Cycloastragenol promotes motor function recovery and cortical neuron regeneration in mice with spinal cord injury

doi:10.12307/2025.694

Abstract

Abstract: BACKGROUND: Previous studies have confirmed that cycloastragenol can promote the repair of sciatic nerve injuries, and activate telomerase reverse transcriptase to further participate in the regulation of axonal regeneration of peripheral sensory neurons. However, it remains unknown whether cycloastragenol can activate the axonal regeneration of cortical neurons, particularly whether it is involved in the restoration of motor function during spinal cord injury recovery.
OBJECTIVE: To investigate the effects of cycloastragenol on the restoration of motor function in mice with spinal cord injury, as well as the related mechanisms by which cycloastragenol promotes cortical neuron regeneration.
METHODS: (1) Twelve ICR mice were randomly divided into a solvent group and a cycloastragenol group, with six mice in each group. Mice were given intraperitoneal injections of PBS containing 2% Tween 20 and 5% dimethyl sulfoxide (DMSO) (the solvent group) and cycloastragenol (20 mg/kg), respectively. The treatment lasted for 12 weeks, and the motor function of the mouse limbs was assessed using the Basso Mouse Scale. (2) E14 fetal mouse cortical neurons were cultured in vitro and divided into a DMSO group and a cycloastragenol group, with the neurons being treated with DMSO and 0.5 μmol/L cycloastragenol for 3 days in vitro. (3) E7 pregnant mice were divided into a solvent group and a cycloastragenol group. After continuous intraperitoneal injections of PBS containing 2% Tween 20 and 5% DMSO and 20 mg/kg cycloastragenol for 7 continuous days, the fetal mouse cortex was harvested. The cortical neurons were then cultured in vitro for 3 days. (4) Tuj1 immunofluorescence staining was performed on fetal rat cortical neurons to analyze the axon length of Tuj1-positive cells, validating the effect of cycloastragenol on axonal regeneration of cortical neurons. Fetal mouse cortical neurons were subjected to dual-label immunofluorescence staining for Tuj1 and telomerase reverse transcriptase, in order to analyze the effect of cycloastragenol on the expression of telomerase reverse transcriptase. Western blot assay was used to detect the relative expression of telomerase reverse transcriptase in cortical neurons of fetal mice. (5) Six ICR mice were randomized into a solvent group and a cycloastragenol group (n=3 per group). After modeling, mice in the two groups received continuous intraperitoneal injections of PBS containing 2% Tween 20 and 5% DMSO and 20 mg/kg cycloastragenol, respectively, for 7 days. The relative expression of telomerase reverse transcriptase protein in spinal cord tissue was detected by western blot assay.
RESULTS AND CONCLUSION: (1) After 4, 6, 8, and 12 weeks of treatment with cycloastragenol, the motor function scores of the mice in the cycloastragenol group were significantly higher than those in the solvent group (P < 0.000 1). The relative protein expression of telomerase reverse transcriptase in the spinal cord tissues of mice in the cycloastragenol group was significantly higher than that in the solvent group after 7 days of cycloastragenol treatment (P < 0.05). (2) The results of Tuj1 immunofluorescence staining showed that the axon length of neurons cultured in vitro in the 0.5 μmol/L cycloastragenol group was significantly greater than that in the DMSO group (P < 0.001), and the axon length of neurons in the 20 mg/kg cycloastragenol intraperitoneal injection group was significantly greater than that in the solvent group (P < 0.01). (3) The results of telomerase reverse transcriptase immunofluorescence staining indicated that the fluorescence intensity of telomerase reverse transcriptase in cortical neurons cultured in vitro in the 0.5 μmol/L cycloastragenol group was significantly higher than that of the DMSO group (P < 0.01). (4) Western blot analysis showed that the relative expression of telomerase reverse transcriptase protein in cortical neurons cultured in vitro in the 0.5 μmol/L cycloastragenol group was significantly higher than that in the DMSO group (P < 0.05). Similarly, the relative expression of telomerase reverse transcriptase protein in cortical neurons in the 20 mg/kg cycloastragenol intraperitoneal injection group was significantly higher than that in the solvent group (P < 0.05). These findings indicate that cycloastragenol promotes axonal growth of cortical neurons and improves motor function in mice after spinal cord injury, which may be associated with elevated expression of telomerase reverse transcriptase.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: cycloastragenol, cortical neurons, axonal growth, telomerase reverse transcriptase, motor function, spinal cord injury, engineered animal model

CLC Number:

Tao Zihan, Saijilafu. Cycloastragenol promotes motor function recovery and cortical neuron regeneration in mice with spinal cord injury [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(20): 4258-4265.

Figures/Tables 7

2.1 实验动物数量分析实验选用ICR小鼠18只，均进入结果分析。 2.2 BMS运动功能评分造模后，各组小鼠BMS运动功能评分均为0分，小鼠后肢完全丧失运动功能，各组无统计学差异。给药4周后，环黄芪醇组小鼠运动功能逐步恢复。自给药4周起，环黄芪醇组小鼠 BMS运动功能评分显著优于溶剂组(P < 0.000 1)，在给药12周时，小鼠一侧后腿实现了灵活运动，而另一侧后腿的踝关节也展现出了较强的活动能力，说明环黄芪醇能够显著改善脊髓损伤小鼠的肢体运动功能，见图1。 2.3 环黄芪醇在体外对神经元活力的影响为了确定环黄芪醇对体外培养的皮质神经元有无潜在的细胞毒性，对细胞进行活死染色，结果显示用0.5 μmol/L环黄芪醇处理皮质神经元3 d，二甲基亚砜组与环黄芪醇组的存活细胞无明显差异，表明环黄芪醇不会导致额外的神经元死亡，见图2。 2.4 环黄芪醇在体外给药对皮质神经元的影响前期研究证实，0.5 μmol/L环黄芪醇能够促进感觉神经元的轴突再生，但是该剂量对于皮质神经元的影响仍是未知[27]。Tuj 1(β-tubulinⅢ)是一种神经元特异性的细胞标记物，通常用于标记早期的神经元前体细胞和成熟神经元中的微管蛋白，可以跟踪神经元的形成和轴突生长[30-31]。神经元轴突生长是神经修复和再生的关键过程。受损的神经元可以通过重新生长轴突从而恢复功能性连接，以实现神经系统的修复和功能恢复[32]。在体外研究中，使用含0.5 μmol/L环黄芪醇的培养基培养E14胎鼠皮质神经元3 d后，对所得神经元进行Tuj1免疫荧光染色并测量轴突长度，发现0.5 μmol/L环黄芪醇组的神经元轴突长度平均值达到约188 μm，而二甲基亚砜组神经元轴突长度平均值约123 μm，两组轴突长度差异有显著性意义(图3A-C)。随机统计Tuj1阳性细胞的数量，结果显示两组皮质神经元的存活无显著差异(图3D)。 2.5 环黄芪醇体外给药对皮质神经元中TERT表达的影响已有研究表明，环黄芪醇能够上调周围神经系统背根神经节中TERT的表达，进一步激活其内在轴突生长能力，并参与外周感觉神经轴突再生的调节[25]，但TERT在中枢神经系统中是否能够继续发挥促进轴突再生的作用仍是未知。在体外用含0.5 μmol/L环黄芪醇的培养基培养E14"

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