| Melatonin (N-acetyl-5-methoxytryptamine) is a small neurohormone that is highly soluble in both lipid and water, its a naturally occurring compound found in animals, plants, and microbes. In animals, circulating levels of the hormone melatonin vary in a daily cycle, thereby allowing the entrainment of the circadian rhythms of several biological functions. Circulating melatonin is synthesized in the pinealgland as well as in peripheral tissues and secreted at high levels in a circadian manner. The control center of daily rhythmicity is in suprachiasmatic nuclei (SCN) of Hypothalamic, and can control the function of Pineal by hyper-synaptic linkage. It can enter the central nervous system (CNS) not only by readily crossing the blood-brain barrier (BBB), but also via the pineal recess, and in damaged brain, such as Parkinson's disease, Alzheimer's disease, and ischemic brain injury. Directly from the circulation because of leaky BBB. Melatonin has a variety of important physiological functions, including regulation of circadian rhythms, as well as visual, reproductive, cerebrovascular, neuroendocrine, and neuroimmunological actions. Recently, it has been reported the expression of melatonin receptor in several region of mice embryo brain, this evidence proofed the involved of melatonin in brain development.Recently, the research are focused on one aspect:melatonin can decrease oxygen radicals that were raised in the hypoxia-ischemia brain damage, and finaly reduce oxidative stress. Especially in CNS, melatonin can decrease oxygen radicals that were raised in the hypoxia-ischemia brain damage, and can reduce the apoptosis of neural stem cell, so melatonin have neuroprotective effective. But wheather melatonin can influence neural stem cell that is hypoxia invitro, and the potential mechanism is unknown. It is proofed that precursor administration can decrease the injury for fetal hypoxia in administration.In addition, melatonin exerts a neuroprotective effect in many pathological conditions of the CNS, Moreover, its antioxidant function has attracted much attention. Recently, it has been reported that melatonin influences cell growth and differentiation of the NSCs. However, its precise roles and the signaling pathway involved in the process, especially under altered conditions have remained unknown.Neural stem cells (NSCs) have the capacity for self-renewal and generation of new neurons and glial supporting cells. They are present not only in the fetal brain but also in the newborn and adult special brain areas in predictable proportions. Furthermore, the proliferation and differentiation activities of the NSCs do not remain static; rather they are dynamically regulated by various humoral and adhesive factors under physiological and pathophysiological conditions. Thus, the identification of different extrinsic factors regulating NSCs activity may contribute to a further understanding of the neural ontogeny as well as toward the development of new therapeutic strategies against neural injury.This study investigated the effect and molecular mechanism of melatonin on mouse NSCs under hypoxic condition in vitro. The effects and mechanism of melatonin on the proliferation, apoptosis and differentiation of NSCs were evaluated. We determined if melatonin could promote NSCs growth especially in hypoxia and, if so, the possibility of its being used as a potential therapeutic agent for the treatment of neonatal hypoxic-ischemic brain damage.一,Isolation,culture and expanding of NSCsNeuroepithelial cells from neural tube are the most primitive NSCs. These neuroepithelial stem cells are a population of undifferentiated cells that are capable of extended self-renewal and multipotential ability to generate multilineage cell types. At mice E12.5d, Neural progenitor cells suspension was successfully isolated by stereoscopic microscope and mechanical blowing, and neural progenitor cells gained in serum-free medium. BrdU labeling technique and Nestin immunofluorescence staining technique were used to detect their self-duplication and self-renewal. Microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunofluorescence staining technique were used to identify their differentiation. The present results suggest that neural progenitor cells from brain cortex of embryonic mice are easily extracted and naturally differentiate into neurocyte and neuroglial cells.