关键词: 环黄芪醇, 皮质神经元, 轴突生长, 端粒酶反转录酶, 运动功能, 脊髓损伤, 工程化动物模型

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