2020 年第 8 期 第 15 卷

银杏内酯对阿尔茨海默病细胞模型细胞活性的影响及其作用机制研究

Effect of ginkgolide on cell viability of Alzheimer′s disease cell model and its mechanism 

作者：殷贺1 许保磊1 牛天童1 杨亭亭1 李晓晴1 刘广志1 李慧琴2 孙毅2

英文作者：Yin He1 Xu Baolei1 Niu Tiantong1 Yang Tingting1 Li Xiaoqing1 Liu Guangzhi1 Li Huiqin2 Sun Yi2

单位：1首都医科大学附属北京安贞医院神经内科100029；2成都百裕制药股份有限公司研发中心610041

英文单位：1Department of Neurology Beijing Anzhen Hospital Capital Medical University Beijing 100029 China;  2Research and Development Centre Chengdu Baiyu Pharmaceutical Co. Ltd. Chengdu 610041 China

关键词：阿尔茨海默病；银杏内酯；核因子κB信号通路；细胞凋亡；细胞因子

英文关键词：Alzheimer′sdisease;Ginkgolide;Nuclearfactor-κBsignalingpathway;Apoptosis;Cytokine 

• 摘要：

• 目的 探讨银杏内酯及其成分（白果内酯及银杏内酯B）对阿尔茨海默病（AD）细胞模型APP-PS1双基因转染HEK293细胞（APP/PS1-HEK293）活性的影响及其作用机制。方法 以不同剂量银杏内酯处理APP/PS1-HEK293，经细胞计数盒8分析筛选出其促进细胞增殖的最佳时间点及最佳浓度。在此基础上，以未经处理的APP/PS1-HEK293作为对照组，将不同浓度银杏内酯及其组分（银杏内酯B、白果内酯）处理的APP/PS1-HEK293作为观察组，体外培养适当时间后，检测上述各组上清液肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β和IL-6水平，同时检测细胞内核因子κB（NF-κB）信号通路组分中的NF-κBp65和NF-κB抑制因子α（IκBα）以及凋亡相关分子B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）的蛋白和mRNA表达水平。结果 APP/PS1-HEK293在经浓度为100 mg/L的银杏内酯处理48 h后细胞活性最高，为（135.1±2.4）%，选取100 mg/L作为最佳药物浓度。银杏内酯高剂量（100 mg/L）组、银杏内酯B组、白果内酯组上清液TNF-α、IL-1β和IL-6的表达均明显低于对照组［（57.8±3.7）、（41.4±2.9）、（48.4±1.7）ng/L比（67.8±2.0）ng/L，（12.45±0.19）、（8.29±0.16）、（10.48±0.16）ng/L比（18.31±0.24）ng/L，（118.4±2.1）、（105.9±0.3）、（112.0±0.7）ng/L比（172.8±4.4）ng/L］，而银杏内酯低剂量（50 mg/L）组上述3项指标均明显高于对照组，差异均有统计学意义（均P<0.01）。低和高剂量银杏内酯组、银杏内酯B组、白果内酯组细胞内NF-κBp65、Bax蛋白表达水平显著低于对照组，而Bcl-2、IκBa蛋白表达水平则显著高于对照组，差异均有统计学意义（P<0.05或P<0.01）。银杏内酯低和高剂量、银杏内酯B、白果内酯组细胞内NF-κBp65、Bax mRNA表达水平均显著低于对照组、IκBa mRNA表达显著高于对照组，而且银杏内酯低和高剂量组Bcl-2 mRNA表达水平亦显著高于对照组，差异均有统计学意义（P<0.05或P<0.01）。结论 银杏内酯能明显提高APP/PS1-HEK293的增殖活性，可能系银杏内酯及其组分（白果内酯及银杏内酯B）抑制NF-κB信号通路的激活，进而或同时促进抗细胞凋亡和减少炎症因子的释放所致。

• Objective To investigate the effect of ginkgolide and its components(ginkgolide B and bilobalide) on cell viability of Alzheimer′s disease(AD) cellular model, APP/PS1 double gene transfected HEK293 cell(APP/PS1-HEK293), and related action mechanisms. Methods APP/PS1-HEK293  was treated with different dose of ginkgolide, the optimal time point and concentration for cell proliferation were screened via cell counting kit-8 assay. Based on these experiments, in vitro study was performed using the untreated APP/PS1-HEK293 as the control group and APP/PS1-HEK293 treated with different dosage of ginkgolide components as the interventional groups. After culture in vitro for appropriate time, the level of supernatant interleukin(IL)-1β， IL-6， tumor necrosis factor-α(TNF-α) in each group was detected, as well as the intracellular protein and mRNA expression of nuclear factor κB(NF-κB)p65, inhibitor of NF-κB α(IκBa) as signaling pathway components and B cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax) as apoptosis-related molecules in each group was detected. Results The activity of APP/PS1-HEK293 cells was highest ［(135.1±2.4)%］ after treatment with ginkgolide at a concentration of 100 mg/L for 48 h, and 100 mg/L was selected as the optimal drug concentration. The supernatant levels of TNF-α, IL-1β and IL-6 in high-dosage ginkgolide(100 mg/L)-, ginkgolide B and bilobalide-treated groups were lower than those in the control group［（57.8±3.7）, （41.4±2.9）, （48.4±1.7）ng/L vs （67.8±2.0）ng/L; （12.45±0.19）, （8.29±0.16）, （10.48±0.16）ng/L vs （18.31±0.24）ng/L; （118.4±2.1）, （105.9±0.3）, （112.0±0.7）ng/L vs （172.8±4.4）ng/L］, but the above three indexes in the low dose ginkgolide group (50 mg/L) were significantly higher than those in the control group（all P<0.01）. The expression levels of intracellular NF-κBp65 and Bax in the low and high dose ginkgolide group, ginkgolide B group and ginkgolide group were significantly lower than those in the control group, while the expression levels of Bcl-2 and IκBa protein were significantly higher than those in the control group (P<0.05 or P<0.01). The expression levels of NF-κBp65 and Bax mRNA in cells of ginkgo lactone B and ginkgolide group were significantly lower than those of control group, IκBa mRNA expression was significantly higher than that of control group, and ginkgolides were low and high Bcl-2 mRNA expression level in the dose group was also significantly higher than the control group (P<0.05 or P<0.01). Conclusion Ginkgolide can significantly increase the proliferation activity of APP/PS1-HEK293. Ginkgolide and its components (ginkgolide and ginkgolide B) inhibit the activation of the NF-κB signaling pathway, which in turn may promote anti-apoptosis and reduce the release of inflammatory factors.