DataSheet1_Total flavonoids of hawthorn leaves protect spinal motor neurons via promotion of autophagy after spinal cord injury.PDF

The death of spinal motor neurons (SMNs) after spinal cord injury (SCI) is a crucial cause, contributing to a permanent neurological deficit. Total flavonoids of hawthorn leaves (TFHL) have been confirmed to have potentially therapeutic for SCI. Nonetheless, the roles and mechanisms of TFHL in recovering neuromotor function and regenerating axons of SMNs have not been fully elucidated. In this study, TFHL was applied to treat rats with SCI and injured SMNs for 7 days. In vivo experiment, rats with SCI were evaluated by a BBB (Basso-Beattie-Bresnahan) score to assess their motor functional recovery. The morphology, microstructure, apoptosis, Nissl bodies, and autophagy of SMNs in spinal cord tissue were detected by Hematoxylin-eosin (HE) staining, transmission electron microscopy, TUNEL staining, Nissl staining, and immunohistochemistry respectively. In vitro experiment, the co-culture model of SMNs and astrocytes was constructed to simulate the internal environment around SMNs in the spinal cord tissue. The cell morphology, microstructure, axonal regeneration, and autophagy were observed via optical microscope, transmission electron microscopy, and immunofluorescence. The content of neurotrophic factors in the cell culture medium of the co-culture model was detected by ELISA. Moreover, the expression of axon-related and autophagy-related proteins in the spinal cord tissue and SMNs was measured by Western Blot. We demonstrated that TFHL improved the neuromotor function recovery in rats after SCI. We then found that TFHL significantly promoted injured spinal cord tissue repair, reduced apoptosis, and improved the functional status of neurons in spinal cord tissue in vivo. Meanwhile, the cell morphology, microstructure, and axonal regeneration of damaged SMNs also obviously were improved, and the secretion of neurotrophic factors was facilitated after treatment with TFHL in vitro. Further, we revealed that TFHL promoted autophagy and related protein expression in vivo and vitro. Taken together, our study suggested that TFHL might facilitate autophagy and have neuroprotective properties in SMNs to enhance the recovery of neuromotor function of rats with SCI.

脊髓损伤（spinal cord injury, SCI）后脊髓运动神经元（spinal motor neurons, SMNs）的死亡是引发永久性神经功能缺损的关键诱因。山楂叶总黄酮（total flavonoids of hawthorn leaves, TFHL）已被证实对脊髓损伤具有潜在治疗价值。然而，山楂叶总黄酮在促进神经运动功能恢复及修复脊髓运动神经元轴突再生中的作用与机制尚未完全阐明。
本研究采用山楂叶总黄酮对脊髓损伤大鼠及体外损伤的脊髓运动神经元进行为期7天的干预。体内实验中，通过BBB（Basso-Beattie-Bresnahan）运动功能评分评估大鼠的运动功能恢复情况；分别采用苏木精-伊红（HE）染色、透射电子显微镜、TUNEL染色、尼氏染色及免疫组织化学检测脊髓组织中脊髓运动神经元的形态、超微结构、凋亡水平、尼氏体表达及自噬状态。
体外实验中，构建脊髓运动神经元与星形胶质细胞的共培养模型以模拟脊髓组织内脊髓运动神经元周围的微环境；通过光学显微镜、透射电子显微镜及免疫荧光观察细胞形态、超微结构、轴突再生情况及自噬水平；采用酶联免疫吸附测定（ELISA）检测共培养模型细胞培养基中的神经营养因子含量。此外，通过蛋白质免疫印迹（Western Blot）检测脊髓组织及脊髓运动神经元中轴突相关及自噬相关蛋白的表达水平。
本研究证实，山楂叶总黄酮可改善脊髓损伤大鼠的神经运动功能恢复。进一步研究发现，体内实验中，山楂叶总黄酮可显著促进受损脊髓组织修复，减少神经元凋亡，并改善脊髓组织内神经元的功能状态；体外实验中，山楂叶总黄酮干预可明显改善受损脊髓运动神经元的细胞形态、超微结构及轴突再生情况，同时促进神经营养因子的分泌。此外，本研究揭示山楂叶总黄酮可在体内及体外促进自噬及相关蛋白的表达。
综上，本研究表明山楂叶总黄酮可能通过促进自噬、发挥脊髓运动神经元神经保护作用，从而增强脊髓损伤大鼠的神经运动功能恢复。