Yao Xue, Sun Chao, Fan Baoyou, Zhao Chenxi, Zhang Yan, Duan Huiquan, Pang Yilin, Shen Wenyuan, Li Bo, Wang Xu, Liu Chang, Zhou Hengxing, Kong Xiaohong, Feng Shiqing
Tianjin Key Laboratory of Spine and Spinal Cord, International Science and Technology Cooperation Base of Spinal Cord Injury, ICMRS Collaborating Center for Spinal Cord Injury, Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin, China.
School of Medicine, Nankai University, Tianjin, China.
J Orthop Translat. 2020 Mar 23;26:74-83. doi: 10.1016/j.jot.2020.02.011. eCollection 2021 Jan.
BACKGROUND/OBJECTIVE: Spinal cord injury (SCI) severely and irreversibly damages the central nervous system. Neurotropin (NTP), a nonprotein extract obtained from inflamed rabbit skin inoculated with vaccinia virus, is a drug that has been used for more than sixty years to alleviate neuropathic pain. It also reportedly exerts a neuroprotective role in peripheral nerves and in response to various central nervous system diseases, such as brain injury and Alzheimer disease. However, whether NTP promotes SCI recovery remains unknown. This study evaluated NTP's effects after SCI and explored its underlying mechanisms in a rat contusion model of SCI.
NTP was intraperitoneally administered to adult female Wistar rats subjected to contusion-induced SCI. Functional recovery was evaluated with behavioural scores and electrophysiological examinations. Tissue recovery was assessed with magnetic resonance imaging as well as histological staining with haematoxylin and eosin and Luxol Fast Blue. Neuronal survival and gliosis were observed after NeuN and glial fibrillary acidic protein immunofluorescence. Levels of apoptosis were demonstrated with TdT-mediated dUTP nick-end labeling (TUNEL) staining, Caspase-3 and B-cell lymphoma-2 (Bcl-2) Western blot, and Annexin V/propidium iodide flow cytometry. A protein antibody chip analysis was performed to evaluate the expression levels of 67 rat cytokines.
NTP treatment improved the hindlimb locomotor recovery of the injured animals as well as their electrophysiological outcomes after SCI. A dosage of 50 NTP units/kg was found to optimize the efficacy of NTP. Magnetic resonance imaging revealed that lesion sizes decreased after NTP treatment. The haematoxylin and eosin and Luxol Fast Blue staining showed significant increases in the amount of spared tissue. The NeuN and glial fibrillary acidic protein immunofluorescence revealed that NTP treatment increased neuronal survival and reduced gliosis in tissue samples obtained from the lesion's epicentre. That NTP inhibited apoptosis was confirmed by the decreased number of TUNEL-positive cells, level of Caspase-3 expression, and number of Annexin V/propidium iodide-positive cells, as well as the increased level of Bcl-2 expression. The protein array analysis identified 28 differentially expressed proteins in the NTP group, and the gene ontology (GO) analysis showed that the enriched differentially expressed proteins implicate janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathways. The expression levels of proinflammatory cytokines such as interleukin 6, thymus chemokine-1(TCK-1), and lipopolysaccharide-induced CXC chemokine (LIX) decreased after NTP treatment, whereas the levels of prorepair cytokine hepatocyte growth factor and adiponectin increased.
Our research provides evidence that NTP can improve functional outcomes and alleviate secondary injury after SCI by inhibiting apoptosis and modulating cytokines.
The multicomponent NTP might have broad target spectra in SCI pathophysiology and halt the secondary injury cascade. As a safe drug that features sixty years of clinical use as an analgesic, translating this demonstrated efficacy of NTP to addressing SCI in human patients may potentially be accelerated.
背景/目的：脊髓损伤（SCI）会严重且不可逆地损害中枢神经系统。神经妥乐平（NTP）是一种从接种痘苗病毒的发炎兔皮中提取的非蛋白质提取物，作为一种药物，它已被用于缓解神经性疼痛六十多年。据报道，它在外周神经以及应对各种中枢神经系统疾病（如脑损伤和阿尔茨海默病）时也发挥神经保护作用。然而，NTP是否能促进SCI恢复仍不清楚。本研究评估了NTP在SCI后的作用，并在大鼠SCI挫伤模型中探究其潜在机制。
对成年雌性Wistar大鼠进行挫伤诱导的SCI后，腹腔注射NTP。通过行为评分和电生理检查评估功能恢复情况。用磁共振成像以及苏木精-伊红染色和卢氏固蓝染色评估组织恢复情况。通过NeuN和胶质纤维酸性蛋白免疫荧光观察神经元存活和胶质细胞增生情况。用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色、半胱天冬酶-3和B细胞淋巴瘤-2（Bcl-2）蛋白质免疫印迹以及膜联蛋白V/碘化丙啶流式细胞术检测细胞凋亡水平。进行蛋白质抗体芯片分析以评估67种大鼠细胞因子的表达水平。
NTP治疗改善了受伤动物的后肢运动恢复情况以及SCI后的电生理结果。发现50 NTP单位/千克的剂量可使NTP的疗效最优化。磁共振成像显示NTP治疗后损伤大小减小。苏木精-伊红染色和卢氏固蓝染色显示 spared组织量显著增加。NeuN和胶质纤维酸性蛋白免疫荧光显示，NTP治疗增加了从损伤中心获得的组织样本中的神经元存活并减少了胶质细胞增生。TUNEL阳性细胞数量减少、半胱天冬酶-3表达水平降低、膜联蛋白V/碘化丙啶阳性细胞数量减少以及Bcl-2表达水平增加，证实了NTP抑制细胞凋亡。蛋白质阵列分析确定了NTP组中有28种差异表达蛋白，基因本体（GO）分析表明，富集的差异表达蛋白涉及janus激酶-信号转导子和转录激活子（JAK-STAT）信号通路。NTP治疗后，白细胞介素6、胸腺趋化因子-1（TCK-1）和脂多糖诱导的CXC趋化因子（LIX）等促炎细胞因子的表达水平降低，而促修复细胞因子肝细胞生长因子和脂联素的水平升高。
我们的研究提供了证据，表明NTP可通过抑制细胞凋亡和调节细胞因子来改善SCI后的功能结果并减轻继发性损伤。
多成分的NTP在SCI病理生理学中可能具有广泛的靶点谱，并能阻止继发性损伤级联反应。作为一种具有六十年临床镇痛应用历史的安全药物，将NTP已证实的疗效转化用于治疗人类SCI患者可能会加速。
