Protective Effects of Melatonin on Methamphetamine-Induced Blood-Brain Barrier Dysfunction in Rat Model.

The specialized brain endothelial cells interconnected by unique junctions and adhesion molecules are distinctive features of the blood-brain barrier (BBB), maintaining the homeostasis of the cerebral microenvironment. This study was designed to investigate the protective effects of melatonin on methamphetamine (METH)-induced alterations of BBB integrity. Wistar rats were randomly distributed into groups and underwent melatonin pretreatment and escalating-high doses of METH treatment. Immunohistochemistry was performed to demonstrate the BBB leakage. Protein and RNA samples were isolated from hippocampal and prefrontal cortical tissues and measured expression levels of molecular markers associated with BBB structural components and inflammatory processes. METH provoked the loss of zonula occludens (ZO)-1, occludin, and claudin-5 tight junction proteins. Furthermore, METH caused an excessive increase in matrix metalloproteinase-9 (MMP-9) enzyme, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) and the increase in NAD(P)H oxidase 2 (NOX2). Melatonin exerted the protective effects by recovering tight junction loss; attenuating excessive MMP-9, NOX2, and cell adhesion molecule expression; and reducing serum albumin in the brain. Our results also showed the protective effects of melatonin against METH neurotoxic profiles, characterized by reactive gliosis: microglia (integrin-αM) and astrocyte (GFAP); an excessive upregulation of primary pro-inflammatory cytokines: interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); activation of neuroinflammatory signaling: nuclear factor-kappa B (NF-κB); and suppression of anti-oxidative signaling: nuclear factor erythroid 2-related factor (Nrf2), that may exacerbate BBB structural impairment. Our results provide insights into the beneficial effects of melatonin against METH-induced BBB disruption and mechanisms that play detrimental roles in BBB impairment by in vivo design.

Long-term administration of melatonin attenuates neuroinflammation in the aged mouse brain.

Aging is often accompanied by a decline in cognitive function in conjunction with a variety of neurobiological changes, including neuroinflammation. Melatonin is a key endogenous indoleamine secreted by the pineal gland that plays a crucial role in the regulation of circadian rhythms, is a potent free radical scavenger, has anti-inflammatory activity and serves numerous other functions. However, the role of melatonin in sterile inflammation in the brain has not been fully investigated. In the present study, we investigated the neuroinflammation status in aged mouse brains. The results showed that the protein levels of integrin αM (CD11b), glial fibrillary acidic protein (GFAP), the major pro-inflammatory cytokines (interleukin-1 beta [IL-1ß], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) and phosphor-nuclear factor kappa B (pNFκB) were significantly increased, while N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and brain-derived neurotrophic factor (BDNF) were down-regulated in the hippocampus and prefrontal cortex (PFC) of 22-months-old (aged) mice compared with 2-months-old (young adult) mice. Melatonin was administered in the drinking water to a cohort of the aged mice at a dose of 10 mg/kg/day, beginning at an age of 16 months for 6 months. Our results revealed that melatonin significantly attenuated the alterations in these protein levels. The present study suggests an advantageous role for melatonin in anti-inflammation, and this may lead to the prevention of memory impairment in aging.

The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide.

Brain inflammaging is considered as one of the underlying factors of neurodegenerative diseases. The present study aimed to investigate the effects of melatonin, an endogenous indoleamine mainly synthesized by the pineal gland, on hydrogen peroxide (H2O2)-induced inflammaging state in SH-SY5Y cells. Our data showed that p21Cip1 and p16INK4a, cell cycle arrest markers, and the number of senescence-associated ß-galactosidase (SA-ßgal) staining increased significantly in H2O2-treated cells. Melatonin treatment could reverse this effect. Flow cytometry analysis showed a significantly higher percentage in the G0/G1 phase and a lower proportion in the S phase of H2O2 treated cells. Cells pretreated with H2O2 showed a dramatic decrease in the formation of Ki67 immunoactivity while the treatment with melatonin increased Ki67-positive cell. Both mRNA and protein expression levels of the pro-inflammatory cytokines, interleukin-1ß (IL-1ß), IL-6 and, tumor necrosis factor-α (TNF-α) which were increased after induction with H2O2, could be attenuated by melatonin. In addition, melatonin decreased the phospho-nuclear factor kappa B (pNF-κB) expression and prevented its nuclear translocation, as well as abrogated the reduction of nuclear factor erythroid 2-related factor 2 (Nrf2) in SH-SY5Y cells exposed to H2O2. The present data suggested the importance of melatonin on ameliorating inflammation in SH-SY5Y cells.

Calcitonin gene-related peptide mediates an inflammatory response in Schwann cells via cAMP-dependent ERK signaling cascade.

