Objective: To investigate the effect of Jisuikang formula-medicated serum for promoting spinal cord injury (SCI) repair in rats and explore the possible mechanism.
Methods: Thirty adult SD rats were randomized into sham-operated group, SCI (induced using a modified Allen method) model group, and Jisuikang formula-medicated serum treatment group. After the operations, the rats were treated with normal saline or Jisuikang by gavage on a daily basis for 14 days, and the changes in hindlimb motor function of the rats was assessed with Basso-Beattie-Bresnahan (BBB) scores and inclined-plate test. The injured spinal cord tissues were sampled from the SCI rat models for single-cell RNA sequencing, and bioinformatics analysis was performed to identify the target genes of Jisuikang, spinal cord injury and glycolysis. In the cell experiment, cultured astrocytes from neonatal SD rat cortex were treated with SOX2 alone or in combination with Jisuikang-medicated serum for 21 days, and the protein expressions of PKM2, p-PKM2 and YAP and colocalization of PKM2 and YAP in the cells were analyzed with Western blotting and immunofluorescence staining, respectively.
Results: The SCI rats with Jisuikang treatment showed significantly improved BBB scores and performance in inclined-plate test. At the injury site, high PKM2 expression was detected in various cell types. Bioinformatic analysis identified the HIPPO-YAP signaling pathway as the target pathway of Jisuikang. In cultured astrocytes, SOX2 combined with the mediated serum, as compared with SOX2 alone, significantly increased PKM2, p-PKM2 and YAP expressions and entry of phosphorylated PKM2 into the nucleus, and promoted PKM2 and YAP co-localization in the cells.
Conclusion: Jisuikang formula accelerates SCI repair in rats possibly by promoting aerobic glycolysis of the astrocytes via activating the PKM2/YAP axis to induce reprogramming of the astrocytes into neurons.
目的: 研究中药脊髓康含药血清联合SRY转录盒因子2（SOX2）诱导星形胶质细胞重编程为神经元的作用机制。
方法: 将30只成年SD大鼠随机分为假手术组（Sham组）、模型组（SCI组）和治疗组，10只/组。SCI组和治疗组均采用改良Allen法构建脊髓损伤大鼠模型，于术后分别给予生理盐水、脊髓康等量灌胃。在术前1 d、术后3、7、14 d进行Basso-Beattie-Bresnahan（BBB）评分和斜板实验。相同方法构建脊髓损伤大鼠模型，提取损伤部位组织进行单细胞RNA测序，再使用生物信息学对“脊髓康”、“脊髓损伤”、“糖酵解”关联基因进行靶点分析。成年SD大鼠以生药量15 g·kg-1·d-1脊髓康灌胃，连续3 d后腹主动脉采血制备脊髓康含药血清。体外培养大鼠幼仔皮层脑组织三代星形胶质细胞，分为SOX2组、SOX2+JSK组。SOX2组加入10 mmol/L SOX2；SOX2+JSK组加入10 mmol/L SOX2、2.5%脊髓康含药血清。培养21 d后，Western blot检测细胞中丙酮酸激酶M2型（PKM2）、磷酸化丙酮酸激酶M2型（p-PKM2）、Yes-相关蛋白（YAP）表达，使用免疫荧光多标染色观测PKM2与YAP共定位情况。
结果: 治疗组大鼠的BBB评分与斜板实验角度明显提升，且各时段均优于SCI组（P < 0.05）。SCI部位PKM2基因在各类细胞中均有高活性表达；生物信息学分析靶点指向HIPPO-YAP信号通路；与SOX2组比较，整个细胞中SOX2+JSK组PKM2表达增高（P < 0.05）、p-PKM2、YAP表达亦升高（P < 0.001），且更多PKM2磷酸化入核。SOX2+JSK组PKM2与YAP共定位情况高于SOX2组。
结论: 中药脊髓康含药血清联合SOX2可能通过提高糖酵解中PKM2与YAP共定位，从而将星形胶质细胞重编程诱导为神经元细胞，促进神经损伤修复。
Keywords: Jisuikang formula; PKM2; YAP; aerobic glycolysis; astrocytes; spinal cord injury.