银杏内酯B通过调控脑内T细胞特性及与胶质细胞间相互作用促进缺血性脑卒中小鼠的神经功能恢复

Ginkgolide B Promotes Neural Function Recovery of Ischemic Stroke Mice by Regulating Characteristics of Brain T Cells and Their Interactions with Glial Cells

目的探究银杏内酯B调控缺血性脑卒中恢复期小鼠的脑内T细胞生物学特性及T细胞与胶质细胞间的作用机制。方法选取36只成年C57BL/6小鼠,随机分为假手术组(Sham组)、对照组(PBS组)和银杏内酯B组(GB组)。Sham组仅给予假手术处理;PBS组和GB组均采用线栓法制备大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)损伤即缺血性脑卒中模型,并于损伤后连续14 d分别鼻饲等体积的PBS和银杏内酯B溶液。采用转棒实验及神经功能评分法评估3组小鼠的神经功能变化;并于实验第15天取PBS组和GB组小鼠脑损伤区及周边皮层、胼胝体及纹状体的新鲜组织,采用单细胞测序方法评估该区域中T细胞及其亚群的生物学特性,并进一步探索T细胞、小胶质细胞和少突胶质细胞之间的相互作用及机制。结果与Sham组相比,PBS组和GB组小鼠的神经功能评分均显著上升(P<0.001),掉落前运动时程均显著降低(P<0.001);与PBS组相比,GB组在缺血性脑损伤后5、10、15d的神经功能评分有下降趋势,掉落前运动时程有上升趋势,尤其15d时的掉落前运动时程显著上升(P<0.05)。与PBS组相比,GB组小鼠在脑损伤后15d时脑内T细胞增殖活性显著升高(P<0.05),增殖性T细胞数量及脂质代谢水平均显著上调(P<0.05),所有T细胞的细胞外基质重塑显著增多(P<0.05);同时,GB组小鼠脑内T细胞与小胶质细胞、少突胶质细胞之间的相互作用以及小胶质细胞自身、小胶质细胞与少突胶质细胞之间的相互作用均显著增强,主要表现为CD74与巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)、集落刺激因子1受体(colony stimulating factor 1 receptor,CSF1R)与集落刺激因子1(colony stimulating factor 1,CSF1)的相互作用增强(P<0.05),但T细胞的炎性水平与PBS组相比均无显著差异。结论采用线栓法MCAO损伤手术可成功构建小鼠缺血性脑卒中疾病模型。银杏内酯B可能通过调控小鼠脑内T细胞生物学特性及其与胶质细胞间的互作关系,促进小鼠脑损伤后的神经功能恢复。

Objective To investigate the regulatory effects of Ginkgolide B on the biological characteristics of brain T cells and their interactions with glial cells during the recovery phase of ischemic stroke in mice.Methods 36 adult C57BL/6 mice were randomly assigned to three groups:sham-operated group(Sham group),control group(PBS group),and Ginkgolide B treatment group(GB group).The Sham group underwent only sham surgeries,whereas the PBS and GB groups were subjected to a middle cerebral artery occlusion(MCAO)model using the filament method,followed by intranasal administration of an equivalent volume of either PBS or Ginkgolide B solution for 14 days post-injury.Neurological function changes were evaluated in all three groups using the rotarod test and a neurological scoring system.On day 15,single-cell sequencing was performed on fresh tissues from the brain injury areas,surrounding cortex,corpus callosum,and striatum of mice in the PBS and GB group to assess the biological characteristics of T cells and their subpopulations,and further explore the interactions and mechanisms among T cells,microglia,and oligodendrocytes.Results Compared with the Sham group,both PBS and GB group exhibited significant improvements in neurological scores and reduced pre-fall motor durations(P<0.001).Compared with the PBS group,the GB group showed a downward trend in neurological scores and an upward trend in pre-fall motor durations on days 5,10,and 15 post-ischemic brain injury,with a significant increase in pre-fall motor duration on day 15(P<0.05).Compared with the PBS group,the GB group exhibited a significant increase in T cell proliferative activity in the brain 15 days post brain injury(P<0.05).The number of proliferative T cells and the levels of lipid metabolism were significantly elevated(P<0.05),and there was a significant increase in extracellular matrix remodeling in all T cells(P<0.05).Additionally,the interactions between T cells and both microglia and oligodendrocytes,as well as among the microglia themselves and between microglia and oligodendrocytes,were significantly enhanced in the GB group.This was primarily evident in the strengthened interactions between CD74 and macrophage migration inhibitory factor(MIF),as well as colony stimulating factor 1 receptor(CSF1R)and colony stimulating factor 1(CSF1)(P<0.05).However,the inflammatory levels of T cells showed no significant differences compared with the PBS group.Conclusion A mouse model of ischemic stroke can be successfully established by MCAO operation.Ginkgolide B may promote neurological recovery post-brain injury in mice by modulating the biological characteristics of T cells within the brain and their interactions with glial cells.