银杏内酯B对肌萎缩侧索硬化细胞模型的保护作用及其机制

目的:探讨银杏内酯B(ginkgolide B,GB)对肌萎缩侧索硬化细胞模型c-Jun氨基末端激酶(JNK)信号通路及细胞凋亡的影响。方法:通过含过表达人突变超氧化物歧化酶1(SOD1)~(G93A)基因(hSOD1~(G93A))和过表达人野生SODWT基因(hSOD1WT)与空质粒的慢病毒感染NSC34细胞,经一定浓度的嘌呤霉素筛选,倒置荧光显微镜下观察慢病毒的转染效率和细胞形态的变化,蛋白免疫印迹法(Western blot)检验感染细胞是否过表达SOD1蛋白,建立hSOD1~(G93A)-NSC34细胞系后给予GB,细胞培养分组为正常组、模型组、不同浓度GB组(25,50,75,100 mg·L-1)组,48 h后噻唑蓝(MTT)比色法检测细胞存活率,筛选出最佳药物浓度,后续实验分组为以正常组,模型组,75 mg?L~(-1) GB组,SP600125组,75 mg?L-1GB+SP600125组,流式细胞术检测各组细胞的凋亡率,蛋白免疫印迹法(Western blot)检测磷酸化(p-)JNK,c-Jun,p-c-Jun,半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白的表达。结果:与正常的NSC34细胞比较,hSOD1~(G93A)NSC34组细胞胞体变圆,突触减少、变短,而hSOD1WT NSC34组细胞和空质粒组细胞形态学未发生明显变化,与正常组比较,hSOD1~(G93A)NSC34组,hSOD1WTNSC3组细胞内SOD1蛋白水平显著升高(P0. 01),肌萎缩侧索硬化(ALS)细胞模型成功建立。与正常组比较,模型组细胞存活率显著降低(P0. 01);与模型组比较,给予不同浓度GB后,细胞存活率均显著升高(P0. 01),药物质量浓度为75 mg?L-1时细胞存活率显著升高(P0. 01)。后续实验,与正常组比较,模型组凋亡率,p-JNK,p-c-Jun,cleaved Caspase-3蛋白表达量显著升高(P0. 01);与模型组比较,75 mg?L-1GB组,SP600125组,75 mg?L-1GB+SP600125组凋亡率,p-JNK,p-c-Jun,cleaved Caspase-3蛋白表达明显降低(P0. 05,P0. 01)。结论:GB对肌萎缩侧索硬化细胞模型具有抑制细胞凋亡的保护作用,这种保护作用可能是通过JNK信号通路实现的。

Protective Effect and Mechanism of Ginkgolide B on Amyotrophic Lateral Sclerosis Cell Model

Objective To investigate the effect of ginkgolide B (GB) on the activation of c-Jun aminoterminal kinase(JNK) signaling pathway and apoptosis in amyotrophic lateral sclerosis cell model.Method NSC34 cells were infected by slow virus containing expression superoxide dismutase1(SOD1)^WT and hSOD1^G93A and empty plasmid, and screened with a certain concentration of puromycin, so as to observe the transfection efficiency of slow virus and cell morphology under inverted fluorescence microscope. Western blot method was used to verify whether infected cells were over-expressing SOD1 target proteins. The hSOD1^G93A-NSC34 cell lines were established and given GB. Cell cultures were divided into normal group, model group and different concentrations of ginkgolide B groups (25, 50, 75, 100 mg?L^-1). After 48 h, methyl thiazolyl tetrazolium (MTT) was used to detect cell survival rates, and select the best drug concentration. Subsequent experimental groups were divided into normal group, model group, 75 mg?L^-1 GB group, SP600125 group, and 75 mg?L^-1 GB + SP600125 group. Flow cytometry was used to detect the apoptosis of each group of cells. Western blot was used to detect the expressions of phosphorylation(p)-JNK, c-Jun, p-c-Jun, and cysteine aspartic acid protease -3(Caspase-3) proteins.Result Compared with normal NSC34 cells, hSOD1^G93A-NSC34 cell body became round, the synapses decreased and shortened, but the cell morphology of hSOD^WT-NSC34 cell and empty plasmid group did not change significantly. Western blot showed that hSOD1^G93A-NSC34, hSOD1^WT-NSC3 intracellular SOD1 protein levels increased significantly (P<0.01), and the amyotrophic lateral sclerosis cell model was established. Compared with the normal group, the cell activity in the model group was significantly reduced (P<0.01). Compared with the model group, the cell activity increased at different concentrations of GB, especially when the drug concentration was 75 mg?L^-1 (P<0.01). In subsequent experiments, compared with the normal group, the apoptosis, and expressions of p-JNK, p-c-Jun, and cleaved Caspase-3 proteins in the model group increased significantly (P<0.01). Compared with the model group, the apoptosis and p-JNK, p-c-Jun, released Caspase-3 protein expressions of 75 mg?L^-1 GB group, SP600125 group, 75 mg?L^-1 GB + SP600125 group decreased significantly (P<0.05, P<0.01).Conclusion GB has a protective effect on the cell model of atrophy lateral sclerosis, which may be realized by JNK signal pathway.