Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara, 252-5201, Japan.
Graduate School of Veterinary Medicine, Azabu University, Fuchinobe, Chuo-ku, Sagamihara, 252-5201, Japan; Division of Pharmacology, National Institute of Health Sciences, Kawasaki, Kanagawa, 210-9501, Japan.
Nitric Oxide. 2019 Dec 1;93:1-5. doi: 10.1016/j.niox.2019.08.008. Epub 2019 Sep 1.
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing βIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as βIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.
丙戊酸（VPA）显著促进脂肪组织来源的干细胞（ASCs）向成熟神经元细胞分化，仅在 3 天内即可实现神经元诱导。在这里，我们研究了 NO 信号在 VPA 促进 ASCs 神经元分化中的作用机制。通过用 2mM 的 VPA 处理培养的大鼠 ASCs 3 天，然后用含有 cAMP 升高剂的神经元诱导培养基（NIM）处理 2 小时，将其分化为富含树突且表达神经元标志物 βIII-微管蛋白的成熟神经元细胞。用荧光 NO 特异性探针二氨基荧光素-FM 二乙酸酯检测到经 VPA 处理的 ASC 分化而来的神经元细胞中细胞内 NO 增加。然而，NO 供体（NOC18）仅将神经元细胞的发生率增加到较小程度，这表明外源性 NO 的不足。用 VPA 处理的 ASC 的 RT-PCR 分析显示，诱导型一氧化氮合酶（iNOS）的 mRNA 表达增加，其相关组蛋白 H3K9 乙酰化。iNOS 抑制剂（1400W 和地塞米松）或可溶性鸟苷酸环化酶（sGC）抑制剂（ODQ）降低了 VPA 处理的 ASC 分化而来的神经元细胞的发生率。这些抑制剂还不同程度地降低了成熟神经元标志物神经丝中等多肽（NeFM）和微管相关蛋白 2（MAP2）以及 βIII-微管蛋白（TUBB3）的 mRNA 表达。考虑到这些结果，VPA 通过 iNOS-NO-sGC 信号通路促进 ASCs 的成熟神经元分化。这为临床应用中 ASCs 的调控性神经元分化提供了思路。
