文献精读｜鼠神经生长因子通过调节磷酸肌醇-3-激酶/丝氨酸/苏氨酸激酶信号通路抑制丙戊酸诱导的自闭症谱系障碍大鼠神经元凋亡
文摘
健康
2024-08-16 11:00
湖北
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题目译名：鼠神经生长因子通过调节磷酸肌醇-3-激酶/丝氨酸/苏氨酸激酶信号通路抑制丙戊酸诱导的自闭症谱系障碍大鼠神经元凋亡
发表期刊：Pharmacogenetics and Genomics （药物遗传学和基因组学）
发表刊号：2023年第33卷第5期
观点提炼：
1. 疗效明确：mNGF改善了VPA（丙戊酸）诱发的ASD（孤独症）大鼠的运动活动、探索行为、社会交流以及空间学习和记忆。
2. 机制探讨：mNGF通过激活PI3K / Akt信号通路抑制VPA诱导的ASD大鼠神经元凋亡并改善异常行为。
3. 以上指标改善体现于试验动物的旷场实验结果、western blot分析以及组织染色后的图片结果。
自闭症谱系障碍（ASD）是一组神经发育障碍，其特征是社交沟通缺陷和限制行为。鼠神经生长因子（mNGF）是一种神经营养因子，对神经元的生长和存活至关重要，mNGF治疗被认为是神经变性的有前途的治疗方法。有鉴于此，研究者旨在评估mNGF对ASD神经功能的影响。自闭症谱系障碍（ASD），也称为自闭症，发生在儿童早期，被归类为与先天性脑部疾病相关的神经发育障碍。ASD的临床诊断主要基于刻板和重复行为、言语交流受损和社交能力受损。
氧化应激被认为在ASD的发病机制中起重要作用。自闭症儿童血清中SOD、过氧化氢酶和谷胱甘肽的活性降低。此外，氧化应激可在多种疾病中诱导细胞凋亡。ASD与神经元成熟、分化、迁移和凋亡有关。目前的证据表明，异常神经元凋亡是ASD病理过程的重要机制之一。抑制ASD大鼠神经元凋亡可改善社会交际功能障碍，改善学习和记忆能力，因此，探讨ASD神经元凋亡的潜在机制具有重要意义。
神经营养因子（NTF）是促进神经元修复和再生的调节蛋白。NTF包括神经生长因子（NGF)、脑源性神经营养因子、神经营养因子-3和神经营养因子-4/5。其中NGF广泛分布于包括大脑在内的全身，可促进中枢和周围神经系统的神经元生长、发育、分化和成熟。在神经损伤中，外源性NGF可通过受损的轴突逆行转运至神经细胞，滋养神经元，促进轴突延伸和髓鞘形成，促进神经纤维再生，降低神经损伤程度，加速神经恢复。NGF的潜在作用机制明确，是治疗全面性发育迟缓（GDD）的合理药物选择，全面性发育迟缓是一种早发性慢性神经系统疾病，常见于婴儿期和幼儿期。一些研究报告说，NGF可以治疗儿童发育障碍并改善运动功能。例如，Xu等发现NGF联合Bobath法治疗运动发育迟缓儿童可促进运动发育和运动功能恢复。Zhao等发现NGF联合康复治疗可有效改善脑瘫（CP）患儿的运动功能，且比单纯的康复训练有更显著的改善效果。鼠神经生长因子（mNGF）注射剂是市场上第一款NGF产品。mNGF与人类NGF具有90%的同源性，具有高安全性和低毒性。并且长期使用mNGF对器官系统没有明显的毒副作用。mNGF已被证明在临床神经损伤的治疗中发挥多种生物学功能，如神经营养和神经保护作用。在CP患儿中，在康复治疗中加用穴位注射mNGF可以更有效地减少血液乳酸水平和改善运动功能。Yin等人的一项研究发现，mNGF治疗可显著促进缺氧缺血性脑损伤新生大鼠的海马神经发生并抑制神经元凋亡；然而，mNGF是否抑制神经元凋亡并缓解ASD尚不清楚。此外 有报道显示，NGF在坐骨神经损伤中起神经保护作用，基于这些发现，本研究旨在探讨mNGF对丙戌酸（VPA）诱导的ASD大鼠神经元凋亡的作用和机制。通过腹腔注射丙戊酸（VPA）建立ASD大鼠模型。测量大鼠的社会行为，学习和记忆。利用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）染色以检测海马体和前额叶皮层中的神经元凋亡和存活。检测细胞凋亡相关蛋白和氧化应激标志物。1.鼠神经生长因子改善自闭症谱系障碍大鼠的社会行为![]()
旷场试验结果显示，与假手术组相比，VPA组减少了越线次数和后肢站立次数（均p＜0.05），与VPA组相比，VPA+mNGF组恢复了这些水平（均p＜0.05，图2a和b）。三箱社交实验显示，与假手术组相比，VPA组幼鼠与陌生大鼠在腔室中的时间更少（p＜0.05），而VPA+mNGF处理逆转了这种变化（p＜0.05，图2c）。此外，VPA+mNGF处理恢复了被VPA处理降低的社交指数（p＜0.05，图2d）。
