Melatonin, a highly lipophilic molecule secreted by the pineal gland in the brain, plays a role in various biological functions. Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic- and adipogenic-lineage. However, the effect of melatonin in neurogenic differentiation in amniotic fluid (AF)-MSCs remains to be explored, thus we investigated the potential role of melatonin on dopaminergic neuron differentiation in AF-MSCs. The results showed that various concentrations of melatonin did not affect cell viability and proliferative effects of AF-MSCs. Increases in the levels of neuronal protein marker (βIII-tubulin) and dopaminergic neuronal markers (tyrosine hydroxylase, TH and NURR1), but decrease in the level of glial fibrillary acidic protein (GFAP), were observed in melatonin-treated AF-MSCs. Melatonin induced alteration in differential expression patterns of mesenchymal stem cell antigens by reducing CD29, CD45, CD73, CD90 and CD105, but no changing CD34 expressing cells. AF-MSCs were sequentially induced in neurobasal medium containing standard inducing cocktails (ST: bFGF, SHH, FGF8, BDNF), 1 μM melatonin, or a combination of ST and melatonin. The levels of TUJ1, TH, MAP2, NURR1 and dopamine transporter (DAT) were significantly increased in all treated groups when compared with control-untreated cells. Pretreated AF-MSCs with non-selective MT1/MT2 receptors antagonist, luzindole and selective MT2 receptor antagonist, 4-P-PDOT diminished melatonin-induced increase in dopaminergic neuronal markers and phosphorylated ERK but did not diminish increase in phosphorylated CaMKII by melatonin. Pretreatment with mitogen-activated protein kinase (MEK) inhibitor, PD98059 and CaMKII inhibitor, KN-93 were able to abolish increase in the levels of dopaminergic markers in melatonin-treated AF-MSCs. These findings suggest that melatonin promotes dopaminergic neuronal differentiation of AF-MSCs possibly via the induction in ERK and CaMKII pathways through melatonin receptor-dependent and -independent mechanisms, respectively.

中文翻译：

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褪黑激素对人羊水间充质干细胞多巴胺能神经元分化的潜在作用及其分子机制。

褪黑激素是大脑中的松果体分泌的高度亲脂性分子，在多种生物学功能中起作用。先前的研究报道，褪黑激素对间充质干细胞（MSC）的存活以及向成骨和成脂谱系的分化具有影响。然而，褪黑素在羊水（AF）-MSCs神经源性分化中的作用仍有待探索，因此我们研究了褪黑素在AF-MSCs中多巴胺能神经元分化中的潜在作用。结果表明，各种浓度的褪黑激素都不会影响AF-MSC的细胞活力和增殖作用。神经元蛋白标志物（βIII-微管蛋白）和多巴胺能神经元标志物（酪氨酸羟化酶，TH和NURR1）的水平增加，但神经胶质纤维酸性蛋白（GFAP）的水平下降，在褪黑素处理过的AF-MSC中观察到。褪黑素通过减少CD29，CD45，CD73，CD90和CD105，但不改变CD34表达细胞，诱导间充质干细胞抗原差异表达模式的改变。在含有标准诱导混合物（ST：bFGF，SHH，FGF8，BDNF），1μM褪黑素或ST和褪黑素的组合的神经基础培养基中依次诱导AF-MSC。与未处理的对照细胞相比，所有处理组的TUJ1，TH，MAP2，NURR1和多巴胺转运蛋白（DAT）的水平均显着增加。具有非选择性MT1 / MT2受体拮抗剂，Luzindole和选择性MT2受体拮抗剂，4-P-PDOT的预处理AF-MSC减少了褪黑激素诱导的多巴胺能神经元标志物和磷酸化ERK的增加，但并未减少褪黑素引起的磷酸化CaMKII的增加。用促分裂原活化蛋白激酶（MEK）抑制剂PD98059和CaMKII抑制剂KN-93进行的预处理能够消除褪黑素治疗的AF-MSC中多巴胺能标志物水平的增加。这些发现表明，褪黑激素可能通过经由褪黑激素受体依赖性和非依赖性机制分别诱导ERK和CaMKII途径来促进AF-MSC的多巴胺能神经元分化。