Wnt/β-catenin信号通路调控脑干出血后少突胶质细胞分化、髓鞘再生的作用机制研究

Brainstem hemorrhage(BSH) complicated with the highest mortality and morbidity among all types of stroke. White matter injury plays an important role in neurological deficits and no efficient rehabilitative treatment is available. Wnt/β-catenin signaling is involved in oligodendrocyte precursor cell (OPC) differentiation and remyelination, but its roles in white matter restoration after stroke remain to be elucidated. In our previous study, we created the brainstem hemorrhage model by autologous blood infusion in rat where diffuse demyelination and oligodendrocyte death were identified which correlated with neurobehavioral deterioration. Additionally, the preliminary experiment found that inhibition of β-catenin promoted OPC differentiation, indicating that Wnt/β-catenin signaling is related to the white matter repair following intracerebral hemorrhage. In this research, we will observe the dynamic expression of key factors of Wnt/β-catenin signaling in the BSH rat model. Then by activating or inhibiting the key protein of the signal pathway, we further define the relationship between Wnt/β-catenin signaling and OPC differentiation and remyelination following BSH through immunohistochemistry, functional MRI and neurobehavioral assessment. This study will reveal the role of Wnt/β-catenin signaling in the remyelination after BSH and provide a potential treatment target to accelerate rehabilitation after stroke.

脑干出血在所有脑卒中类型中死亡率及致残率居于首位，出血后脑白质损伤在神经功能废损中起重要作用，缺乏有效的神经康复措施。Wnt/β-catenin信号通路参与少突胶质细胞前体细胞（OPC）分化、髓鞘再生，但在脑卒中后对脑白质的修复作用不清楚。前期研究中我们构建自体血脑干出血大鼠模型，发现血肿周围存在广泛脱髓鞘及少突胶质细胞死亡，与行为学障碍相关。此外，预实验发现抑制β-catenin促进出血后OPC分化，减轻神经功能废损，提示Wnt/β-catenin通路与脑出血后白质修复有关。本课题拟在脑干出血模型中动态观察Wnt通路关键分子的表达，通过激活或抑制通路关键蛋白，采用组织学、分子生物学、影像学、行为学等手段明确Wnt/β-catenin通路与髓鞘再生关系，揭示Wnt/β-catenin在脑干出血后髓鞘再生中的作用机制，为神经康复治疗提供新的靶点, 为研究在其他类型脑卒中的作用奠定基础。

脑干出血（BSH）后幸存患者神经功能障碍与脑白质损伤(WMI)有关，促进神经纤维髓鞘再生有利于神经康复。Wnt/β-catenin信号通路参与少突胶质细胞前体细胞（OPC）分化、髓鞘再生，前期研究中我们脑干出血后血肿周围存在广泛脱髓鞘及少突胶质细胞死亡，与行为学障碍相关；预实验也提示Wnt/β-catenin通路与脑出血后白质修复有关。本项目首先通过构建大鼠脑干出血模型，利用Western blot、实时荧光定量RT-PCR等技术动态定量血肿周围脑白质中Wnt/β-catenin通路分子β-catenin、GSK-3β蛋白及基因表达情况，发现Wnt/β-catenin信号通路与脱髓鞘改变及神经功能障碍时序性明显相关，即GSK-3β表达在出血3天降至低谷，与脱髓鞘改变负相关，而β-catenin表达在7天时上升至高峰，与脱髓鞘改变呈正相关；同时发现β-catenin以少突胶质细胞表达为主。提示经典Wnt信号通路中，β-catenin表达可能与脑干出血后脱髓鞘及髓鞘再生有关。随后，构建β-catenin RNA干扰慢病毒抑制Wnt信号通路，使用GSK-3β特异性抑制剂AR-A014418抑制GSK-3β表达，激活β-catenin依赖的Wnt通路，结合超场强MRI（7T）多参数动态检测血肿周围脱髓鞘改变，发现β-catenin RNA慢病毒干扰后OPC细胞分化明显受阻（OL阳性细胞数明显减少），出现明显的脱髓鞘改变（FA值及λ⊥值均明显下降，MBP蛋白表达显著下降），出现明显的神经功能障碍；AR-A014418注射组OPC分化数明显增加且表现为显著的髓鞘再生过程（FA值及λ⊥值均明显增加，MBP蛋白表达显著增加），神经功能障碍明显改善。上述结果证实经典Wnt通路对脑干出血后OPC细胞分化及髓鞘再生具有正性调控作用，GSK-3β通过磷酸化降解β-catenin抑制OPC细胞分化及髓鞘再生。激活经典Wnt通路可以促进脑干出血后髓鞘再生，改善神经功能预后。