Involvement of nNOS in the antinociceptive activity of melatonin in inflammatory pain at the level of sensory neurons.

OBJECTIVE: The efficacy of melatonin as an analgesic agent has been well documented in animals and humans. However, the underlying mechanisms by which melatonin exerts antinociceptive effects on inflammatory pain are poorly understood. Here, we investigated the potential of melatonin to ameliorate inflammatory pain. MATERIALS AND METHODS: In vitro, ND7/23 neurons were treated with capsaicin. We used PCR and Western blot analyses to detect the expression of neuronal nitric oxide synthase (nNOS) in response to melatonin. Orofacial inflammatory pain was induced by 4% formalin administration on the right whisker pad of Sprague Dawley (SD) rats. The analgesic effect of melatonin was evaluated using mechanical threshold analyses. The expression level of nNOS in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (Vc) neurons was assessed by RNAscope and immunohistochemistry. RESULTS: In vitro, capsaicin upregulated the expression of nNOS, which was dose-dependently reversed by melatonin pretreatment (p < 0.001). In a rat model of orofacial inflammatory pain, melatonin pretreatment significantly attenuated mechanical allodynia in both the acute and chronic phases (p < 0.05). Furthermore, melatonin decreased the formalin-evoked elevated nNOS mRNA and protein levels in the TG and Vc neurons in the acute and chronic phases (p < 0.05). CONCLUSIONS: Taken together, these results suggest that nNOS may play an active role in both peripheral and central processing of nociceptive information following orofacial inflammatory pain induction. The regulatory effect of melatonin on nNOS in inflammatory pain may have potential implications for the development of novel analgesic strategies.

Melatonin regulates mitochondrial function and biogenesis during rat dental papilla cell differentiation.

OBJECTIVE: The aim of this study was to investigate the effect of melatonin on mitochondria of dental papilla cells (DPCs) during the odontogenic differentiation process. MATERIALS AND METHODS: Primary DPCs were obtained from the first molar dental papilla of neonatal rats and cultured in osteogenic (OS) or basal medium supplemented with melatonin at different concentrations (0, 1 pM, 0.1 nM, 10 nM, and 1 µM) for differentiation in vitro. Effects of melatonin on differentiation, mitochondrial respiratory function, and mitochondrial biogenesis of DPCs were analyzed. RESULTS: Upon odontogenic induction, Alkaline phosphatase (ALP) activity, dentin sialophosphoprotein (DSPP), and dentin matrix protein (DMP1) expression were significantly enhanced, with a peaked expression at 10 nM of melatonin treatment. During DPCs differentiation, 10 nM melatonin could significantly induce the increase of intracellular Adenosine triphosphate (ATP), the decrease of the oxidized form of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and reactive oxygen species (ROS). The mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM) were significantly increased, and the peak level of expression was found in cells treated with 10 nM of melatonin. Furthermore, the mitochondria DNA (mtDNA) copy number was significantly decreased during DPCs differentiation. CONCLUSIONS: These findings suggest that melatonin can promote the differentiation of rat DPCs and regulate mitochondrial energy metabolism, ROS scavenging, and mitochondrial biogenesis.

Electrophysiological effects of melatonin on rat trigeminal ganglion neurons that participate in nociception in vitro.

OBJECTIVE: Melatonin (MT) is a hormone mainly produced by the pineal gland. It may be involved in the regulation of nociception, the mechanisms of which remain unclear. In the present study, electrophysiological effects of MT on neurons were studied. MATERIALS AND METHODS: The cultured neurons were isolated from Sprague-Dawley rats trigeminal ganglia (TG). The neuron was voltage clamped using the whole cell patch clamp technique. We recorded resting membrane potential, action potential threshold and number, action potential duration and GABA-activated inward currents in the presence of 0.01 µM, 10 µM MT, and in the absence of MT. RESULTS: In the presence of high concentration of MT, the spontaneous action potential disappeared and action potential threshold was significantly increased. GABA-activated inward currents were recorded and blocked by GABAA receptor antagonist, bicuculline, in the majority of TG neurons (91% 40/44). Continuous perfusion of MT could cause a decrease of GABA-activated currents. The inhibiting effect was dose-dependent and irreversible. CONCLUSIONS: The results suggest that MT has several electrophysiological effects on TG neurons, which may be involved in the peripheral mechanisms of orofacial pain.