Department of Children Rehabilitation, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders (Chongqing), China; International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing Engineering Research Center of Stem Cell Therapy, Chongqing Key Laboratory of Child Health and Nutrition, Children's Hospital of Chongqing Medical University, Chongqing, China.
Wuhan Children's Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430016, Hubei, China.
Neurosci Lett. 2020 May 14;727:134922. doi: 10.1016/j.neulet.2020.134922. Epub 2020 Mar 20.
Our previous experiments found that a suitable dose of vitamin A (VA) can affect neuronal apoptosis after hypoxic-ischemic brain damage (HIBD) by binding to RARα to activate the PI3K/AKT signaling pathway; however, the other neuroprotective effects of VA after HIBD, for example, whether it promotes neural stem cell (NSC) proliferation, remain unclear. In this study, in vivo and in vitro experiments revealed that VA regulates β-catenin signaling through RARɑ to affect NSC proliferation after HIBD and to improve neurocognitive outcomes. Because of the accumulation and suspended growth characteristics of NSCs, we performed in vitro experiments with PC12 cells to mimic NSCs. Flow cytometry, CCK8, EdU staining, immunofluorescence and behavioral tests were performed to explore the effects of retinoic acid (RA) on NSC proliferation and post-HIBD function. The expression of RARα and β-catenin pathway components were measured by real-time PCR and Western blotting. We found that the learning and memory of the VA-deficient (VAD) group was more seriously damaged than that of the VA normal (VAN) group. The proliferation of hippocampal NSCs was significantly decreased in the VAD group compared with the VAN group. The mRNA and protein expression of RARɑ, AKT, GSK-3β, β-catenin and Cyclin D1 were significantly lower in the VAD group than in the VAN group. In vitro, too high and too low of an RA intervention resulted in decreased proliferation, while an appropriate RA concentration (1-5 μmol/L) significantly promoted proliferation, S phase cells and high β-catenin pathway expression. These results suggested that VA can exert a neuroprotective effect by promoting the proliferation of hippocampal NSCs after neonatal HIBD injury at the appropriate concentration. VA activates RARɑ, which regulates the β-catenin signaling pathway, which in turn upregulates Cyclin D1 expression, promotes NSC proliferation, and finally plays a role in the neuroprotective effect.
我们之前的实验发现，适当剂量的维生素 A（VA）可以通过与 RARα 结合来激活 PI3K/AKT 信号通路，从而影响缺氧缺血性脑损伤（HIBD）后的神经元凋亡；然而，VA 在 HIBD 后的其他神经保护作用，例如是否促进神经干细胞（NSC）增殖，尚不清楚。在这项研究中，体内和体外实验表明，VA 通过 RARɑ 调节 β-连环蛋白信号通路，影响 HIBD 后 NSC 的增殖，并改善神经认知结果。由于 NSCs 的积累和悬浮生长特性，我们使用 PC12 细胞进行了体外实验来模拟 NSCs。通过流式细胞术、CCK8、EdU 染色、免疫荧光和行为测试来探索维甲酸（RA）对 NSC 增殖和 HIBD 后功能的影响。通过实时 PCR 和 Western blot 测量 RARα 和 β-连环蛋白通路成分的表达。我们发现，VA 缺乏（VAD）组的学习和记忆损伤比 VA 正常（VAN）组更严重。与 VAN 组相比，VAD 组海马 NSCs 的增殖明显减少。VAD 组的 RARα、AKT、GSK-3β、β-连环蛋白和 Cyclin D1 的 mRNA 和蛋白表达均明显低于 VAN 组。体外实验表明，过高和过低的 RA 干预都会导致增殖减少，而适当浓度的 RA（1-5 μmol/L）则能显著促进增殖、S 期细胞和高β-连环蛋白通路表达。这些结果表明，VA 可以通过促进新生 HIBD 损伤后海马 NSCs 的增殖来发挥神经保护作用。VA 激活 RARα，调节 β-连环蛋白信号通路，从而上调 Cyclin D1 表达，促进 NSC 增殖，最终发挥神经保护作用。