二,The effected of melatonin on NSCs hypoxia in vitroThe effects of various concentrations of melatonin on NSCs viability were first assessed. NSCs were incubated in the growth medium in the presence of increasing concentra-tions of melatonin (0,1 nm,10 nm,100 nm, 1μm, 10μm, 100μm). MTT results showed that melatonin treatment for 24 hr obviously improved the cell viability of NSCs in a dose-dependent manner 10 nm,100 nm, 1μm melatonin was in comparison with cells not treated with melatonin. Furthermore,100 nm melatonin yielded the optimal effect on cell viability.We then examined the effect of melatonin on the proliferation of hypoxic NSCs in vitro. The cell viability was first determined at 1-5 days after hypoxia and the result showed that hypoxia decreased the cell viability. Melatonin treatment promoted the cell viability both in normal and hypoxic conditions at all time points, notably at 3 days after hypoxia. At 3 days after hypoxia, the ratio of BrdU/nestin positive cells against total nestin-positive NSCs decreased. However, after melatonin treatment, the frequency of BrdU/nestin positive cells was significantly increased compared with the untreated cells.For cell differentiation analysis, NSCs were exposed to hypoxic condition for 12 hr. The cells were then differentiated in differentiation medium (containing 2% FBS) in normal condition or treated with melatonin to determine whether it would influence the 2% FBS-induced NSC differentiation into MAP2-immunoreactive neurons and GFAP-expressing astrocytes by immunocytochemistry at 3,5,7, and 9 days after hypoxia. The percentage of neurons in relation to the total cell number was decreased in hypoxia group compared with the controls at all time points. Melatonin reversed this and increased the percentage of MAP2-positive cells in all melatonin treated groups, especially at 7 days differentiation as confirmed by Western blot analysis. When compared with the controls, hypoxia did not appear to affect the differentiation of NSCs into astrocytes. In melatonin-treated groups, NSCs differentiation into GFAP-positive astrocytes remained unchanged.三,The mechanism of melatonin on NSCs hypoxia in vitroNext, we explored the possible molecular mechanisms that might be linked to the proliferation of NSCs after hypoxia by melatonin. Expression of phosphorylation of the main MAPK (ERK1/2) was found to change significantly at different time points. Phosphorylation of ERK 1/2 elicited by melatonin was evident as early as 15 min, peaking at 60 min and declined at the 75 min. Melatonin receptor-MT1 mRNA and protein were detected in NSCs by RT-PCR and Western blot, respectively. To determine whether the melatonin-induced increase in NSCs proliferation acts via the melatonin receptor, NSCs were pretreated with 10μm luzindole, a melatonin receptor antagonist, prior to the addition of 100 nm melatonin. Phosphorylation of ERK1/2 induced by melatonin was significantly reduced. BrdU incorporation assay showed that cell proliferation in the presence of melatonin and luzindole was similar to the control group.To determine the mechanism of melatonin by which it can regulate differentiation of NSCs, we investigated some bHLH transcription factors which determine the neuron and astrocyte fates from NSCs. NSCs were cultured under hypoxic condition for 12 hr. The mRNA expression levels of Mashl, Neurogl, NeuroD2, Hesl, Hes5, Idl, and Id2 in differentiated cells were analyzed at 7 days after differentiation. The mRNA levels of Mash1, Neurog1, and NeuroD2 were significantly increased in melatonin treatment groups, while the mRNA levels of Hesl, Hes5, Id1, and Id2 had no significant differences in all groups.四,The mechanism of melatonin on NSCs apoptosis in cell hypoxia in vitroTo determine whether melatonin could affect the cell death of NSCs in hypoxia, NSCs were stained with Hoechst at 3 days after hypoxia. NSCs showing pyknotic nuclei indicative of cell death were enumerated. The rate of cell death in hypoxia was significantly higher than that in the control. In hypoxic cells treated with melatonin, the frequency of pyknotic nuclei decreased.We examined changes in hypoxia-induced caspase-3 activity following melatonin treatment of NSCs for 3 days. Melatonin treatment was found to suppress the caspase-3 activity which was increased in hypoxia. To clarify the mechanism underlying the protective effect of melatonin, Western blot analysis was performed to assess the expression of Bcl-2 and Bax in all groups at 3 days after hypoxia. Treatment of melatonin was shown to induce Bcl-2 expression and increase Bcl-2/Bax ratio. This suggests that melatonin inhibits caspase-3 activity and induces Bcl-2 expression, thereby inhibiting apoptotic cell death in NSCs.Conclusion:Taken together, our findings suggest that melatonin treatment for NSCs in hypoxia is a powerful strategy that can increase cell proliferation, reduce cell death, and enhance neuronal differentiation without affecting the astroglial differentiation. Because melatonin can easily enter the CNS, this intrinsic modulator might be beneficially used for stimulating endogenous NSCs. It is suggested that a better clarification of the sites and mechanisms underlying the induction and modulation by melatonin may lead to development of novel strategies for purposes of expansion and differentiation of endogenous NSCs in hypoxia injury disease. |
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