to stimulate melatonin production by the pineal gland via beta and alpha-1 adrenoceptor activation [ 173 174 ] ( Figure 5 ). 174, The constant excitatory output of the PVN is intermittently inhibited by GABAergic innervation from the SCN [ 175 ]. Melatonin release from the pineal gland, which is triggered by the absence of light impulses [ 113 ], could modulate the central sympathetic system on several levels: melatonin may bind to its receptors in the SCN or area postrema, both with a high melatonin receptor density [ 139 173 ]. Moreover, melatonin enhances GABAergic signaling, which is involved in the inhibition of PVN by the SCN and in inhibition of the RVLM [ 176 ]. Additionally, melatonin enhances NO availability, which was shown to potentiate the GABAergic inhibitory effects in the PVN and RVLM [ 177 178 ]. Indeed, in L-NG-nitro arginine methyl ester (L-NAME)-induced hypertension, peak night-time pineal melatonin concentration in the pineal gland was higher in L-NAME-treated rats than in controls, supposedly by the L-NAME-decreased inhibitory effect of NO on melatonin production in the pineal gland [ 179 ]. It seems that melatonin may have a negative feedback effect on the SNS by GABAergic inhibitory signaling on the PVN via the SCN, and NO may potentiate this action. The endogenous melatonin may thus represent a counterregulatory mechanism against excessive sympathetic stimulation [ 173 180 ] ( Figure 5 ). The melatoninergic sympatholytic effects have been confirmed in a number of experiments. In spontaneously hypertensive rats (SHRs), acute melatonin administration reduced BP and norepinephrine serum levels [ 181 ]. The antihypertensive effect of regular melatonin treatment was associated with reduced plasma norepinephrine concentration and a lower number of beta receptors in the heart [ 182 ] and improved baroreflex responses [ 183 ]. In neurogenic hypertension induced by clipping one renal artery, melatonin reduced BP, attenuated sympathoexcitation to the ischemic kidney and improved baroreflex control of the heart rate (HR) along with ROS reduction in the brainstem regions regulating BP [ 184 ]. In clinical conditions, melatonin attenuated cardiac remodeling and brain–heart sympathetic hyperactivation after myocardial infarction [ 185 ] and improved autonomic control in pinealectomized patients [ 186 ]. Heart rate is under tight control of the SNS, and an elevated heart rate reliably reflects the dominance of the sympathetic over the parasympathetic nervous system tone. Melatonin’s ability to reduce HR both in animals and humans [ 187 ] indicates the sympatholytic nature of this indolamine and underlies the potential protection by melatonin not only in cardiovascular pathologies but also in mood disturbances related to overt sympathetic stimulation, such as anxiety ( Figure 5 ).
3.4.2. Potential Interference of Melatonin with the Renin–Angiotensin–Aldosterone System The available data indicate that the melatoninergic system may interact with the RAAS: 174,189,112,173,191,193,194,195,196,193,194,195,196, (1) Angiotensin II and melatonin seem to have opposite roles in cardiovascular pathophysiology. While Ang II increases BP and pathologic remodeling of a hypertensive and failing heart [ 188 ], melatonin has been shown to reduce BP in hypertensive patients [ 111 190 ] and in a number of animal models of hypertension [ 101 192 ] and to exert antifibrotic effects in hypertensive [ 101 197 ] and failing heart [ 198 ] and in kidney [ 52 199 ], similar to the ACE inhibitor captopril [ 52 199 ]. (2) In patients with non-dipping BP, the reduced excretion of 6-sulfatoxymelatonin in the urine was observed [ 200 ]. The chronic subcutaneous infusion of a subpressor dose of Ang II to rats induced a shift in circadian BP rhythm in terms of induction of non-dipping hypertension, as also seen in patients [ 201 ]. Tissue angiotensin was earlier detected in the pinealocytes of the pineal gland and locally synthesized angiotensin modified melatonin synthesis in the pineal [ 202 ]. Recently, insulin-regulated aminopeptidase (IRAP) in the pineal gland has been detected as a receptor target for Ang IV. Ang IV increased the norepinephrine-induced melatonin synthesis in pinealocytes to a similar degree as Ang II [ 203 ]. It was suggested that