摘要： 目的 探讨黄芪甲苷对1-甲基-4-苯基吡啶离子（MPP⁺）诱导的帕金森病SK-N-SH细胞损伤的作用机制。方法 人神经母细胞瘤细胞系SK-N-SH培养至对数生长期，随机予以常规培养（对照组）、MPP⁺诱导（MPP⁺组）、黄芪甲苷10 mg/ml+MPP⁺诱导（黄芪甲苷10 mg/ml组）、黄芪甲苷30 mg/ml+MPP⁺诱导（黄芪甲苷30 mg/ml组）、黄芪甲苷30 mg/ml+MPP⁺诱导+Janus激酶2（JAK2）抑制剂AG490 （JAK2抑制剂组），噻唑蓝法检测细胞存活率，流式细胞术检测细胞凋亡率，DCFH-DA荧光探针测定活性氧（ROS）含量，酶法测定乳酸脱氢酶（LDH）和超氧化物歧化酶（SOD）活性，Western blotting法测定磷酸化JAK2（pJAK2）和磷酸化信号转导与转录激活因子3（pSTAT3）蛋白相对表达量。结果 不同处理组SK-N-SH细胞存活率（P=0.000）、凋亡率（P=0.000）、ROS含量（P=0.000）、LDH活性（P=0.000）、SOD活性（P=0.003）、pJAK2（P=0.000）和pSTAT3（P=0.000）蛋白相对表达量差异有统计学意义。与对照组相比，MPP⁺组、黄芪甲苷10 mg/ml组和30 mg/ml组、JAK2抑制剂组细胞存活率（P=0.000，0.001，0.049，0.000）和SOD活性（P=0.000，0.002，0.012，0.000）、pJAK2（P=0.003，0.006，0.036，0.002）和pSTAT3（P=0.001，0.002，0.024，0.001）蛋白相对表达量降低，细胞凋亡率（P=0.001，0.001，0.001，0.000）、ROS含量（P=0.000，0.001，0.002，0.000）和LDH活性（P=0.000，0.002，0.038，0.000）升高；与MPP⁺组相比，黄芪甲苷10 mg/ml组和30 mg/ml组细胞存活率（P=0.016，0.000）和SOD活性（P=0.003，0.001）、pJAK2（P=0.013，0.002）和pSTAT3（P=0.018，0.002）蛋白相对表达量升高，细胞凋亡率（P=0.021，0.008）、ROS含量（P=0.031，0.003）和LDH活性（P=0.001，0.000）降低；与黄芪甲苷10 mg/ml组相比，黄芪甲苷30 mg/ml组细胞存活率（P=0.002）和SOD活性（P=0.027）、pJAK2（P=0.007）和pSTAT3（P=0.006）蛋白相对表达量升高，ROS含量（P=0.019）和LDH活性（P=0.011）降低，JAK2抑制剂组细胞凋亡率（P=0.016）、ROS含量（P=0.030）和LDH活性（P=0.004）升高，SOD活性（P=0.004）、pJAK2（P=0.001）和pSTAT3（P=0.005）蛋白相对表达量降低；与黄芪甲苷30 mg/ml组相比，JAK2抑制剂组细胞存活率（P=0.001）、SOD活性（P=0.001）、pJAK2（P=0.000）和pSTAT3（P=0.001）蛋白相对表达量降低，凋亡率（P=0.004）、ROS含量（P=0.002）和LDH活性（P=0.001）升高。结论 黄芪甲苷可以减轻MPP⁺诱导的帕金森病SK-N-SH细胞损伤，其机制可能与激活JAK2-STAT3信号转导通路有关。

