| 秦子昕1#, 邵雪花2#, 梁 赫1, 温雪梅1, 路 伟1*.双香豆素诱导斜纹夜蛾卵巢细胞程序性死亡的机理研究[J].植物保护,2023,49(1):96-107. |
| 双香豆素诱导斜纹夜蛾卵巢细胞程序性死亡的机理研究 |
| Mechanism of dicoumarin-induced programmed cell death in Spodoptera litura ovarian cells |
| 投稿时间：2022-08-18 修订日期：2022-09-27 |
| DOI：10.16688/j.zwbh.2022497 |
| 中文关键词: 双香豆素 细胞毒性 斜纹夜蛾 细胞凋亡 |
| 英文关键词:dicoumarin cytotoxicity Spodoptera litura apoptosis |
| 基金项目:新疆维吾尔自治区重点研发专项(2022B02033-1)；国家重点研发计划(2017YFD0201903)；新疆农业科学院科技创新重点培育专项(xjkcpy-2020004) |
| 作者 | 单位 | E-mail | | 秦子昕1#, 邵雪花2#, 梁 赫1, 温雪梅1, 路 伟1* | 1. 新疆农业大学农学院, 棉花教育部工程研究中心, 自治区农林有害生物监测与安全防控重点实验室, 乌鲁木齐 830052
2. 广东省农业科学院果树研究所, 农业农村部南亚热带果树生物学与遗传资源利用重点实验室, 广东省热带亚热带果树研究重点实验室, 广州 510640 | teerakon@sina.com |
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| 中文摘要: |
| 为探索双香豆素(dicoumarin, DIC)抑制斜纹夜蛾 Spodoptera litura 卵巢细胞SL-221增殖及作用机制, 首先, 采用CCK-8法测定双香豆素等21个酚类化合物对SL-221细胞的毒性; 其次, 通过倒置显微镜观察4 μg/mL双香豆素处理细胞48 h后其形态变化, 并结合流式细胞术研究双香豆素对SL-221的细胞周期、线粒体膜电位及凋亡的影响; 最后, 通过实时荧光定量PCR技术(RT-qPCR)探索磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)/雷帕霉素靶蛋白(target of rapamycin, TOR)营养信号通路的下游关键基因的表达变化。结果表明, 21个酚类化合物中双香豆素对SL-221细胞的增殖抑制活性最显著, 且呈现明显的浓度依赖性, 其48 h的IC50为0.85 μg/mL, 对细胞的增殖抑制活性是阳性对照印楝素(IC50为7.20 μg/mL)的8.47倍。4 μg/mL双香豆素处理SL-221细胞48 h后, 显微镜下可见细胞肿胀、膜破裂及大量凋亡小体; 流式细胞术分析发现, 双香豆素处理48 h, SL-221的细胞周期被阻滞在G2/M期, 同时线粒体膜电位相比0 h下降99.62%, 细胞凋亡, 且凋亡率与处理时间呈正相关。RT-qPCR检测发现, 双香豆素可显著下调营养信号通路(PI3K/TOR)上的关键基因 SL-PI3K 和 SL-TOR 的表达, 同时诱导自噬标志基因 SL-Atg8 及凋亡指示基因 SL-Cytochrome C 和 SL-P53 上调表达, 且抑制抗凋亡基因 SL-Bcl-2 的表达。综上, 双香豆素对斜纹夜蛾卵巢细胞具有优异的抑制增殖活性, 同时阻断细胞周期并通过抑制营养信号途径(PI3K/TOR)诱导细胞程序性死亡, 该化合物具备开发成为新型植物源杀虫剂的潜力。 |
| 英文摘要: |
| To investigate the inhibition of dicoumarin (DIC) on the proliferation of Spodoptera litura ovarian SL-221 cells and its mechanism, firstly the cytotoxicities of 21 phenolic compounds including DIC to SL-221 cells were detected by CCK-8 method; secondly, the morphological changes of SL-221 cells treated with 4 μg/mL dicoumarin were observed by inverted microscope, and the effects of dicoumarin on the cell cycle, mitochondrial membrane potential and apoptosis of SL-221 cells were studied by flow cytometry; finally, the expression changes of nutrient signaling pathways (PI3K/TOR) and its downstream key genes were explored by real-time quantitative PCR (RT-qPCR). The results showed that among the 21 phenolic compounds, DIC had the most significant inhibitory activity against the proliferation of SL-221 cells in a concentration-dependent manner. The IC50 value was 0.85 μg/mL after treatment for 48 h, and its activity was 8.47 times that of the positive control azadirachtin (IC50=7.20 μg/mL). The SL-221 cells treated with 4 μg/mL DIC for 48 h, showed swelling, membrane rupture, and apoptotic bodies. Flow cytometry analysis showed that the cell cycle of SL-221 cells was blocked in G2/M phase; the mitochondrial membrane potential decreased by 99.62% compared with 0 h, leading to apoptosis, after DIC treatment for 48 h, and the apoptosis rate was positively correlated with the treatment time. RT-qPCR analysis showed that DIC could significantly down-regulate the expression of key genes SL-PI3K and SL-TOR in the nutrient signaling pathway (PI3K/TOR), while up-regulating the autophagy marker gene SL-Atg8 and apoptosis indicator genes SL-Cytochrome C and SL-P53, and inhibiting the expression of anti-apoptotic gene SL-Bcl-2. Our study suggested that DIC has an excellent anti-proliferation activity against Spodoptera litura ovarian cells, and can block the cell cycle and induce programmed cell death by inhibiting nutrient signaling pathways. The compound has the potential to be developed into a new type of plant-derived insecticide. |
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