Melatonin

Melatonin (N-acetyl-5-methoxy tryptamine) is a molecule predominantly produced by the pineal gland (Reiter, 1991). The chemical structure is shown in Figure 1.

Melatonin regulates the coupling of circadian rhythms, especially core temperature and sleep-wake cycle but also influences physiological functions such as immune function, tumor growth inhibition, antioxidant protection and redox homeostasis (Carrillo-Vico, Guerrero, Lardone, & Reiter, 2005; Hardeland, Madrid, Tan, & Reiter, 2012; Mauriz, Collado, Veneroso, Reiter, & Gonzalez-Gallego, 2013; Mills, Wu, Seely, & Guyatt, 2005).

The synthesis and release are activated during darkness and inhibited by light.

Information on light/dark environments is transmitted via the retinohypothalamic tract to the suprachiasmatic nucleus (SCN). After that, an electrical signal is transferred to the pineal gland via complex autonomic neural circuitry, which is ultimately conveyed by postganglionic sympathetic fibers. The postganglionic terminals release norepinephrine, which activates melatonin production via β1 adrenergic receptors in the pinealocyte.

Pinealocytes take up tryptophan from the blood and convert it to serotonin by hydroxylation and decarboxylation. In the darkness, serotonin is converted into

N-acetyl-serotonin by the enzyme N-acetyltransferase (NAT). Subsequently, the enzyme hydroxyindole-O-methyl transferase acts on N-acetylserotonin causing its methylation and forming melatonin (Gomez-Moreno, Guardia, Ferrera, Cutando, & Reiter, 2010).

Melatonin is also produced in the brain, retina, gastrointestinal tract, bone marrow, skin, reproductive organs and lymphocytes, from which it may have similar physiological functions through paracrine, autocrine and antioxidant actions (Pandi-Perumal et al., 2008;

Reiter et al., 2016).

As a medicine, it is used to prevent phase shifts from jet lag and little improvements in insomnia. It is categorized by the US Food and Drug Administration (FDA) as a dietary supplement and is sold over-the-counter in both the US and Canada (Costello et al., 2014).

According to current evidence, melatonin has a very slight side effect profile and limited evidence of habituation and tolerance.

1.5.1 Mechanisms of melatonin action

Melatonin performs its functions using dependent and receptor-independent pathways (Fig. 2). Melatonin can diffuse through biological membranes easily and acts as a direct scavenger in subcellular compartments. Intracellularly, melatonin can serve as a direct free radical scavenger in all subcellular compartments as well as bind the cytosolic calcium-binding messenger protein calmodulin, cytosolic quinone oxidoreductase 2 (NQO2, also known as MT3) (Nosjean et al., 2000) and the nuclear retinoic acid receptor subfamily including retinoid-related orphan receptors (RORs) and retinoid Z receptors (RZRs). Furthermore, melatonin can also bind and activate two membrane melatonin receptors, MT1 and MT2.

Both of MT1 and MT2 receptors belong to the G protein-coupled receptor superfamily with seven transmembrane domains (Jockers, Maurice, Boutin, &

Delagrange, 2008). The alpha subunits of G protein which coupled with MT1 and MT2 receptors can directly inhibit adenylyl cyclase activity. This inhibition lowers cAMP levels and suppresses protein kinase A (PKA) activity, which influences phosphorylation and activity of the cellular transcription factor CREB (cAMP response element-binding protein) (Dubocovich, Rivera-Bermudez, Gerdin, & Masana, 2003; Witt-Enderby, Bennett, Jarzynka, Firestine, & Melan, 2003).

Besides, MT1 and MT2 receptors can also couple to the beta/gamma subunit of G protein and activate phospholipase C (PLC), which leads to increase inositol-(1,4,5)-triphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC), which modulates the activity of the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway, again ultimately influencing the activity of transcription factors including CREB. Moreover, IP3 also stimulates the release of intracellular calcium and activate signaling by Calmodulin (Slominski, Reiter, Schlabritz-Loutsevitch, Ostrom, & Slominski, 2012).

Regarding the distribution and expression of MT1 and MT2, the published studies are largely on MT1. MT1 receptors are extremely widespread in various tissues of the body, while MT2 receptors are relatively less expressed (Pandi-Perumal et al., 2008). In several tissues, the co-expression of both receptor types has been reported, and the formation of MT1-MT2 heterodimer has also been found (Ekmekcioglu, 2006; Jockers et al., 2008).

As for the MT3 receptor, the existence was theorized at one time. However, this biological target of melatonin was found to actually be a cytosolic enzyme, quinone oxidoreductase 2 (NQO2) (Nosjean et al., 2000). This enzyme catalyzes the reduction of quinones.

1.5.2 Melatonin in tissue engineering and regenerative medicine

Among the numerous functions of melatonin, the regulatory effect on the viability, proliferation, and differentiation of MSCs has recently been reported. Numerous studies demonstrate that melatonin regulates the differentiation of MSCs into chondrogenic, osteogenic, and myogenic lineages. Studies carried out on osteogenic differentiation suggest that melatonin enhances osteogenesis through the activation of Wnt/β-catenin and MAPK signaling pathways and increasing the expression of osteoblast-related markers (Luchetti et al., 2014). Other studies suggest that melatonin, by decreasing PPARγ in MSCs, directly inhibits adipogenic differentiation and enhances osteoblastogenesis to gaining bone formation in tissue repair (Takada, Kouzmenko, & Kato, 2009; Zhang et al., 2010). Recently, a study describes the melatonin’s stimulation of osteoblastogenesis and inhibition of osteoclastogenesis under the mechanisms of OPG and RANKL (Maria et al., 2018). As for odontogenic differentiation, a study demonstrates the effects of melatonin on the differentiation of human dental pulp cells (hDPCs). The hDPCs cultured in osteogenic induction medium with melatonin presented an increase of ALP activity, expression of DSPP, mRNA levels of ALP and DSPP, and mineralization nodules formation (Liu et al., 2017).

Melatonin also protects MSCs against oxidative stress-induced apoptosis as well as has the beneficial effects in wound healing. It involves as an antioxidant and anti-inflammatory agent in the suppression of the chronic wound severity and enhancement of the wound contraction (Pugazhenthi, Kapoor, Clarkson, Hall, & Appleton, 2008).

Owing to its vast biological activities, melatonin emerges as a novel and potential molecule use in tissue engineering and regenerative medicine. For this reason, it is wondering if the protective/regenerative effects of melatonin can be applied in

regenerative endodontics. However, there have been no studies reported biological activity of melatonin in human apical papilla cells.