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结果还表明，VPA组中的幼鼠在陌生大鼠的房间里比在熟悉的大鼠房间里花费的时间更少。而VPA+mNGF组部分逆转了这些变化（p＜0.05，图2e）。同时，VPA+mNGF处理使VPA诱导的社会偏好指数下降增加（p＜0.05，图2f）。Y-迷宫测试结果表明，与VPA组相比，VPA+mNGF处理增加了自发变化（p＜0.05，图2g）.此外，Morris水迷宫测试结果显示，VPA组的逃避潜伏期没有显著变化，而VPA+mNGF组的逃避潜伏期呈时间依赖性下降（p＜0.05，图2h）.此外，与VPA组相比，VPA+mNGF处理增加了在目标方块中花费的时间（p＜0.05，图2i）2.鼠神经生长因子抑制丙戌酸诱导的自闭症谱系障碍大鼠神经细胞凋亡
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TUNEL结果显示VPA促进大鼠海马和前额皮质细胞凋亡，而VPA+mNGF处理抑制这种作用（图3a和b ）
尼氏染色结果显示，假手术组海马和前额叶皮层神经元轮廓清晰，结构紧凑，胞质和体丰富，神经元细胞活力高，而VPA组神经元活力受损，细胞轮廓模糊不清。重要的是，VPA+mNGF处理改善了VPA诱导的神经元损伤（图3c和d）.
Western blotting结果表明，VPA诱导降低了大鼠海马和前额叶皮质中Bcl-2, p-PI3K 和 p-Akt 的水平，并增加了Bax和cleaved caspase-3的水平，而VPA+mNGF处理逆转了这些变化（图3e和f）。同时，VPA+mNGF处理显著逆转了VPA诱导的氧化应激和炎症的反应。VPA+mNGF处理还降低了VPA处理升高的丙二醇水平，降低了p-MAPK水平的升高。
3.鼠神经生长因子通过调节磷酸肌醇-3-激酶/Akt信号通路减少丙戌酸诱导的神经细胞凋亡研究者进一步使用PI3K抑制剂LY294002 来评估mNGF是否通过激活PI3K/Akt 信号通路来抑制VPA诱导的ASD大鼠细胞凋亡。![]()
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试验结果表明，与VPA组相比，VPA+mNGF处理增加了越线次数和后肢站立行为，而LY294002消除了这些影响（（p＜0.05，图5a）b）TUNEL结果显示VPA+mNGF 处理阻止了VPA诱导的海马体和前额皮质细胞凋亡，而LY294002消除了这种作用（图5c）。尼氏染色结果表明，VPA组的细胞在海马体和前额皮质中表现出活力受损和细胞轮廓模糊，很可能为VPA+ mNGF处理改善了这种神经元损伤（图5d）
在这项研究中，研究者发现mNGF治疗改善了VPA诱导的ASD大鼠的运动活动、探索行为、社会交流以及空间学习和记忆。与VPA相比，mNGF抑制神经元凋亡，增加大鼠海马和前额叶皮质神经元数量。mNGF增加SOD和谷胱甘肽的水平，降低海马和前额叶皮质中ROS、一氧化氮、TNF-α,和IL-1β的水平。此外mNGF增加了Bcl-2,p-PI3K和p-Akt的水平，并降低了Bax和裂解的caspase-3的水平，而PI3K抑制剂LY294002逆转了这些影响。这些数据表明mNGF通过激活p-PI3K/Akt信号通路抑制神经元凋亡并改善VPA诱导的ASD的异常行为。mNGF是一种相对分子质量为26.5KDa的生物活性蛋白，主要提取自小鼠颌下腺。mNGF的发现对神经生物学的建立具有重要意义。目前，mNGF常用于神经损伤的临床治疗。总之这项研究表明，mNGF改善了VPA诱发的ASD大鼠的运动活动、探索行为、社会交流以及空间学习和记忆。进一步的结果表明，mNGF通过活p-PI3K/Akt信号通路抑制神经元凋亡，mNGF在ASD中调节PI3K/Akt信号通路的确切机制还需要进一步的研究来证实。1 Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum
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