Effect and mechanism of fasudil on proliferation and differentiation of neural stem cells

doi:10.3969/j.issn.2095-4344.1638

Abstract

Abstract:

BACKGROUND: The self-repairing ability of damaged brain and spinal cord is limited. It is important to search for potential therapeutics to promote the proliferation and differentiation of neural stem cells.
OBJECTIVE: To investigate the effect of fasudil on the proliferation and differentiation of primary cultured neural stem cells and its potential mechanism.
METHODS: Neural stem cells were obtained from the brain tissue of 15-day-old fetal rats in vitro. The expression of Nestin in the cells was detected by immunofluorescence. After treatment with fasudil at different concentrations (50, 100, 200 µmol/L) for 24, 48 and 72 hours, the proliferation rate of neural stem cells was detected by MTT, and the apoptosis rate was detected by flow cytometry. After further treatment with autophagy inhibitor, necrosis inhibitor, apoptosis inducer and ferroptosis inducer, the proliferation rates of neural stem cells were detected by MTT and the levels of malondialdehyde were detected by biochemical method. The expression of Nestin, doublecortin, microtubule- associated protein and glial fibrillary acidic protein in neural stem cells were detected by western blot and immunofluorescence after treatment with 200 µmol/L fasudil for 10 days.
RESULTS AND CONCLUSION: The positive expression of Nestin protein in primary cultured neural stem cells was observed. The proliferation rate of neural stem cells increased gradually with the increase of fasudil concentration as well as with the prolongation of action time (P < 0.05). Both apoptosis inhibitor and ferroptosis inhibitor can increase the proliferation rate of neural stem cells (P < 0.05). Fasudil increased the proliferation rate of neural stem cells treated by apoptosis inducer and ferroptosis inducer (P < 0.05). Fasudil and ferroptosis inhibitors both decreased the level of malondialdehyde in neural stem cells, while ferroptosis inducers increased the level of malondialdehyde in neural stem cells (P < 0.05). After treatment with fasudil, the expression of doublecortin and glial fibrillary acidic protein protein in neural stem cells increased, and the expression of Nestin decreased (P < 0.05). To conclude, fasudil can improve the survival of neural stem cells by inhibiting apoptosis and ferroptosis, and moreover, it can promote the proliferation and differentiation of neural stem cells into neuron-like cells and glial cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neural Stem Cells, Cell Proliferation, Apoptosis, Cell Differentiation, Tissue Engineering

CLC Number:

Sun Wanying, Tang Ying, Chen Meijuan. Effect and mechanism of fasudil on proliferation and differentiation of neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1336-1341.

Figures/Tables 1

2.1 胎鼠神经干细胞的形态和鉴定结果原代分离的神经干细胞培养1 d后，多数细胞呈球形悬浮生长，单个散在分布，见图1A；培养3 d后，细胞聚集成团，体积较小，悬浮生长，见图1B；培养7 d后，细胞聚集呈体积较大的神经球，悬浮生长，细胞排列紧密，球体中央细胞边界不清，未见明显突起形成，见图1C；细胞免疫荧光检测神经干细胞标志物Nestin呈阳性表达，见图1D，提示分离培养的细胞是未分化的神经干细胞。 2.2 法舒地尔提高神经干细胞的增殖活性随着法舒地尔浓度升高及作用时间延长，神经干细胞的增殖率逐渐增高，见图2，提示法舒地尔可能通过减少凋亡或增加活性提高神经干细胞的增殖率，且呈剂量与时间依赖性。 2.3 法舒地尔抑制神经干细胞凋亡和铁死亡随着法舒地尔浓度增高和作用时间延长，神经干细胞的凋亡率没有明显变化，见图3A。凋亡抑制剂z-vad-fmk和铁死亡抑制剂ferrostatin-1可提高神经干细胞的增殖率，而坏死抑制剂与自噬抑制剂不发挥作用，见图3B。法舒地尔可提高凋亡诱导剂CCCP和铁死亡诱导剂Erastin处理的神经干细胞增殖率，提示法舒地尔可能抑制神经干细胞的凋亡和铁死亡，见图3C。 2.4 法舒地尔通过降低脂质过氧化水平提高神经干细胞增殖活性法舒地尔和铁死亡抑制剂Ferrostatin-1均能降低神经干细胞中丙二醛水平，铁死亡诱导剂Erastin则使神经干细胞中丙二醛水平升高，见图4，提示法舒地尔可能通过降低脂质过氧化水平提高神经干细胞增殖活性。 2.5 法舒地尔促进神经干细胞向神经元与神经胶质细胞分化 Western blot检测结果显示，法舒地尔作用后，神经干细胞中DCX和GFAP的表达升高，Nestin的表达降低(P < 0.05)；免疫荧光染色结果也显示，法舒地尔组DCX和GFAP阳性细胞比例高于对照组，Nestin阳性细胞比例低于对照组(P < 0.05)，见图5。以上结果提示法舒地尔可能促进神经干细胞向神经元与神经胶质细胞分化。"

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