关键词: 帕金森病, 黄芪, Janus激酶2, 转录激活因子3, 肿瘤细胞, 培养的

Abstract: Objective To investigate the effect of astragaloside Ⅳ on the injury of SK-N-SH cell in Parkinson's disease (PD) induced by 1-methy-4-phenylpyridine (MPP⁺) and its mechanism. Methods Human neuroblastoma cell line SK-N-SH was cultured to logarithmic growth stage, which were randomized to routine culture (control group), MPP⁺ induction (MPP⁺ group), astragaloside Ⅳ 10 mg/ml + MPP⁺ induction (astragaloside Ⅳ 10 mg/ml group), astragaloside Ⅳ 30 mg/ml + MPP⁺ induction (astragaloside Ⅳ 30 mg/ml group), astragaloside Ⅳ 30 mg/ml + MPP⁺ induction + Janus kinase 2 (JAK2) inhibitor AG490 (JAK2 inhibitor group). Cell survival was detected by methyl thiazolyl tetrazolium (MTT) assay, cell apoptosis was detected by flow cytometry, reactive oxygen species (ROS) level was detected by DCFH-DA fluorescence probe, lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities were detected by enzyme method. And the relative expression levels of phosphorylated Janus kinase 2 (pJAK2) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) proteins in JAK2-STAT3 signal transducer pathway were detected by Western blotting. Results There were significant differences in cell survival rate (P=0.000), apoptosis rate (P=0.000), ROS content (P=0.000), LDH activity (P=0.000), SOD activity (P=0.003), pJAK2 (P=0.000) and pSTAT3 (P=0.000) proteins relative expression levels among different SK-N-SH groups. Further pairwise comparison showed that compared with the control group, the cell survival rate (P=0.000, 0.001, 0.049, 0.000), SOD activity (P=0.000, 0.002, 0.012, 0.000), relative expression levels of pJAK2 (P=0.003, 0.006, 0.036, 0.002) and pSTAT3 (P=0.001, 0.002, 0.024, 0.001) proteins were decreased in MPP⁺ group, astragaloside Ⅳ 10 mg/ml and 30 mg/ml groups, JAK2 inhibitor group, but the cell apoptosis rate (P=0.001, 0.001, 0.001, 0.000), ROS content (P=0.000, 0.001, 0.002, 0.000) and LDH activity (P=0.000, 0.002, 0.038, 0.000) were increased. Compared with MPP⁺ group, the survival rate (P=0.016, 0.000), SOD activity (P=0.003, 0.001), relative expression of pJAK2 (P=0.013, 0.002) and pSTAT3 (P=0.018, 0.002) proteins increased in astragaloside Ⅳ 10 mg/ml and 30 mg/ml groups, which the apoptosis rate (P=0.021, 0.008), ROS content (P=0.031, 0.003) and LDH activity (P=0.001, 0.000) decreased. Compared with astragaloside Ⅳ 10 mg/ml group, the cell survival rate (P=0.002), SOD activity (P=0.027), relative expression of pJAK2 (P=0.007) and pSTAT3 (P=0.006) proteins were increased in astragaloside Ⅳ 30 mg/ml group, but ROS content (P=0.019) and LDH activity (P=0.011) decreased, while apoptosis rate (P=0.016), ROS content (P=0.030) and LDH activity (P=0.004) increased in JAK2 inhibitor group, and SOD activity (P=0.004), the relative expression of pJAK2 (P=0.001) and pSTAT3 (P=0.005) proteins were decreased. Compared with astragaloside Ⅳ 30 mg/ml group, cell survival rate (P=0.001), SOD activity (P=0.001), the relative expression of pJAK2 (P=0.000) and pSTAT3 (P=0.001) proteins in JAK2 inhibitor group were decreased, but the apoptosis rate (P=0.004), ROS content (P=0.002) and LDH activity (P=0.001) were increased. Conclusions Astragaloside Ⅳ can reduce MPP⁺ induced SK-N-SH cell injury in PD, which may be related to the activation of JAK2-STAT3 signal transducer pathway.

Key words: Parkinson disease, Astragalus membranaceus, Janus kinase 2, Activating transcription factor 3, Tumor cells, cultured