in rats with Ang II-induced non-dipping BP, peripheral RAAS may interact with the brain RAAS. Ang II produced by glial cells may act on AT1b receptors on pinealocytes, thus stimulating tryptophan hydroxylase, the rate-limiting enzyme of melatonin synthesis. BP alteration was supposedly induced by chronodisruption in BP regulation either via Ang II or through melatonin-induced shift of clock gene expression in the SCN or in the circadian clock of several cardiovascular organs [ 204 ]. (3) In 53 patients with chronic kidney disease (CKD), impaired night-time excretion of melatonin metabolite urinary 6-sulfatoxymelatonin tightly correlated with altered urinary excretion of angiotensinogen [ 205 ], previously detected as a marker of renal renin–angiotensin system activation and renal damage in CKD patients [ 206 ]. Moreover, in a 5/6 nephrectomy rat model of progressive CKD characterized by stimulation of the intrarenal RAAS in terms of enhanced level of angiotensinogen, Ang II and AT1 receptor density, melatonin attenuated intrarenal RAAS activation and renal injury via its antioxidant effect [ 207 ]. Melatonin-induced attenuation of the mutual RAAS and free radical stress activation has been suggested to ameliorate CKD progression by this indolamine [ 208 ]. On the other hand, in L-NAME hypertension, melatonin reduced BP and prevented fibrotic remodeling of the left ventricle without any effect of the renin–angiotensin system and increased serum aldosterone level [ 209 ]. Thus, melatonin might exert a different impact on circulatory and tissue RAAS. These prevailing protective effects of melatonin in relation to the RAAS may supposedly participate in the anxiolytic impacts of melatonin.
3.4.3. Melatonin vs. Glucocorticoids in Anxiety Melatonin’s blunting effect on glucocorticoid actions was suggested to be associated with its putative anxiolytic properties. In fact, melatonin was shown to reduce density, affinity or nuclear translocation of glucocorticoid receptors in different tissues, including the brain [ 210 211 ]. Furthermore, melatonin treatment counteracted glucocorticoid-induced dysregulation of the HPA axis in rats [ 212 ] and protected rat hippocampus from glucocorticoid-induced neurotoxicity [ 213 ]. Finally, melatonin suppressed anxiety-like behavior and reduced serum corticosterone level in both chronic immobilization stress in rats [ 214 ] and sleep deprivation in mice [ 215 ]. In patients with fibromyalgia, melatonin supplementation was associated with decreased urinary cortisol level and anxiety, as assessed by state–trait anxiety test [ 216 ].
3.4.4. Melatonin Interaction with Oxidative and Nitrosative Stress in Anxiety Melatonin and its derivates are considered to be potent free radical scavengers and broad-spectrum antioxidants [ 135 217 ]. Moreover, melatonin was shown to modulate NO synthase expression and activity in different tissues, including the brain [ 218 ]. Several studies showed that melatonin supplementation reduces oxidative/nitrosative stress, improves antioxidant defense and reduces lipid peroxidation markers in health and disease [ 219 ]. Indeed, a meta-analysis of 12 randomized control trials encompassing 521 unhealthy subjects showed that melatonin supplementation was associated with an increase in total antioxidant capacity, elevated activities of glutathione, superoxide dismutase and glutathione peroxidase and a reduction in malondialdehyde (a marker of lipid peroxidation) levels [ 220 ]. Interestingly, melatonin’s antioxidant properties were shown to be associated with its putative anxiolytic effects. In fact, melatonin supplementation prevented sleep deprivation-induced anxiety-like behavior in mice, associated with reduced oxidative stress, as determined by increased levels of superoxide dismutase and depressed levels of malondialdehyde in amygdala and serum [ 221 ]. The attenuation of sleep deprivation-induced anxiety-like behavior and oxidative stress was also associated with amelioration of neuroinflammation, apoptosis and autophagy [ 215 ]. In rats with streptozotocin-induced diabetes, melatonin treatment ameliorated anxiety-like behavior and reversed the increase in malondialdehyde levels and the decrease in reduced glutathione level in both the hippocampus and prefrontal cortex [ 222 ].