| 摘要: |
| ［摘要］ 目的 探讨银杏叶提取物EGb761改善结直肠癌（CRC）细胞对顺铂（DDP）敏感性的机制。方法 选择人CRC细胞RKO进行实验，根据不同的干预方法设置对照组、EGb761组、DDP组及EGb761+DDP组。采用CCK-8法检测EGb761、DDP对RKO细胞活力的影响。通过实时荧光定量聚合酶链反应（RT-qPCR）和Western blot实验检测鞘氨醇激酶1（SphK1）和凋亡相关因子半胱氨酸天冬氨酸蛋白酶3（Caspase 3）、B淋巴细胞瘤2（BCL2）的表达。采用流式细胞术实验检测RKO细胞的凋亡水平。通过慢病毒转染构建SphK1低表达的RKO细胞株，分析敲低SphK1和EGb761干预对凋亡相关因子表达水平的影响。在中药系统药理学数据库与分析平台（TCMSP）检索银杏叶的主要活性成分，通过分子对接探讨它们与SphK1的结合能力。结果 CCK-8实验结果显示，EGb761可抑制RKO细胞的增殖，并增强RKO细胞对DDP的敏感性。RT-qPCR实验和Western blot实验结果显示，与对照组相比，EGb761组、DDP组和EGb761+DDP组Caspase 3的mRNA和蛋白表达水平显著升高（P<0.05），且EGb761+DDP组的表达水平较EGb761组、DDP组更高（P<0.05）。与对照组相比，EGb761组、DDP组和EGb761+DDP组BCL2的mRNA和蛋白表达水平显著下降（P<0.05），且EGb761+DDP组的表达水平较EGb761组、DDP组更低（P<0.05）。流式细胞术实验结果显示，与对照组相比，EGb761组、DDP组和EGb761+DDP组的凋亡率显著升高（P<0.05），且EGb761+DDP组的凋亡率较EGb761组、DDP组更高（P<0.05）。EGb761可抑制RKO细胞SphK1蛋白表达，且作用效果呈剂量依赖性（P<0.05）。经慢病毒转染法成功构建SphK1低表达的RKO细胞株，敲低SphK1能显著提升RKO细胞对DDP的敏感性，而EGb761增强RKO细胞对DDP敏感性及促进凋亡的作用依赖于对SphK1的抑制。分子对接结果表明，银杏叶主要活性成分白果内酯、毛茛素、芝麻素、（+）-儿茶素、（-）-儿茶素和异鼠李亭与SphK1具有较强的结合能力。结论 银杏叶提取物EGb761通过抑制SphK1表达促进细胞凋亡，从而增强CRC细胞对DDP的敏感性。 |
| 关键词: 银杏叶提取物 结直肠癌 鞘氨醇激酶1 顺铂 化疗敏感性 |
| DOI：10.3969/j.issn.1674-3806.2025.04.07 |
| 分类号: |
| 基金项目:国家自然科学基金项目（编号：82260579）；广西中医药适宜技术开发与推广项目（编号：GZSY21-56）；广西研究生教育创新计划项目（编号：YCSW2023240） |
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| A study on the mechanism of Ginkgo biloba leaf extract EGb761 improving sensitivity of colorectal cancer cells to cisplatin |
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NING Qiting, CHEN Xingmei, HUANG Shanpei, ZHU Liye, QIU Xinze, LIU Shiquan
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Department of Gastroenterology, the Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, China
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| Abstract: |
| ［Abstract］ Objective To explore the mechanism of Ginkgo biloba leaf extract EGb761 improving sensitivity of colorectal cancer(CRC) cells to cisplatin(DDP). Methods Human CRC cells RKO were selected for experiment, and control group, EGb761 group, DDP group and EGb761+DDP group were set up according to different intervention methods. The cholecystokinin-8(CCK-8) method was used to detect the effects of EGb761 and DDP on the viability of RKO cells. Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR) and Western blot experiments were used to detect the expressions of sphingosine kinase 1(SphK1) and apoptosis-related factor cysteinyl aspartate-specific proteinase 3(Caspase 3), and B-cell lymphoma 2(BCL2). Flow cytometry experiment was used to detect the apoptosis level of RKO cells. The RKO cell lines with low expression of SphK1 were constructed through lentiviral transfection, and the effects of knocking down SphK1 and EGb761 intervention on the expression levels of apoptosis-related factors were analyzed. The main active ingredients of Ginkgo biloba leaves were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and their abilities to bind to SphK1 were explored through molecular docking. Results The results of CCK-8 experiment showed that EGb761 could inhibit the proliferation of RKO cells and enhance the sensitivity of RKO cells to DDP. The results of RT-qPCR experiment and Western blot experiment showed that compared with those in the control group, the mRNA and protein expression levels of Caspase 3 in the EGb761 group, the DDP group and the EGb761+DDP group were significantly increased(P<0.05), and the mRNA and protein expression levels of Caspase 3 in the EGb761+DDP group were higher than those in the EGb761 group and the DDP group(P<0.05). Compared with those in the control group, the mRNA and protein expression levels of BCL2 in the EGb761 group, the DDP group and the EGb761+DDP group were significantly decreased(P<0.05), and the mRNA and protein expression levels of BCL2 in the EGb761+DDP group were lower than those in the EGb761 group and the DDP group(P<0.05). The results of flow cytometry experiment showed that compared with the control group, the apoptosis rates in the EGb761 group, the DDP group and the EGb761+DDP group were significantly increased(P<0.05), and the apoptosis rates in the EGb761+DDP group were higher than those in the EGb761 group and the DDP group(P<0.05). EGb761 could inhibit the expression of SphK1 protein in the RKO cells, and the inhibitory effect was dose-dependent(P<0.05). The RKO cell lines with low expression of SphK1 were successfully constructed by lentiviral transfection method. The experimental results showed that knocking down SphK1 could significantly enhance the sensitivity of RKO cells to DDP, while the actions of EGb761 on enhancing the sensitivity of RKO cells to DDP and promoting apoptosis depended on the inhibition of SphK1. The results of molecular docking showed that the main active ingredients of bilobalide, ranunculin, sesamin, (+)-catechin, (-)-catechin and isorhamnetin in Ginkgo biloba leaves had strong abilities to bind to SphK1. Conclusion Ginkgo biloba leaf extract EGb761 promotes apoptosis by inhibiting SphK1 expression, thereby enhancing the sensitivity of CRC cells to DDP. |
| Key words: Ginkgo biloba leaf extract Colorectal cancer(CRC) Sphingosine kinases 1(SphK1) Cisplatin(DDP) Chemosensitivity |
