福建片通过调节BDNF剪切酶介导的proBDNF/mBDNF平衡，促进MCAO大鼠皮质脊髓束重塑，改善运动功能

摘要(中文译文)

福建片（FJT）是一种中药制剂，在中医理论的背景下，已被临床用于与缺血性脑损伤相关的神经系统疾病的康复。然而，其促进缺血性卒中后运动功能恢复的分子机制，特别是通过调节皮质脊髓束（CST）重塑——运动控制的关键结构——仍然不清楚。本研究旨在探讨FJT对失神经半球CST重塑和大脑中动脉闭塞（MCAO）大鼠运动功能恢复的影响，并通过关注前体脑源性神经营养因子（proBDNF）和成熟BDNF（mBDNF）之间的平衡来探索其潜在机制，后者受BDNF裂解酶（Pcsk1和Furin）的严格调节。采用管腔内细丝法建立MCAO大鼠模型。模型大鼠随机分为四组：MCAO模型组、FJT低剂量组、FJET中剂量组和FJT高剂量组。通过猫步步态分析（评估平均速度、步长和站立时间）评估运动功能。分别通过生物素化葡聚糖胺（BDA）神经追踪和运动诱发电位（MEP）检测来确定CST重塑和传导效率。使用定量实时聚合酶链式反应（RT-qPCR）和Western blot测量脑组织中BDNF、切割酶（Pcsk1、Furin）和相关受体（TrkB、p75NTR、Sortilin）的mRNA和蛋白质表达。通过BDNF沉默实验验证BDNF在FJT诱导效应中的作用。此外，使用体外神经元培养来观察FJT、外源性mBDNF和Pcsk1/Furin抑制剂对神经元生长的影响。与MCAO模型组相比，中剂量FJT的治疗效果最为显著。具体而言，FJT显著改善了步态参数，将平均速度从20.77 mm/s（MCAO）提高到25.71 mm/s（FJT），步长提高了约21.14%。此外，FJT增强MEP传导效率并促进CST重塑，其特征是失神经半球BDA阳性神经纤维增加5.26%，生长相关蛋白43（GAP43）表达升高。在分子水平上，FJT上调Pcsk1和Furin的mRNA和蛋白质表达，增加BDNF及其功能受体TrkB的水平，下调proBDNF偏好受体p75NTR和Sortilin的表达，最终使proBDNF/mBDNF比值向mBDNF优势转变。BDNF沉默显著减弱了这些改善，逆转了FJT诱导的运动恢复和CST重塑。在体外，FJT促进的神经元生长被外源性mBDNF模拟，但被Pcsk1/Furin抑制剂逆转。与MCAO模型组相比，中剂量FJT的治疗效果最为显著。具体而言，FJT显著改善了步态参数，将平均速度从20.77 mm/s（MCAO）提高到25.71 mm/s（FJT），步长提高了约21.14%。此外，FJT增强MEP传导效率并促进CST重塑，其特征是失神经半球BDA阳性神经纤维增加5.26%，生长相关蛋白43（GAP43）表达升高。在分子水平上，FJT上调Pcsk1和Furin的mRNA和蛋白质表达，增加BDNF及其功能受体TrkB的水平，下调proBDNF偏好受体p75NTR和Sortilin的表达，最终使proBDNF/mBDNF比值向mBDNF优势转变。BDNF沉默显著减弱了这些改善，逆转了FJT诱导的运动恢复和CST重塑。在体外，FJT促进的神经元生长被外源性mBDNF模拟，但被Pcsk1/Furin抑制剂逆转。

Abstract

Fujian Tablets (FJT), a traditional Chinese medicinal (TCM) preparation, has been clinically used in the rehabilitation of neurological disorders related to ischemic brain injury in the context of TCM theory. However, its molecular mechanism underlying the promotion of post-ischemic stroke motor function recovery, especially via regulating corticospinal tract (CST) remodeling-a key structure for motor control-remains unelucidated. This study aimed to investigate the effect of FJT on CST remodeling in the denervated hemisphere and motor function recovery in middle cerebral artery occlusion (MCAO) rats, and to explore its potential mechanism by focusing on the balance between precursor brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF), which is tightly regulated by BDNF-cleaving enzymes (Pcsk1 and Furin). The MCAO rat model was established using the intraluminal filament method. Model rats were randomly divided into four groups: MCAO model group, FJT low-dose group, FJT medium-dose group, and FJT high-dose group. Motor function was evaluated by Catwalk gait analysis (assessing average speed, step length, and standing time). CST remodeling and conduction efficiency were determined via biotinylated dextran amine (BDA) neural tracing and motor evoked potential (MEP) detection, respectively. The mRNA and protein expressions of BDNF, cleaving enzymes (Pcsk1, Furin), and related receptors (TrkB, p75NTR, Sortilin) in brain tissues were measured using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot. BDNF silencing experiment was performed to verify the role of BDNF in FJT-induced effects. Additionally, in vitro neuronal culture was used to observe the effects of FJT, exogenous mBDNF, and Pcsk1/Furin inhibitors on neuronal growth. Compared with the MCAO model group, medium-dose FJT exhibited the most significant therapeutic effects. Specifically, FJT notably improved gait parameters increasing average speed from 20.77 mm/s (MCAO) to 25.71 mm/s (FJT) and step length by approximately 21.14 %. Furthermore, FJT enhanced MEP conduction efficiency and promoted CST remodeling, characterized by a 5.26 % increase in BDA-positive nerve fibers and elevated growth-associated protein 43 (GAP43) expression in the denervated hemisphere. At the molecular level, FJT upregulated the mRNA and protein expressions of Pcsk1 and Furin, increased the levels of BDNF and its functional receptor TrkB, and downregulated the expressions of proBDNF-preferring receptors p75NTR and Sortilin, ultimately shifting the proBDNF/mBDNF ratio toward mBDNF dominance. BDNF silencing significantly attenuated these improvements, reversing FJT-induced motor recovery and CST remodeling. In vitro, FJT-promoted neuronal growth was mimicked by exogenous mBDNF but reversed by Pcsk1/Furin inhibitors. Compared with the MCAO model group, medium-dose FJT exhibited the most significant therapeutic effects. Specifically, FJT notably improved gait parameters, increasing the average speed from 20.77 mm/s (MCAO) to 25.71 mm/s (FJT) and step length by approximately 21.14 %. Furthermore, FJT enhanced MEP conduction efficiency and promoted CST remodeling, characterized by a 5.26% increase in BDA-positive nerve fibers and elevated Growth-Associated Protein 43 (GAP43) expression in the denervated hemisphere. At the molecular level, FJT upregulated the mRNA and protein expressions of Pcsk1 and Furin, increased the levels of BDNF and its functional receptor TrkB, and downregulated the expressions of proBDNF-preferring receptors p75NTR and Sortilin, ultimately shifting the proBDNF/mBDNF ratio toward mBDNF dominance. BDNF silencing significantly attenuated these improvements, reversing FJT-induced motor recovery and CST remodeling. In vitro, FJT-promoted neuronal growth was mimicked by exogenous mBDNF but reversed by Pcsk1/Furin inhibitors.

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