山楂叶总黄酮调控星形胶质细胞治疗脊髓损伤的分子机制

Molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury

背景:星形胶质细胞对脊髓损伤后神经元的修复具有重要作用,有研究证明山楂叶总黄酮可促进大鼠脊髓损伤后运动功能恢复,山楂叶总黄酮是否影响星形胶质细胞促进神经元修复仍不清楚。目的:探索山楂叶总黄酮调控星形胶质细胞治疗脊髓损伤的分子机制。方法:构建脊髓星形胶质细胞与脊髓神经元transwell共培养模型,对照组为正常脊髓星形胶质细胞和正常脊髓神经元;损伤组为正常脊髓星形胶质细胞和损伤脊髓神经元;药物组为125μg/mL山楂叶总黄酮干预的正常脊髓星形胶质细胞和损伤脊髓神经元。荧光探针DCFH-DA检测脊髓神经元内活性氧的生成;实时荧光定量PCR检测脑源性神经营养因子、神经生长因子、转录因子4的表达;Western blot检测脑源性神经营养因子、神经生长因子、Wnt/β-catenin通路关键蛋白GSK-3β、phsopho-GSK-3β(ser9)、β-catenin及转录因子4的表达;免疫荧光观察Wnt/β-catenin通路关键蛋白β-catenin的表达。结果与结论:(1)损伤组活性氧生成远高于对照组(P<0.001);药物组活性氧生成低于损伤组(P<0.01),高于对照组(P<0.001);(2)药物组脑源性神经营养因子、神经生长因子基因及蛋白表达高于对照组与损伤组(P<0.01);(3)与对照组、损伤组相比,药物组phsopho-GSK-3β(ser9)蛋白表达增加(P<0.001),β-catenin蛋白表达增加(P<0.001),转录因子4基因及蛋白表达增加(P<0.01);(4)结果表明,山楂叶总黄酮可以减轻脊髓神经元的氧化损伤,激活星形胶质细胞Wnt/β-catenin通路,结合转录因子4启动转录,促进其分泌神经营养因子,实现对脊髓神经元的修复。

BACKGROUND:Astrocytes play an important role in neuronal repair after spinal cord injury.Studies have shown that total flavonoids of hawthorn leaves can promote motor function recovery after spinal cord injury in rats.Whether total flavonoids of hawthorn leaves can affect astrocytes to promote neuronal repair remains unclear.OBJECTIVE:To explore the molecular mechanism of total flavonoids of hawthorn leaves regulating astrocytes to repair spinal cord injury.METHODS:Transwell was used to construct the co-culture model of injured spinal cord neurons and spinal cord astrocytes.The control group consisted of normal spinal astrocytes and normal spinal neurons.The injured group consisted of normal astrocytes and injured spinal neurons.The drug group consisted of 125μg/mL total flavonoids of hawthorn leaves-intervened normal spinal astrocytes and injured spinal neurons.The generation of reactive oxygen species in spinal cord neurons was detected by the fluorescence probe DCFH-DA.Real-time fluorescent quantitative PCR was used to detect the expression of brainderived neurotrophic factor,nerve growth factor and transcription factor-4.The expression levels of brain-derived neurotrophic factor,nerve growth factor,Wnt/β-catenin pathway key proteins GSK-3β,phsopho-GSK-3β(ser9),β-catenin and transcription factor-4 were detected by western blot assay.The expression of key proteinβ-catenin of the Wnt/β-catenin pathway was observed by immunofluorescence method.RESULTS AND CONCLUSION:(1)The production of reactive oxygen species in the injury group was much higher than that in the control group(P<0.001).The production of reactive oxygen species in the drug group was lower than that in the injury group(P<0.01)and higher than that in the control group(P<0.001).(2)The gene expression of brain-derived neurotrophic factor and nerve growth factor in the drug group was higher than that in the control group and injury group(P<0.01).(3)Compared with the control group and the injury group,the protein expression of phsopho-GSK-3β(ser9)increased(P<0.001);β-catenin protein expression increased(P<0.001);the protein and gene expression of transcription factor 4 was increased(P<0.01)in the drug group.(4)The results showed that the total flavonoids of hawthorn leaves could alleviate the oxidative damage of spinal cord neurons,activate the Wnt/β-catenin pathway of astrocytes,combine with transcription factor 4 to start transcription,promote its secretion of neurotrophic factors,and realize the repair of spinal cord neurons.