AIMS: Calcitonin gene-related peptides (CGRP), an endogenous neuropeptide, play an important role in the development of neuroinflammation by acting upon its receptor. The CGRP receptor immunoreactivity was identified on Schwann cells. However the effects of CGRP on Schwann cells are unknown and the exact signaling mechanisms associated with CGRP receptor activation related to Schwann cells inflammatory responses are not well understood. We investigated the effect of CGRP on CGRP receptor activation mediates a proinflammatory signaling response in Schwann cells. MAIN METHODS: CGRP-induced ERK-MAPK phosphorylation and proinflammatory cytokines, interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) expressions were measured by immune blotting. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including CGRP (8-37), SQ 22536 and H-89. KEY FINDINGS: Treatment with CGRP demonstrated a significant generation of IL-1ß and IL-6 but not in the level of TNF-α. In addition, there was a temporal increase in the activated form of ERK caused by CGRP that was prevented after pretreatment with CGRP (8-37), SQ 22536 and H-89. Furthermore, use of the CGRP (8-37), ERK inhibitor PD 98059, SQ 22536 or H-89 abolished the CGRP mediated increase in IL-1ß. SIGNIFICANCE: This investigation provides evidence for a novel CGRP activation on Schwann cells that mediates inflammatory response by increasing of IL-1ß and IL-6 expression. CGRP activates the cAMP-PKA-ERK signaling cascade leading to IL-1ß production. These results support the notion that CGRP may play a direct role to initiate inflammatory processes in the peripheral nervous system.

The anti-inflammatory effect of melatonin on methamphetamine-induced proinflammatory mediators in human neuroblastoma dopamine SH-SY5Y cell lines.

Methamphetamine (METH) is a highly addictive drug that is commonly abused worldwide. This psychostimulant drug causes the disturbances in the dopaminergic and serotonergic neurons of several brain areas. Exposure to METH has been shown to induce oxidative stress, reactive oxygen species, reactive nitrogen species, and neuroinflammation. However, the mechanism underlying METH-induced inflammation in neurons is still unclear. In this study, we investigated whether METH caused inflammatory effects in human dopaminergic neuroblastoma SH-SY5Y cells and whether this effect involved the nuclear factor-κB (NF-κB) transcription factor pathway. The present results showed that METH significantly increased inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner and significantly increased the levels of tumor necrosis factor (TNF)-α mRNA and phosphorylated NF-κB, which is translocated into the nucleus. Moreover, our results also show that METH downregulated another transcription factor, the nuclear factor erythroid 2-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Furthermore, we also examined the anti-inflammatory effect of melatonin against these METH-induced neuroinflammatory functions. The results show that melatonin significantly decreases the iNOS protein expression and TNF-α mRNA levels caused by METH. The activation and the level of pNF-κB were decreased while Nrf2 expression was increased when cells were pre-incubated with 100 nM of melatonin. In order to show the relationship between cell death and the increase of iNOS, 100 µM of L-NAME, an iNOS inhibitor pretreatment significantly prevented cell death caused by METH. These results demonstrate, for the first time, that METH directly induces inflammation in neurons via an NF-κB-dependent pathway and that the anti-neuroinflammatory effects of melatonin result from the inhibition of activated NF-κB in parallel with potentiated antioxidant/detoxificant defense by activated Nrf2 pathway.

Noradrenergic depression of neuronal excitability in the entorhinal cortex via activation of TREK-2 K+ channels.

The entorhinal cortex is closely associated with the consolidation and recall of memories, Alzheimer disease, schizophrenia, and temporal lobe epilepsy. Norepinephrine is a neurotransmitter that plays a significant role in these physiological functions and neurological diseases. Whereas the entorhinal cortex receives profuse noradrenergic innervations from the locus coeruleus of the pons and expresses high densities of adrenergic receptors, the function of norepinephrine in the entorhinal cortex is still elusive. Accordingly, we examined the effects of norepinephrine on neuronal excitability in the entorhinal cortex and explored the underlying cellular and molecular mechanisms. Application of norepinephrine-generated hyperpolarization and decreased the excitability of the neurons in the superficial layers with no effects on neuronal excitability in the deep layers of the entorhinal cortex. Norepinephrine-induced hyperpolarization was mediated by alpha(2A) adrenergic receptors and required the functions of Galpha(i) proteins, adenylyl cyclase, and protein kinase A. Norepinephrine-mediated depression on neuronal excitability was mediated by activation of TREK-2, a type of two-pore domain K(+) channel, and mutation of the protein kinase A phosphorylation site on TREK-2 channels annulled the effects of norepinephrine. Our results indicate a novel action mode in which norepinephrine depresses neuronal excitability in the entorhinal cortex by disinhibiting protein kinase A-mediated tonic inhibition of TREK-2 channels.