HDAC4在大鼠脑梗死后骨骼肌及神经肌肉接头继发性改变中的作用机制研究

Secondary changes in skeletal muscle and neuromuscular junctions after stroke are closely related to the recovery of motor function, but the mechanisms are not clear enough. Denervation is one of the major causes of muscle atrophy after stroke. The applicant confirmed the pathological damage of the spinal cord and anterior root after cerebral infarction previously, and will further focus on the secondary changes of denervated skeletal muscle and neuromuscular junction and the related mechanisms. Histone deacetylase 4 (HDAC4) is a key regulator of denervated muscle atrophy and neuromuscular junction changes caused by motor neuron disease and peripheral nerve injury, possibly plays an important role in secondary changes of skeletal and neuromuscular junctions post-stroke. In the pre-experiment, the denervated morphological changes of muscle fibers and neuromuscular junctions in rat MCAO were observed, so did the upregulation of the expression of HDAC4 and its downstream molecules. Based on this, this study intends to observe the secondary changes of skeletal muscle and neuromuscular junction after MCAO in rats dynamically, and further explore the mechanisms of HDAC4 involved by gene suppression/overexpression, looking for new targets for stroke treatment and rehabilitation.

卒中后骨骼肌及神经肌肉接头继发性改变与运动功能损伤恢复密切相关，但发生机制不够清晰。失神经支配是卒中后肌萎缩的重要原因，申请人前期研究明确了动物脑梗死后脊髓及脊神经前根的病理损害，拟进一步关注失神经支配骨骼肌与神经肌肉接头继发性改变及相关机制。组蛋白去乙酰化酶4（HDAC4）是运动神经元病及周围神经损伤所致失神经支配肌萎缩及神经肌肉接头改变的关键调节因子，可能在卒中后骨骼肌与神经肌肉接头继发性改变中也发挥重要作用。预实验中大鼠MCAO后肌纤维及神经肌肉接头发生失神经支配病理改变，同时骨骼肌HDAC4及其下游分子表达上调。本研究拟在此基础上动态观察大鼠MCAO后骨骼肌与神经肌肉接头的继发性改变，并通过基因抑制/过表达深入探索HDAC4在其中的作用机制，为卒中治疗与康复寻找新的分子靶点。

卒中后骨骼肌及神经肌肉接头继发性改变与运动功能损伤恢复密切相关，但发生机制不够清晰。失神经支配是卒中后肌萎缩的重要原因。组蛋白去乙酰化酶4（histone deacetylases-4，HDAC4）是运动神经元病及周围神经损伤所致失神经支配肌萎缩及神经肌肉接头改变的关键调节因子，可能在卒中后骨骼肌与神经肌肉接头继发性改变中也发挥重要作用。本研究动态观察了大鼠大脑中动脉闭塞（middle cerebral artery occlusion，MCAO）后骨骼肌与神经肌肉接头的继发性改变，以及HDAC4与下游dach2-myogenin、MAP-AP1及UP1通路分子，肌萎缩标志蛋白MuRF1和Atrogin-1，以及AChR各亚基表达变化；并通过基因抑制/过表达HDAC4进一步观察其对下游分子及肌肉和NMJs的影响。研究发现：（1）与非瘫痪侧比较，脑梗死后1、4、8、12周，瘫痪侧肌肉重量、肌纤维横截面积均减小，失神经支配NMJs占比增多，差异均具有显著性意义，且失神经支配NMJs占比与神经功能评分具有相关性；（2）与非瘫痪侧比较，脑梗死后1、4周，瘫痪侧肌肉中HDAC4与下游dach2-myogenin，以及AChRγ表达均增加；脑梗死后8、12周，双侧肌肉中HDAC4、dach2-myogenin、AChRγ表达无显著性差异；（3）与对照腺相关病毒组比较，脑梗死后1周HDAC4shRNA组Longa评分减低，且瘫痪侧肌肉肌肉重量及肌纤维横截面积增大，失神经支配NMJs占比减少，HDAC4、dach2-myogenin、AChRγ表达减少，均具有显著性差异；（4）与对照腺相关病毒组比较，脑梗死后1周HDAC4过表达组Longa评分增高，瘫痪侧肌肉肌肉重量及肌纤维横截面积减小，失神经支配NMJs占比增多，HDAC4、dach2-myogenin、AChRγ表达增加，均具有显著性差异。综上，脑梗死后瘫痪侧肌肉发生失神经支配性肌萎缩，HDAC4在脑梗死后失神经支配性肌萎缩及运动功能障碍中发挥重要负性作用，抑制其表达可减少其相关下游分子表达，并减轻肌萎缩及运动功能障碍。其有望成为卒中治疗与康复的新的分子靶点。

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