银杏平颤方对帕金森病模型小鼠多巴胺神经元丢失及其凋亡的影响

Effects of Ginkgo biloba Pingchan Recipe on loss and apoptosis of dopamine neurons in mouse models of Parkinson's disease

背景:现代药理研究证明银杏叶及其提取物生物具有抗氧化作用以及刺激神经生长因子产生等保护多巴胺神经元的存活,减少其凋亡。目的:验证银杏平颤方对帕金森病小鼠多巴胺神经元丢失及其凋亡的影响以及的神经母细胞瘤衍生细胞株SH-SY5Y的增殖情况的影响。方法:(1)C57BL小鼠随机分成3组。正常组10只不处理。其他2组小鼠每天腹腔注射MPTP建立小鼠帕金森病的模型,连续6周;每次腹腔注射前30 min进行灌胃处理,模型组10只灌胃生理盐水;治疗组10只灌胃中药银杏平颤方。分别在造模开始后的第15天和第30天取脑组织,免疫组织化学观察中脑黑质多巴胺神经元丢失和细胞凋亡情况;(2)体外培养神经母细胞瘤衍生细胞株SH-SY5Y并用银杏平颤方处理细胞,MTT法检测人SH-SY5Y的增殖,流式细胞术检测细胞凋亡情况;Real-time PCR和Western blotting检测细胞PARP、PTEN mR NA和蛋白质表达。结果与结论:(1)动物实验结果:正常组小鼠的中脑黑质致密部酪氨酸羟化酶阳性神经元的数目多于模型组,并存在时间依赖关系;银杏平颤方中药治疗组小鼠中脑黑质致密部酪氨酸羟化酶阳性神经元的数目多于模型组(P<0.05);(2)体外细胞实验结果:银杏平颤方处理的细胞凋亡率低于模型组;银杏平颤方处理细胞的PARP、PTEN的mR NA及蛋白水平均低于模型组。(3)结果说明,银杏平颤方治疗可在一定程度上抑制中脑神经元细胞的凋亡;其分子机制可能是银杏平颤方通过抑制PTEN表达进而降低细胞凋亡水平,保护多巴胺神经元,阻止其丢失,起到防治帕金森病的作用。

BACKGROUND： Current pharmacological research has administrated that Ginkgo biloba and its extracts can eliminate loss and apoptosis of dopamine neurons by antioxidation and nerve growth factor activation. OBJECTIVE： To confirm the effects of Ginkgo biloba Pingchan Recipe on loss and apoptosis of dopamine neurons as well as proliferation of neuroblastoma derived cell lines SH-SY5 Y in mice with Parkinson＇s disease. METHODS：（1） C57 BL mice were randomly divided into three groups： 20 mice were modeled into Parkinson＇s disease by intraperitoneal injection of MPTP for 6 weeks, and at 30 minutes before each intraperitoneal injection, mice in model group were given gavage of normal saline, mice in treatment group given Ginkgo biloba Pingchan Recipe. The other 10 mice received no any interventions as normal group. The loss and apoptosis of dopamine neurons were observed by immunohistochemistry at 15 and 30 days after modeling.（2） In vitro cultured SH-SY5 Y cell lines were treated with Ginkgo biloba Pingchan Recipe. Subsequently, the cell proliferation and apoptosis were detected using MTT assay and flow cytometry, respectively; PARP and PTEN m RNA and protein levels were measured through real-time PCR and western blot assay. RESULTS AND CONCLUSION：（1） Compared with the model group, the number of tyrosine hydroxylase-positive neurons at the substantia nigra compacta of midbrain was significantly increased in a time-dependant manner in the normal and treatment groups（P 〈0.05）.（2） The apoptosis rate and m RNA and protein levels of PARP and PTEN in the treatment group were lower than those in the model group. These results suggest that Ginkgo biloba Pingchan Recipe can partly inhibit midbrain neuron apoptosis that may be by decreasing PTEN level. As a result, it becomes possible to prevent and treat Parkinson＇s disease via protecting dopamine neurons from loss.