| 苏芳,徐家宝,徐烨,等.附苓方通过DRP1调控糖代谢重编程抑制卵巢癌细胞转移的机制研究[J].浙江中医药大学学报,2023,47(12):1363-1373. |
| 附苓方通过DRP1调控糖代谢重编程抑制卵巢癌细胞转移的机制研究 |
| Mechanism of Compound Fuling Granule Regulating Glucose Metabolism Reprogramming and Inhibiting Ovarian Cancer Metastasis through DRP1 |
| DOI：10.16466/j.issn1005-5509.2023.12.001 |
| 中文关键词: 附苓方 人卵巢癌HEY-T30细胞 DRP1 糖代谢重编程 转移 线粒体形态 |
| 英文关键词: Compound Fuling Granule ovarian cancer cell HEY-T30 DRP1 reprogramming of glucose metabolism metastasis mitochondrial morphology |
| 基金项目:国家自然科学基金项目(82074391)；浙江省自然科学基金项目(LY21H270004)；浙江省中医药管理局青年项目(2019ZQ013) |
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| 中文摘要: |
| [目的] 研究附苓方(Compound Fuling Granule，CFG)对卵巢癌细胞糖代谢及转移的影响，并探讨其作用机制。[方法] 体外培养人卵巢癌HEY-T30细胞，采用细胞计数试剂盒(cell counting kit-8，CCK-8)检测不同浓度的CFG(0.25、0.5、1、1.5、2 mg·mL-1)干预24 h后各组细胞活力；以Transwell实验检测CFG对HEY-T30细胞迁移的影响；采用乳酸测定试剂盒及葡萄糖检测试剂盒检测CFG对HEY-T30细胞乳酸生成量及葡萄糖消耗量的影响；采用免疫印迹法检测动力相关蛋白1(dynamin-related protein 1，DRP1)、糖酵解及转移相关蛋白的表达；以Mito-Tracker Red标记线粒体，观察线粒体形态。采用慢病毒转染技术，构建DRP1稳定敲低的人卵巢癌HEY-T30细胞，以实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction，Real-time qPCR)检测DRP1的mRNA表达，以乳酸测定试剂盒及葡萄糖检测试剂盒检测CFG对DRP1敲低的HEY-T30细胞乳酸生成量及葡萄糖消耗量的影响，Transwell实验检测CFG对DRP1敲低的HEY-T30细胞迁移的影响，免疫印迹检测CFG对DRP1敲低的HEY-T30细胞转移及糖代谢相关蛋白表达的影响。[结果] 与对照组比较，CFG浓度在0.5、1、1.5、2 mg·mL-1时细胞存活率明显下降(P<0.01)；0.5、1 mg·mL-1浓度组细胞线粒体平均长度明显增大(P<0.01)，DRP1蛋白表达量明显下降(P<0.05，P<0.01)，细胞乳酸生成量及葡萄糖消耗量明显下降(P<0.01)；0.25、0.5、1 mg·mL-1浓度组细胞迁移数量明显减少(P<0.05，P<0.01)。敲低DRP1后，卵巢癌细胞的糖酵解水平及迁移能力减弱(P<0.05，P<0.01)，糖酵解及转移相关蛋白表达有不同程度的下降(P<0.05，P<0.01)，CFG对卵巢癌细胞糖酵解及转移的抑制作用也被减弱。[结论] CFG可能通过DRP1调控糖代谢重编程，从而抑制卵巢癌细胞转移。 |
| 英文摘要: |
| [Objective] To investigate the mechanism of Compound Fuling Granule(CFG) in inhibiting the glucose metabolism and metastasis of ovarian cancer cells. [Methods] Ovarian cancer cell HEY-T30 were cultured in vitro and incubated with different concentration(0.25，0.5，1，1.5，2 mg·mL-1) of CFG for 24 h. Cell counting kit-8(CCK-8) assay was performed to detect the effect of CFG on the cell proliferation， Transwell assay was used to investigate cell migration ability， lactic acid detection kit and glucose detection kit were used to detect lactic acid production and glucose consumption， the expressions of dynamin-related protein 1(DRP1)， some key glycolysis-related proteins and metastasis-related proteins were detected by Western blot， Mito-Tracker Red was used to label mitochondria to observe mitochondria morphology. Lentivirus transfection technique was used to achieve the stable DRP1 knockdown HEY-T30 cells. Real-time quantitative polymerase chain reaction(Real-time qPCR) was used to detect the expression of DRP1 mRNA， the effect of CFG on lactic acid production and glucose consumption of HEY-T30 after DRP1 knockdown was detected by lactic acid detection kit and glucose detection kit， Transwell assay was used to investigate the migration ability of HEY-T30 with DRP1 knockdown after treated with CFG， the effect of CFG on the expression of glycolysis-related proteins and metastasis-related proteins in HEY-T30 with DRP1 knockdown was detected by Western blot. [Results] Compared with control group， the cell survival rate in 0.5，1，1.5，2 mg·mL-1 CFG groups reduced significantly(P<0.01). The average length of mitochondria in 0.5，1 mg·mL-1 ?group was markedly increased(P<0.01)， the protein expression of DRP1 was significantly reduced(P<0.05， P<0.01)， and the lactate production and glucose consumption in 0.5 and 1 mg·mL-1 ?groups were significantly decrease(P<0.01). The number of migration cell was significantly reduced in 0.25，0.5 and 1 mg·mL-1 concentration groups(P<0.05， P<0.01). After knockdown of DRP1， the glycolysis level and migration of HEY-T30 were decreased(P<0.05， P<0.01)， and the expressions of glycolysis-related proteins and metastasis-related proteins were decreased(P<0.05， P<0.01). The inhibitory effect of CFG on glycolysis and metastasis of ovarian cancer cells was also weakened. [Conclusion] By targeting DRP1 to regulate glucose metabolism reprogramming， CFG could inhibit the metastasis of ovarian cancer. |
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